Bioscience Blog Page
Powerful tools to achieve valuable results. Learn here about the latest news on our technologies and customer applications.
Bioscience Blog Page
Powerful tools to achieve valuable results. Learn here about the latest news on our technologies and customer applications.
-
Bioscience Blog
Powerful tools to achieve valuable results. Learn here about the latest news on our technologies and customer applications -
Learn more about the clinical trials: What are they exactly? What types of trials are there? How many phases are there? What happens in each phase?
Speeding up the PyroCell® MAT Testing
The monocyte activation test (MAT) is an in-vitro test system used to detect pyrogens in pharmaceutical preparations. It serves as replacement of the rabbit pyrogen test (RPT) that is consuming experimental animals. As compared to the Bacterial Endotoxin Test (BET) the MAT is capable of detecting non-endotoxin pyrogens in a sensitive manner.
The PyroCell® MAT System consists of cryopreserved, pooled human peripheral blood mononuclear cells (PBMC), an optimized supplement either based on fetal bovine serum (FBS) or human serum (HS), and a human IL-6 ELISA.
The Rapid ELISA Kit comprises two kit boxes, i.e. the PeliKine IL-6 ELISA Rapid Set A, containing binding antibodies, IL-6 reference standard and the HPE dilution buffer and the Pelikine IL-6 ELISA Rapid Set B (formerly known as Pelikine Toolset) that now includes the pre-coated plates, buffers, substrate and stop solution.
The new PeliKine human IL-6 Rapid ELISA assay is validated with the PyroCell® MAT Kit and PyroCell® MAT HS Kit for comparable performance. The two kits are available side-by side until Q1 2024. The performance report comparing the two ELISA kits and the customer notification letter can be retrieved upon request. Please reach out to scientific.support.eu@lonza.com (for Europe, Africa, and APAC) or to scientific.support@lonza.com (for AMERICAS).
Written by Ingo
Senior Scientific Support Specialist
Review on the Genome Editing of Immune Cells CRISPR Virtual Event 2022
Did you know that we had a great CRISPR virtual event in September 2022?
There were two sessions and a roundtable with interesting insights from our CRISPR experts.
The first session, entitled “CRISPR on its way to the clinic”, was kicked-off by Dr. Dimitrios Wagner, head of R&D at BeCAT from Charité - Universitätsmedizin Berlin. He gave a brilliant talk about the generation of tumour-specific T cells using virus-free CRISPR-Cas9 gene editing and linear dsDNA as a promising platform for rapid and scalable engineering of adoptive T cell therapies. The second talk of this session was from Tobias Bexte (MD student of the Children's Hospital, University Hospital Frankfurt am Main) who showed impressive results on transfection of natural killer (NK) cells. He concluded that primary CD19-CAR-NK cells demonstrate safe anti-tumour effect and have a huge potential for therapeutic approaches.
In the second session with a focus on CRISPR screenings, Dr. Theodore Roth (Resident, Stanford Pathology; Co-Founder, Arsenal Biosciences) gave a fantastic talk about scalable discovery systems for human cellular therapies and showed that it is possible to transfect human T cells with a 2-3 kb DNA sequence for a knock in.
Then Dr. Benedetta Carbone (Scientific Investigator from GSK) gave us new insights into the Phenotypic CRISPR screening in human iPSC-derived macrophages what she uses as a platform for drug discovery.
Roundtable questions:
What were your biggest CRISPR challenges and how did you overcome these?
Theodore Roth: “A big challenge is, if you vary one parameter e.g. how much DNA, finding the optimal one and then next week go on and vary another one. So, the interaction between different optimization parameters can be a challenge. Being able to test in high-throughput helps a lot to find local, but also global optimal combinations. This was super helpful and efficient, especially in the 96-well unit.”
Kevin Holden: “Started in 2015 with plasmids and was challenging, using RNPs really does help and literally gives you a standardized way to work with. If you can standardize transfection by using RNPs, this really helps and Nucleofection® was a huge difference maker, it always works!”
Where are we going to be with CRISPR in 5 or 10 years from now?
Benedetta Carbone: “From a sort of clinical slash, public perspective I am hoping to see a bit more acceptance, so less hocus-pocus vibe from the general regulation. Hoping that the public is more exposed to the CRISPR technology, introducing the technology more easily to the clinic with less hesitancy and restrictions.”
Dimitrios Wagner: “I think what CRISPR therapeutics really allows to dream about is the future, where we can achieve a solution for every person. Even for ultra-rare diseases, I hope that in 5 years, we have had seen a large amount of clinical trials with that early technologies, maybe we can see some clinical trials with the new technologies like base editing or we will see prime editing in the clinics.”
We thank all the speakers for their interesting talks and answering all the questions. Furthermore, we thank our moderators Melanie Homberg, Kathleen Burke, Luke Pratsis, Elke Lorbach as well as the whole event team for their effort to make this virtual event happen.
We are also looking forward to our next virtual event focusing on CRISPR in drug discovery at the 26 September 2023 – You can already register for the CRISPR event. In the meantime, you can also watch the 2022 CRISPR event on demand.
Written by Camilla
Scientific Support Specialist
The statements made by each of presenters does not necessarily represent the opinions of his or her employer.
Arrayed CRISPR screening of primary lung small airway epithelial cells with 384-well Nucleofector® System
Did you know that SAECs (small airway epithelial cells) are a crucial model for target identification and validation in drug discovery? During our virtual event about CRISPR in Drug Discovery Anna Dickson and Alessia Serrano (Senior Research Scientists at AstraZeneca) were two of our fantastic speakers.*
Recently, Dickson et al. published a scalable and automated CRISPR screening workflow for SAECs combined with a disease-specific endpoint assay in SLAS Discovery.
As primary cells such as SAECs are hard to transfect cells, CRISPR/Cas9 gene editing is often performed by ribonucleoprotein (RNP) nucleofection. An established and robust CRISPR screen is not only a powerful tool for drug target identification, but also for the target validation associated with disease phenotypes of primary cell types. The detailed screening protocol utilizes the advantages of both; a disease-relevant immortalised cell line can be utilized for a larger scale screen (e.g. genome wide) and then the top hits can be carried forward into a smaller, targeted validation screen using disease-relevant primary cells such as SAECs.
As it is impractical to assess the efficiency of editing of each individual gene in a screening, it is critical to include editing controls and genes which drive or reduce the desired endpoint phenotype to ensure confidence in the whole automation workflow. It is also crucial to add a neutral editing control for normalisation instead of unedited cells as CRISPR gene editing itself can affect cell health or growth.
We put together a short summary of the SAEC CRISPR screen workflow with the use of RNPs (for further details see Dickson et al.), 384-well Nucleofector® System and P3 Primary Cell 384-well Nucleofector® Kit.
Workflow
First step: Cell culture of SAECs (thawing and expansion for 8 days to achieve 30 Mio. cells)
Second step: Preparation of RNP plate and complex formation (2h)
Third step: CRISPR editing of SAECs via 384-well Nucleofector® System(4h)
Fourth step: Cell handling post Nucleofection® (2h)
Fifth step: Dispensing the edited cells into assay plates
Sixth step: Endpoint assay and analysis (4-5 days after CRISPR gene editing)
The publication also gives relevant information regarding critical steps, troubleshooting (e.g. errors due to bubbles) and can be easily adapted to other cell types.
If you are interested or planning CRISPR screening experiments, we highly recommend to read through this great publication.
We thank Anna Dickson and Alessia Serrano for their brilliant talks at our virtual event CRISPR in Drug Discovery. Of note, also other speakers spoke about setting up CRISPR screens – watch it on demand.
Written by
Camilla Scientific
Support Specialist
References:
Dickson A, Mullooly N, Serrano A, Escudero-Ibarz L, Wiggins C, Gianni D. Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells. SLAS Discov. 2023 Mar;28(2):29-35
Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses, related to performing the screening workflow, including using CRISPR/Cas9.
*The statements made by each of the presenters does not necessarily represent the opinions of his or her employer.
Lonza Joins the B2Run in Cologne: Running for a Cause
Cologne, known for its rich history, stunning architecture, and vibrant culture, witnessed a different kind of spectacle recently as 19,000 participants from 700 companies came together for the annual B2Run event. This exciting event, which promotes corporate health, teamwork, and community engagement, brought colleagues from diverse industries onto the streets to participate in a 5.3-kilometer run. Among the multitude of companies, Lonza proudly stood out as one of the participants, showing its commitment to employee well-being and social responsibility.
Lonza Cologne Presence
With 25 registered participants, Lonza made its presence felt at the B2Run in Cologne. It's remarkable that Lonza employees not only started the race but also successfully crossed the finish line, showcasing their determination and enthusiasm for both the event and the company's values.
Running for a Cause
The B2Run in Cologne wasn't just about running; it was also about giving back to the community. Each participant contributed 5 Euros to DKMS, a German nonprofit organization dedicated to the fight against blood cancer. This collective effort by thousands of participants from various companies demonstrates the power of corporate social responsibility and the positive impact it can have on society.
Green Initiatives
Apart from supporting a charitable cause, the B2Run in Cologne also had an eco-conscious touch. In line with their commitment to sustainability, the event organizers planted one tree for each participating company. Lonza, as one of the 700 participating companies, contributed to this green initiative, helping to make the world a greener and healthier place. The B2Run in Cologne not only provided a platform for employees to bond, promote physical activity, and give back to the community but also allowed companies like Lonza to underscore their commitment to employee well-being and sustainability. It's an excellent example of how corporate events can be meaningful, fun, and impactful on both individual and societal levels. As Lonza continues to foster a culture of health, teamwork, and social responsibility, participating in events like the B2Run in Cologne becomes an integral part of their corporate identity. It's not just about running; it's about running with purpose and making a difference in the world.
Written by
Peter
Associate Director Scientific Support Bioscience
Source: Text-label and text logo are from B2Run
The Effect of Light on Media Performance
Several types of Lonza media contain labels stating, “protect from light,” but why is this? Can light exposure really be that harmful to the media?
The answer is yes! In fact, cell culture media contains various components that undergo a process called photooxidation, generating reactive oxygen species (ROS) that are toxic to cells. Scientists from the University of Strathclyde in Glasgow, Scotland found that violet-blue light in the region of 405 nm has antimicrobial activity, but at the same time can become cytotoxic to mammalian cells (1). This is an important finding, considering many labs still use ultraviolet radiation as means to sterilize culture surfaces.
Not only does light exposure influence cell viability, but it can also activate induce cell signaling. Scientists from the Institute of Environmental Medicine determined that a tryptophan-induced photoproduct called 6-Formylindolo [3,2-b] carbazole or FICZ, acts as a ligand to the aryl hydrocarbon receptor (AhR), a transcription factor that induces expression of CYP1A1 (2). This CYP enzyme is involved in many metabolic processes in the liver.
Furthermore, the effects of light exposure to culture media is not limited to bacterial and animal cells. Scientists have also found UV and blue wavelengths in unfiltered light inhibits growth of three different plant species (3).
What type of cell media components are facilitating this process? There are many, but I will mention only a few here (4).
Riboflavin (vitamin B2): Involved in redox processes for metabolism. Upon exposure to light, riboflavin becomes highly reactive, assisting in redox reactions that generate harmful ROS.
Folic acid (vitamin B9): Acts as a coenzyme in many metabolic reactions. Degradation of folic acid is accelerated by riboflavin in the presence of UV light.
Photoeffects can be reduced if antioxidants are added to the medium and more obviously, if the medium is protected from light as much as possible. It is important to consider these details when planning your experiments so that you do not waste time or effort and can protect your precious cells!
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Quick Tips for Successful Nucleofection Experiments
When it comes to science and research I think we can all agree that the path to reliable and consistent results begins with the basics. These may not be the most exciting things to discuss but they are the foundation of designing and conducting quality experiments. We all know that running the proper controls are important yet this step often gets skipped in the interest of time or maybe because we feel we don’t need them. So with that in mind here are some basic tips to follow for successful NucleofectionTM experiments. We’ve covered the first topic already: Always run your positive and negative controls.
Next up is passage number. Cells of a lower passage number typically respond better to transfection and will have higher transfection efficiencies and viabilities than those of higher passage numbers. For the most efficient gene transfer, we recommend using cells that are in logarithmic growth phase and at a passage number lower or less than 10 – 15 (from the time of thaw).
When working with adherent cells for transfection, the cells should be grown to a confluency level of
70 – 85%. Higher or lower than this will give you sub-optimal results. Nobody, including us, wants that.
When using suspension cells, they should be transfected when they are in the logarithmic growth phase. Generally, this corresponds to a density of 2 – 5 x 105 cells per ml.
For both adherent and suspension cells, it is important to make sure that the culture is growing properly and that the cells have the proper morphology. If they do not, this could indicate contamination with bacteria, fungi, or mycoplasma.
Cell harvesting and handling comprises about 95% of the troubleshooting I do regularly and proper handling leads to better results. Before harvesting your cells, wash the monolayer to get rid of any residual growth medium. Always refer back to the optimized protocol to see what is required and never include any extra wash or centrifugation steps that are not mentioned in the protocol.
Never vortex or scrape your cells. Suspension cells just need to be centrifuged to remove the growth media and then directly resuspended in NucleofectorTM solution (no extra wash required!)
Lastly, a word about centrifugation. You have probably noticed that we do not use RPM’s. Our standard is 90xg (there are some exceptions based on the cell type so be sure to check your protocol). The speed you select to obtain 90xg will depend on your rotor so consult the operation manual for your centrifuge.
Centrifugation speeds and g-forces may not be as critical with other transfection methods but they are with Nucleofection so be sure to calculate those g’s! Here’s a handy website you might find helpful:
Centrifuge Rotor Speed Calculator
I wish you good luck with all of your experiments and we are always just a phone call or email away.
Written by Sean
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Summary of Strengths: CAR-NK Cells and Automated Production for Immunotherapy
Although anti-cancer therapies via pharmaceutical approaches have seen much improvement in the last several decades, the high recurrence rate of malignancy remains an unsolved problem. As the therapies continue to evolve, a new class of cell-based therapeutics has emerged, with autologous CAR-T cells leading the field and driving unprecedented outcomes in patients with relapsed/refractory malignancies. Natural Killer (NK) cells, given their intrinsic abilities to identify and kill tumor cells, have become an attractive source for anti-cancer cell therapy. In comparison to CAR-T cells, the application of CAR-NK is reported to have a number of additional benefits.
• Large doses of T cells can induce cytokine release syndrome (CRS). NK cells have a distinct cytokine release profile which reduces the occurrence of CRS and therefore strengthens the safety of use.
• Whereas CAR-T cells will mostly kill target cells by a CAR-specific mechanism, CAR-NK cells can leverage NK-cell-intrinsic cytotoxicity mechanisms to induce target cell death. This boosts the killing effect and may help to maintain an anti-tumor response even when target cells down-regulate specific antigens in both CAR-dependent and CAR-independent manners.
• Allogeneic-grafted CAR-NK cells have a profound killing effect. These cells hold great potential to become an ‘’off-the-shelf product’’, as therapeutic manufacturers can obtain potent NK cells from healthy donors, thus expanding the material availability for cell therapy manufacturing. Additionally, clinical evidence has shown that CAR-NK cells present a low risk of inducing graft-versus-host-disease.
• Based on current regulations, CAR-NK infusions do not require hospitalization due to the low potential toxicity, reducing the high clinical costs associated with cell therapy administration.
However, the pursuit of high-quality, clinical-grade manufacturing consumes excessive resources. An automated cell therapy production platform can help reduce the production cost and hence make the therapy more accessible to the public. Further, there are more advantages to implementing automated cell production:
• Parameters in an automation system are traceable: a fundamental requirement in a GMP environment.
• Automation reduces the requirement for a highly-trained workforce and supports protocol reproducibility.
• Implementation of automation at early stage supports smooth scalability during and after regulatory approval.
• Potential for reducing overall project cost through streamlined development.
Though much progress has been made, the application of CAR-NK cell therapy is still at an early stage. In spite of encouraging news from ongoing clinical trials for both hematological and solid tumors, further investigations are required to complete the safety profile for individual CAR-NK cell therapies. As CAR-NK therapies continue to progress through clinical development, choosing an automated manufacturing platform can help reduce the complexity of these processes. Utilizing best-in-class automation instrumentation to link individual closed manufacturing processes is one option for facilitating the expansion of clinical manufacturing. The Cocoon® Platform from Lonza, for example, can be a reliable choice for production for emerging immunotherapies, such as CAR-NK cells. This automated, functionally closed system can reduce hands-on requirements for cell isolation, activation, genetic modification (transduction or transfection) and expansion of cells.
Written by Ming, Scientific Support Specialist, together with Tamara and Peter from the Personalized Medicine team
References:
Khawar MB, Sun H. CAR-NK Cells: From Natural Basis to Design for Kill. Front Immunol. 2021 Dec 14;12:707542.doi: 10.3389/fimmu.2021.707542. PMID: 34970253; PMCID: PMC8712563.
The information contained herein is believed to be correct. However, no warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information. Lonza disclaims any liability for the use of this presentation and the use of the information contained herein is at your own risk. All trademarks belong to Lonza or its affiliates or to their respective third party owners and are only being used for informational purposes. The products, methods, and processes described in this presentation may be covered by one or more granted patents or pending patent applications. For a complete listing please visit our IP site. ©2023 Lonza. All rights reserved.
How to create your own quote on our website
You are looking to purchase products on our website, but first you need a quote. You can generate the quote yourself in a few simple steps, here’s how:
1) Add all the product(s) you need to your shopping cart
2) Go to your cart
3) Click “Generate Quote” at the bottom of your list (see first image on the left)
4) In case you are not logged in, please login or register
5) Your quote will be generated straight away and displayed on the Quote Details page (see second image on the left).
Download the quote in pdf format under “Download”, save and share with others, e.g. for PO generation
Written by
Peter
Associate Director, Scientific Support
Scaling Up for Clinical Use - CAR-T Cell Expansion
What is the status quo of CAR-T cell therapies? In the last three years, CAR-T cell therapy has stepped into the world of medicine, directly driving in the fast lane. Supported by FDA approvals, several companies started clinical trials and the outcome was amazing.
However, individualized treatments go along with high costs, a topic that needs to be covered…
In this context, I came across the publication of Morita et al. who started investigating how to optimize and enhance the expression of Anti-CD19 CARs in CAR-T cells.
Just for information, the actual costs for a one-time treatment are around $400,000. This definitely needs to be diminished in order to make CAR-T cell therapies accessible to patients in real life.
The starting point of these researchers was the use of CD19.CAR-T cells transfected with the piggyBac transposon system. This method already features reduced costs compared to viral transduction. However, in order to further shorten time lines and costs, cell cultivation must be improved to end up with the required number of functional CD19.CAR-T cells for clinical adoptive transfer.
Parameters for optimization in this publication were:
- The use of autologous activated T cells as feeder cells
- Stimulating the T cells via viral antigens instead of anti-CD3/CD28 mAb
- Using a CH2CH3-free CD19.CAR construct
Using these modifications, 51 % CAR-T positive cells were achieved with a 2,8 fold expansion after 14 days of culture. - Good approach. It’s just the beginning…of the next “Industrial” Revolution.
I hope to read more about successful optimizations of large-scale transfections, medium improvements and stimulation techniques - all applicable for clinical use of course - in the near future.
Read the full article here.
Written by Isabella
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Senescent Zombie in Primary Cell Culture
The state of senescence is also known as the zombie state. Swollen cells in a state of senescence have lost their original features, function and affect nearby cells. This state is irreversible and there’s currently no effective reversal. Various cellular stresses are reported as the main cause inducing cellular senescence, including telomere exhaustion, oncogene expression and DNA damage. Cellular senescence has been recognized not only as a hallmark of in vitro cell culture decay but also as an important contributor to age-related disorders in a wide variety of tissues and organs.
An increase of senescent cells in culture reduces proliferation capacity and gradually decays the culture. Despite this, the zombie cells can affect other cells through a specific secretome: the senescence‐associated secretory phenotype (SASP). This includes chemokines, as well as inflammatory, pro-angiogenesis and growth regulators, serving as communication signals between cells (paracrine and autocrine signaling). The paracrine signaling of the SASP extends the malfunction of senescent cells from intracellular to tissue level, leading to changes in intracellular microenvironment.
Senescent cells generally have an enlarged and flattened cell morphology. However the appearance can differ between cell types. To more precisely verify a state of senescence, there are multiple markers that can be utilized to identify these zombie cells in vitro and in vivo. The commonly used markers include γ-H2AX, cyclin dependent kinase (CDK) inhibitors (Cdkn1a, Cdkn2a and Cdkn2d), nuclear Lamin B1 protein and lysosome abundance detected by senescence associated-β-galactosidase (SA-β-gal) staining. However, none of these markers can provide evidence the senescent state on its own. All these markers have their own cellular characteristics and participate in other biological pathways. Therefore, the zombie state needs to be evaluated by multiple markers in parallel to formulate a comprehensive judgment of the cell senescence.
For primary cultures, manufacturer protocols optimize culture confluency, reduces reactive oxygen species (ROS) propagation and therefore can minimize cellular senescence (following a well-designed instruction is important!). However primary cells have their limitation in vitro. The zombies can eventually appear with high cell passage. As all organs go through aging, utilizing senescent culture and exploring the field of senescence are becoming the latest research trend, eg. selective senescent cells removal through pharmaceutical approaches (fight the zombies!).
Written by
Ming
Scientific Support Specialist
References:
- King. Zombie cells hold clues to COPD progression. Nature. 2020 May 13
- Kumari R, Jat P. Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. Front Cell Dev Biol. 2021 Mar 29
- Liao CM, Wulfmeyer VC, Chen R, Erlangga Z, Sinning J, von Mässenhausen A, Sörensen-Zender I, Beer K, von Vietinghoff S, Haller H, Linkermann A, Melk A, Schmitt R. Induction of ferroptosis selectively eliminates senescent tubular cells. Am J Transplant. 2022 May 23
Detection of CRISPR Off-Target Effect with DISCOVER-Seq
Although the CRISPR/Cas9 system has many advantages for use in gene editing including efficiency, simplicity and target specificity, the potential for off-target effect (via Cas9 enzyme binding to unintended genomic sites for cleavage and inducing mutations) also has a certain influence on clinical applications. Therefore, it is important to consider potential off-target effects and to verify if off-target cleavage has occurred.
To address testing for off-target cleavage, did you know that it is possible to use a DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) approach when using CRISPR/Cas9 technology for gene editing?
A detailed experimental protocol using the 4D-Nucleofector® Device and analysis pipeline was recently published for the use of ribonucleoproteins (RNPs), but in theory, it can be used with any type of CRISPR-Cas delivery vehicle (e.g. lipofection, viral transduction). Furthermore, any form of editing reagent (e.g. RNPs, plasmids, mRNAs) can be used with the DISCOVER-Seq workflow.
This method is based on tracking the precise recruitment of MRE11 to double-strand breaks (DSBs) by chromatin immunoprecipitation and subsequent next-generation sequencing. BLENDER (blunt end finder) as a customized open-source bioinformatics pipeline is then able to identify off-target sequences genome-wide in primary cells and in situ. There are three advantages of this testing approach including low false-positive rates, the application of the method to a wide range of systems like cells from patients and animal models, and the rapidity of the test method in that the whole protocol can be completed within 2 weeks.
Please keep in mind, it is quite important to consider the off-target effect and to select your sgRNAs wisely with a low off-target effect and high on target effect.
If you need help with your CRISPR experiments, for more information concerning sgRNA design tools and troubleshooting/optimizing your transfection efficiency, you can reach Lonza scientific support using the regional e-mail adresses: US e-mail address or EU / international e-mail address.
Looking forward to your results in knock-ins, outs, base editors, epigenetic editing, screenings, etc. by the use of the CRISPR tool like a ‘Swiss army knife’.
Written by
Camilla
Scientific Support Specialist
References:
Wienert B, Wyman SK, Yeh CD, Conklin BR, Corn JE. CRISPR off-target detection with DISCOVER-seq. Nat Protoc. 2020 May;15(5):1775-1799
Endotoxin Detection Certificates of Analysis – A General Guide
Compendial endotoxin detection assays contain LAL lysate made from the blood of horseshoe crabs (Limulus polyphemus) which measures sample endotoxin potency. That measurement is based on correlating the sample response to an analyst prepared standard curve. The FDA requires that the lysate and endotoxin used for the standard curve be matched. This matching happens when the lysate/endotoxin combination is standardized against the USP Reference Standard Endotoxin (RSE). Any company purchasing an LAL assay must use matching reagents in order to be compliant.
A benefit to purchasing Lonza endotoxin detection assays is that for most of the assay kits, the matching lysate and control standard endotoxin (CSE) vials are contained inside the kit box. The associated Certificate of Analysis (CoA) contains the lot number of the vials, the reconstitution potency for the CSE, and is the link that demonstrates the vials are matched if questioned by an auditor.
As a member of Lonza’s Scientific Support Team, I often receive customer questions regarding the COA. For example, “Where can I find the CoA? It wasn’t in the kit box.” or “Why isn’t my COA coming up on the website?” are very common inquiries.
A few CoA points to consider:
- All Lonza CoAs can be found at our CoA webpage
- The key to searching for the kit’s CoA is to enter the kit catalog number and lot number – not the vial catalog number and lot number. While many times the vial CoA may be available, it will not contain the matching information needed to perform the assay. However, the kit’s CoA will contain this information.
- Save the printed CoA in a binder or save the CoA PDF to a desktop folder for easy viewing during an audit or when needed as a quick reference for performing the assay.
- Always reference the CoA prior to setting up the standard curve to ensure that the properly matched vials are being used for that test.
One final thought: if you ever have an issue accessing the Lonza website or cannot find the matching CoA, please contact scientific support for assistance!
You can reach Lonza scientific support using the regional e-mail adresses: US e-mail address or EU / international e-mail address.
Written by Travis
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
The Basics of Clinical Trials
With the advent and development of new and novel therapeutics, more and more questions arise about clinical trials. What are they exactly? What types of trials are there? How many phases are there? What happens in each phase?
While the topic is more complex and involved that we can fully cover here, the hope is that this can answer some of the more basic questions.
So, what are Clinical Trials? In short, they are voluntary research studies that are conducted in people and designed to answer various questions about the safety and/or efficacy of drugs, vaccines, or other therapies.
We are all probably familiar with the idea of there being different phases in Clinical Trials. We will often hear the terms Phase I or Phase II for example. But before we get to that, did you know there are also different types of trials?
Let’s review those now.
- Preventative Trials: These are for testing new medications and vaccines. This type of trial is used to determine if the medication or vaccine in question can lower someone’s risk of disease.
- Screening Trials: These trials focus on finding ways to detect and diagnose diseases before patients experience the symptoms of those diseases. Typically the point here is to see if earlier detection will lower the risk of disease.
- Diagnostic Trials: These are similar to Screening Trials but they focus on detection and diagnosis after the patient begins to experience symptoms
- Treatment Trials: The are the trials that most of us probably think of when it comes to trials. These test new medications, medical devices, and other therapies in order to collect and determine safety and effectiveness.
- Genetic Studies: These are done to learn new ways to predict various genetic disorders earlier.
Now, let’s move on to the different phases of a trial.
Phase One: I should point out that prior to entering into a Phase One Trial, you should have collected enough laboratory data to indicate that your product is worth investigating further. During Phase I a small group of participants that represent those that would be ideal patients for your product (a medication or therapy) actually use the product and report any results including side effects or other symptoms back to you. All information must be recorded.
Phase Two: If the results from Phase One show that your product was safe for use, you can now test it on a larger number of patients. If the product is a medication for example, appropriate dosage should have been determined in Phase One. If, after Phase Two, you have tested the product on a large enough group and the FDA is reasonably sure that it can be effective on a larger scale, you can move the Phase Three.
Phase Three: In this phase your product is tested on larger groups of people and you will monitor the side effects and efficacy for each patient. If your product is a medication, you can utilize placebos which act as a control. Essentially, they help reduce the effects of any perception or expectations that your patients may have.
Phase Four: If you’ve made it this far, congratulations! Your product, be it a medication, therapy, surgery, or medical device has received FDA approval. Studies done during this phase focus on the longer-term effects of your product and gathering more data.
Again, this is a very simplified overview. Any intention to take a product into trials requires a lot of work, years in many cases, and huge amounts of research. This is in no way a definitive guide. I do hope it helps explain the differences in the types of trials and the basics of the trial phases.
Written by Sean
Senior Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
- K Rich. An overview of clinical trials. J Vasc Nurs. 2004 Mar;22(1):32-4
- CA Umscheid, DJ Margolis, CE Grossman. Key concepts of clinical trials: a narrative review. Postgrad Med. 2011 Sep;123(5):194-204
Why We are Here
During an internal meeting to mark the end of the year, a colleague from our Nucleofector® Sales Team shared a story which touched our hearts deeply. This story really shows why we love our jobs and therefore we would like to share this with you. And it goes like this:
"My maternal grandmother died from complications of diabetes when I was a junior in high school. Six months later, when I started my senior year of high school, my paternal grandmother died after her colon cancer metastasized. So, I have a vested interest in helping move research forward, especially cancer research…"
Further on this, the colleague reported that in the last few days she was in close contact with one of her customers having issues with their 4D-Nucleofector® LV Unit failing to transfect cells from a patient. Currently, this lab is running an important clinical trial for cancer treatment, for patients who have not responded to earlier therapies. Luckily, the patient yielded enough cells so that there was a "spare" billion cells to try again. Just one more try.
At this point it was more than important to connect to our Engineering Team to check for their experience and feedback on the issue observed in the first attempt. One of our engineers analyzed the results from the first trial and explained that the observed issue seemed to be more related to a handling issue rather than to the device itself and she wound up Face Timing with the researchers as they ran the remaining cells immediately after the call to support them in their second trial. The cells were transfected successfully and could be used for treatment.
"Which means that our technology – our engineering of the 4D-Nucleofector® LV Unit, all of the training we’ve received, each of us, were part of giving this patient another shot at life.
I cannot tell you how much it means to me that we have been entrusted with this responsibility. Nor can I express my pride, doubt, fear, and excitement at being part of this!
I have watched cancer eat away people I love and it sucks! It is very fulfilling to be a part of the solution in being able to help someone fight, even though we won’t meet that person. And I just want to say thank you! Because someday, it might be me and it’s very comforting to know that there’s an army behind the frontline of healthcare workers working towards making a possible cure."
To protect the privacy of everyone involved, some details have been adapted.
Story shared by the Sales Team and written by SST and Marketing
Lonza Pharma-Bioscience Solutions at Lonza
Ph. Eur. announces end for Rabbit Pyrogen Test
Rabbits, rejoice: The end is nigh!
Specifically, the end for the Rabbit Pyrogen Test (RPT). First introduced to regulatory documents in the 1940s, it has been the official test to detect pyrogen contaminations in pharmaceuticals for decades. However, science marches on, and other tests have become available. The first was the Bacterial Endotoxins Test, which since has risen to be the “go-to” test in most cases. It’s not always applicable, though, as it only detects endotoxins and not all pyrogens. Therefore, a major step was the invention of the Monocytes Activation Test (MAT) in the 1990s, which is a direct replacement for the RPT.
Nowadays, animal testing is being increasingly frowned upon. European authorities have been critical about the continued use of RPT for years, especially as an alternative in-vitro-test exists and is recognized as a compendial assay as per the European Pharmacopoeia (Ph. Eur., Chapter 2.6.30. Monocyte-Activation Test, since 2009).
On 28 June 2021, the Ph. Eur. issued a press release announcing that they are finally putting a stop to the RPT. Within the next five years, so the approximated timeline, references to pyrogen testing with RPT in monographs will be replaced “with a suitable in-vitro alternative, ultimately leading to the complete elimination of the RPT”. The aim is to push companies to adopt the MAT instead.
With this, the Ph. Eur. actively supports yet again efforts towards the goal of “3R” – i.e. to replace, reduce, or refine use of animal for testing everywhere.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Written by
Peter
New cellular models to study Parkinson disease
Parkinson’s disease (PD) is the second most common neurodegenerative disease, after Alzheimer’s disease. It is a progressive disorder primarily affecting dopaminergic neurons of the substantia nigra and is characterized by progressive development of motoric symptoms such as tremors and bradykinesia. PD is often associated with other symptoms unrelated to movement such as loss of smell and cognitive impairments. Despite multiple studies, the exact mechanism of what causes PD remains currently unclear. Genetic analyses of familial cases have implicated involvement of several genes and pathways in the pathophysiology of PD.
CRISPR in the Classroom
The CRISPR (clustered regularly interspaced short palindromic repeats) – CAS (CRISPR associated protein) system is a prokaryotic adaptive immunity mechanism. Emmanuelle Charpentier and Jennifer A. Doudna adapted this system as a tool for gene editing experiments, for which they became the winners of the Nobel Prize 2020 in Chemistry. This new revolutionary technology has become a valuable tool in cell biology.
Empowering NK Cell Immunotherapy Research Using a Novel Genome Editing Protocol
I was wondering why Natural Killer (NK) cells lag behind the scene of the blossoming immunotherapeutic targets and agents, although they naturally scan and clean our body from stressed cells, either infected or transformed. They limit metastasis and are increasingly recognized to orchestrate inflammation and immune infiltration in tumors. Additionally, NK-mediated cytotoxicity operates in a mode that has desirable qualities for cancer immunotherapy.
NK cells express a number of activating and inhibitory receptors. Deletion of diverse inhibitory receptors dramatically enhanced their anti-tumor immunity, making them good targets for empowering the killing potency of NK cells with inhibitory drugs or gene silencing / knockdown.
If you already felt the pain of trying to gene edit primary human NK cells, you will appreciate this forward work of Prof. Nick Huntington’s team from Australia (Rautela et al, Efficient genome editing of human natural killer cells).
It underlines some of the field difficulties:
- Differences in NK cell phenotypes and frequencies between species
- Low NK cell transduction efficiencies with retro/lentivirus
- Poor survival of NK cell post-electroporation
Researchers have been waiting for rapid and efficient gene editing tools to better understand the biology of NK cells and use them for immunotherapies. This work address exactly this aspect and could potentially reshape the field.
The authors optimized transfection conditions of human NK cells using the 4D-NucleofectorTM Device, with different NucleofectionTM solutions and substrates. They identified DN-100, CM-137 and CN-114 programs to result in 80-95% transfection efficiency of a 70kDa FITC-labelled dextran with reasonable to great cell viabilities. For targeted genome editing, they transfected CRISPR-Cas9 ribonucleoprotein (RNP) complexes, using a NLS-tagged Cas9. The Cas9 RNP cell delivery outcome was largely superior to Cas9 plasmid transfection. In fact, RNP enable genome editing independently of plasmid transcription / translation, circumvent NK cell sensitivity to DNA, and has a boosted nuclear delivery through NLS.
In a gene knockout attempt, Nicks’ team observed loss of both copies of PTPRC gene (CD45-/-) in 75% of primary NK cells. They deleted the cytotoxicity triggering receptor NKp46 or the cytokine-induced checkpoint gene Cish in primary human NK cells and validated their key roles in regulating the anti-tumor function of human NK cell in vivo. Interestingly, they observed significantly higher gene deletion when editing was done after 2 weeks of NK cell expansion and stimulation (85% NKp46 negative) compared to fresh NK cell (about 25% NKp46 negative) from same donor.
This approach paves the way for large-scale genetic modification of NK cells. With further optimization of guide sequences, multiplexing using different guides RNA and CRISPR Cas source, most genes would be knocked-out at a high efficacy to clarify their implication in NK cell biology. I trust this method will highly serve immunotherapy drug discovery including rapid gene-editing of autologous or allogenic NK cell therapy products for personalized medicine.
Written by Nazim
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Improving Bioprocess Applications with Stable Glutamine
L-glutamine is an amino acid commonly used as an auxilary energy source in cell culture. Specific metabolic roles include, but are not limited to:
- Providing a non-toxic nitrogen source to cells
- Providing a nitrogen source for nucleotide synthesis and transamination
- Providing an alternative energy source when glucose levels are low and energy demands are high
L-glutamine is typically supplied to a cell culture in excess, approximately 3 to 8-fold greater than other amino acids. Therefore, some degradation of L-Glutamine can be tolerated without adverse effects. Typical concentrations range from 2-10 mM.
However, it is important to note that L-glutamine can be a relatively unstable molecule depending on the temperature at which it is stored. Lonza data for long-term storage indicates that the L-glutamine concentration is reduced by approximately 3% per month in cell culture media stored at 2-8°C. When stored at 35-37°C, approximately 50% L-glutamine remained after only 3 days. When L-glutamine spontaneously breaks down in solution, it generates ammonia, a cytotoxic metabolite, and pyrrolidine carboxylic acid .
The more stable counterpart of L-glutamine is the dipeptide L-alanyl-glutamine which Lonza produces under the name UltraGlutamineTM I Supplement . UltraGlutamineTM I Supplement can be autoclaved (121°C, 20 min) with no significant breakdown. UltraGlutamineTM I Supplement can substitute L-glutamine on an equimolar basis with little to no adaptation for adherent and suspension cultures. Growth performance of most cell types is comparable to that obtained with L-glutamine without toxic ammonia accumulation in the culture. Some cells are sensitive to ammonia even at non-toxic levels.
Applications that benefit from UltraGlutamineTM I Supplement are:
- Ammonia sensitive systems (bioreactors)
- Long term toxicity studies
- Long incubations without feeding (cloning assays)
In fact, customer feedback indicates a preference for alanyl-glutamine (UltraGlutamine► I Supplement) over L-glutamine for increased stability in their media and more robust performance of their bioprocesses. Be sure to try it out for yourself.
For technical questions related to this product, please contact our Scientific Support Team
US: 1-800-521-0390 , scientific.support@lonza.com
EU: +49 221 99199 400, scientific.support.eu@lonza.com
For a product quote, please contact the Sales Support Team
EU: LBSSalesSupportEU.Verviers@lonza.com
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References:
A. Chevriaux, T. Pilot, V. Derangère, H. Simonin, P. Martine, F. Chalmin, F. Ghiringhelli, C. Rébé. Cathepsin B Is Required for NLRP3 Inflammasome Activation in Macrophages, Through NLRP3 Interaction. Front Cell Dev Biol. 2020 Mar 31;8:167.doi: 10.3389/fcell.2020.00167. eCollection 2020.
M. Marzotto, F. Arruda-Silva, P. Bellavite. Fibronectin Gene Up-regulation by Arnica montana in Human Macrophages: Validation by Real-Time Polymerase Chain Reaction Assay. Homeopathy. 2020 Aug;109(3):140-145. doi: 10.1055/s-0040-1708044. Epub 2020 Apr 20.
J. Mock, C. Pellegrino, D. Neri. A universal reporter cell line for bioactivity evaluation of engineered cytokine products. Sci Rep. 2020 Feb 24;10(1):3234. doi: 10.1038/s41598-020-60182-4.
P. Martine, A. Chevriaux, V. Derangère, L. Apetoh, C. Garrido, F. Ghiringhelli, C. Rébé. HSP70 is a negative regulator of NLRP3 inflammasome activation. Cell Death Dis. 2019 Mar 15;10(4):256. doi: 10.1038/s41419-019-1491-7.
S. Irmscher, S. R. Brix, S. L. H. Zipfel, L. D. Halder, S. Mutlutürk, S. Wulf, E. Girdauskas , H. Reichenspurner, R. A. K. Stahl , B. Jungnickel, T. Wiech, P. F. Zipfel, C. Skerka. Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies. Nat Commun. 2019 Jul 4;10(1):2961. doi: 10.1038/s41467-019-10766-0.
Allergy Injections and Endotoxins
A few years ago it was determined that allergy injections were necessary to reduce my suffering from the flora of the changing seasons, household dust and the dander from common household pets. Per the ACAAI.org website, “allergy shots, also known as subcutaneous immunotherapy (SCIT), are the most commonly used and most effective form of allergy immunotherapy. This is the only treatment available that actually changes the immune system, making it possible to prevent the development of new allergies and asthma.”1
I don’t receive just one or two injections; I receive four allergy injections every other week. Each group of allergens is allocated to one of four vials and injected into my arms at four different points. These vials are prepared at an immunologist’s office in a very similar fashion as a pharmaceutical compound is prepared. Even though this is a sterile environment and the stock solutions are sterile, is there a concern about endotoxin contamination? Will endotoxins affect the allergen immunotherapy?
In 2003, three NIH scientists examined this exact topic. Fourteen different allergen vaccines were tested for endotoxin levels using the 0.06 EU/ml sensitivity gel clot assay2. Beta glucans and protease interferences were inactivated by dilution and heat-inactivation.
A total of 58 lots of vaccines were tested. The endotoxin content for the samples of note that trigger my allergic response is as follows:
- Cat hair – 2883 EU/ml
- Grass – 160 EU/ml
- Ragweed pollen – 341 EU/ml
This data is fairly startling to me as the USP endotoxin limit for parenteral drugs is 5 EU/kg/maximum dose (per kg). Generally 70 kg is used for the average adult body mass and my personal injections are ~1.0 ml, which equates to a 0.014 ml/kg dose. The above mentioned endotoxin result for the cat hair sample far exceeds what I would have expected based on the USP limit calculation:
Endotoxin limit = 5.0 EU/kg / 0.014 ml/kg = 357 EU/ml (versus 2883 EU/ml)
Unfortunately, the authors determined that the endotoxin content of standardized allergen vaccines is extremely variable and differs greatly from manufacturer to manufacturer. A proposed additional study will look at the effects of the high levels of endotoxin in some vaccines on the immunomodulatory changes associated with allergen immunotherapy.
Written by Travis
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References
- “Allergy Immunotherapy” ACAAI Public Website, 14 Nov. 2018
- Trivedi, B., Valerio, C., and Slater, J. (2003) Endotoxin content of standardized allergen vaccines. Journal of Allergy and Clinical Immunology, 111(4), 777-783
Testing Unique Samples for Endotoxin. Where to Begin?
As many already know, the primary use for the endotoxin detection assay is to determine the potency of LPS in injectables. However, every year I am contacted by customers inquiring about specific treatments that can be used to test their unique sample type. For our purposes moving forward, a “unique sample” will be one that is not a typical injectable and has no release limit established in the compendium.
Assay Selection
Before you can even begin to perform an endotoxin assay you may be deciding which assay is best to test your sample. That decision can be made based on a few criteria – sensitivity required, available lab equipment, known sample interferences and budget. Lonza offers a full portfolio of endotoxin detection assays that will meet your needs. The LAL assays range from the classic gel clot method to the more sensitive kinetic assays. The PyroGeneTM rFC Assay is a non-animal alternative to the LAL method and is the best solution for samples containing beta glucans. Last, but not least, let me introduce you to our newest product, the PyroCellTM Monocyte Activation Test (MAT), which is an in vitro assay for pyrogen testing. Quick Guides are available for all of these methods to ensure that your endotoxin testing runs smoothly.
Sample Interference
LAL Reagent Water (LRW), also known as Water for BET, is the gold standard for endotoxin detection assays – as it is endotoxin-free and non-interfering - and endotoxin spiked LRW is used for system and assay validation studies. Therefore, any sample that isn’t LRW could interfere with the assays. This interference will appear as a false negative (inhibition) or a false positive (enhancement). With any recommendation given for sample testing, we remind the analyst that Positive Product Control (PPC) sample spiking is required in order to determine if the sample is inhibiting or enhancing the endotoxin result. The specification for the PPC is 50-200% and the lowest dilution with a passing PPC result in product screening is the result chosen for product validation and routine testing.
Common interfering factors include heavy metals, high osmolality, pH, proteins, sample color, liposomes, beta glucans, chelating agents and protease inhibitors. Whereas most of the interferences are remedied with simple dilution in LRW, sometimes sample pretreatment is necessary. Lonza’s “Overcoming Assay Inhibition or Enhancement” technical tip document discusses this topic in much greater detail.
Solubility Concerns
Keep in mind that any sample that is soluble can be tested with the LAL and rFC assays. Alternatively, samples that are insoluble can also be tested with these assays as medical devices. These samples can be immersed, flushed, rinsed or soaked in LRW and the extract is tested for endotoxin content. Additional information can be found in Lonza’s “Medical Device – Quick Guide”.
Endotoxin Release Limits (ERL)
For many unique samples an official/compendial release limit has not been established, so a limit will have to be set through sample validation and testing. The ERL for a medical device is a little different. In this case the ERL formula is K × N V, where K = allowable endotoxin per device 20.0 EU / device (2.15 EU / device for cerebrospinal contact), N = number of devices tested and V = total volume of extraction solution.
Establishing Maximum Valid Dilution (MVD)
As stated earlier, simple dilution in LRW will overcome many sample interfering factors. However, when is too much “too much” for dilutions? Once the ERL is determined, the MVD (Maximum Valid Dilution) can be calculated. The MVD is used to determine at what point dilution has exceeded the level of accuracy – i.e. when the endotoxin is diluted past the point of detection. MVD is calculated by dividing the endotoxin release limit (ERL) by the assay sensitivity.
In conclusion, when a new or unique sample comes into your lab, there is a methodology for establishing a testing plan and overcoming any interferences the sample may contribute to the endotoxin assay.
Written by Travis
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Which cells should I use to research COVID-19?
Due to the global impacts of the virus, SARS-CoV-2 (COVID-19) is currently a popular topic of research. A logical experimental design to study the transmission of the disease is to model viral infection utilizing primary cells to represent the human body in an in-vitro setting. When selecting a lineage of cell for this purpose, it is important to select a product that faithfully represents a part of the body that experiences initial exposure to the virus and faithfully illustrates the responsible biology.
COVID-19 is thought to employ a liquid droplet suspended in the air as a vector.1 The most common way that these droplets enter the body is via inhalation, making cells of the respiratory tract the first barrier to internal entry for the virus. Lonza offers two products that represent these cells. Our normal human bronchial epithelial (NHBE) cells, are isolated from the trachea and upper bronchial tube. Alternatively, our human small airway epithelial cells (SAEC), are isolated via enzymatic digestion of the small lower airways of the lungs (bronchioles of >1 mm). SAECs include both the type-I and the type-II pneumocytes that make up the alveolar region of the lung.
COVID-19 viral particles bind tightly to angiotensin converting enzyme-II (ACE-II) utilizing the novel "spike protein" that characterizes the virus. Upon exposure, the virus uses this attachment to infiltrate the cell and undergo viral replication.2 Any cellular model attempting to replicate this process must express the ACE-II receptor at high enough levels to sufficiently illustrate this biology. Fortunately, due to data collected from previous similar viruses, the expression patterns of ACE-II in the respiratory tract are well characterized. ACE-II can be found in both the lower respiratory tract, largely expressed by type-II pneumocytes, and in cells throughout the trachea.3 Lonza has tested both our NHBE and SAEC products for ACE-II expression. For both products, we tested multiple donors for expression of ACE-II post-cryopreservation and found repeatable positive results.
Because overall expression of ACE-II is thought to be higher in the lungs than in the trachea, Lonza recommends SAEC for immunostaining experiments. However, because the ratio of type-II pneumocytes to type-I pneumocytes is quite low, bulk RNA extractions can result in deceptively low expression levels. For these applications, Lonza recommends CC-2540 as a potential alternative.
Written by Jonathan
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References
- Heshu Sulaiman Rahman, Masrur Sleman Aziz, Ridha Hassan Hussein, Hemn Hassan Othman, Shirwan Hama Salih Omer, Eman Star Khalid, Nusayba Abdulrazaq Abdulrahman, Kawa Amin, Rasedee Abdullah. The transmission modes and sources of COVID-19: A systematic review. International Journal of Surgery Open. Volume 26, 2020, Pages 125-136
- Xu, H., Zhong, L., Deng, J. et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 12, 8 (2020)
- Lorenzo CasalinoLorenzo Casalino, Zied Gaieb, Jory A. Goldsmith, Christy K. Hjorth, Abigail C. Dommer, Aoife M. Harbison, Carl A. Fogarty, Emilia P. Barros, Bryn C. Taylor, Jason S. McLellan, Elisa Fadda, and Rommie E. Amaro. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein. ACS Cent. Sci. 2020, 6, 10, 1722–1734
Reprogramming of PMBCs into iPSCs for disease research
Reprogramming of patient derived cells is becoming a more and more common methodology for disease studies, especially peripheral blood mononuclear cells (PBMCs). PBMCs are easy to collect and well-established protocols for reprogramming into iPSCs by using Lonza’s NucleofectorTM Devices exist.
Three recent examples for reprogramming of patient derived PBMC into iPSCs are presented here. Cell line CSUASOi004-A was derived from a 15-year-old female patient suffering from a PRPF6 mutation causing Retinitis Pigmentosa (Zhou et al., 2020). Cell line SDQLCHi017-A was derived from a 1-month-old patient with mutations in the NEB gene (Ma et al., 2020), and cell line SDQLCHi007-A was derived from a 4-year-old male patient diagnosed with Duchenne muscular dystrophy caused by mutations in the dystrophin gene (Guan et al., 2020). All these iPSCs were generated by delivering episomal vectors to the patient derived cells. All three generated cell lines still carry the disease mutations and show an iPSC-like morphology, expression of pluripotency markers, and a normal karyotype. Differentiation potential into all three germ layers was confirmed for all lines by qRT-PCR.
These results show that reprogramming of human somatic cells into iPSC by using NucleofectorTM Devices is an easy and helpful method to gain insights into disease mechanisms by using cells collected from patients suffering from these diseases.
Written by SST
References
- Jingyun Guan, Xinnong Liu, Haiyan Zhang, Xiaomeng Yang, Yanyan Ma, Yue Li, Zhongtao Gai, Yi Liu. Reprogramming of human Peripheral Blood Mononuclear Cell (PBMC) from a Chinese patient suffering Duchenne muscular dystrophy to iPSC line (SDQLCHi007-A) carrying deletion of 49-50 exons in the DMD gene. Stem Cell Res. 2020 Jan;42:101666.
- Yanyan Ma, Haiyan Zhang, Xiaomei Li, Xiaomeng Yang, Yue Li, Jingyun Guan, Yuqiang Lv, Zhongtao Gai, Yi Liu. An integration-free iPSC line (SDQLCHi017-A) derived from a patient with nemaline myopathy-2 disease carrying compound heterozygote mutations in NEB gene. Stem Cell Res. 2020 Mar;43:101729.
- Yalan Zhou 1, Yutong Jing 2, Shengru Mao 3, Jian Liu 4, Zekai Cui 5, Yini Wang 6, Juan Chen 7, Hon Fai Chan 8, Shibo Tang 9, Jiansu Chen. Establishment of induced pluripotent stem cell line CSUASOi004-A by reprogramming peripheral blood mononuclear cells of a PRPF6-related dominant retinitis pigmentosa patient. Stem Cell Res. 2020 May;45:101793.
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Endotoxin Testing for Cell and Gene Therapy Products
Cell and gene therapy products, also called “advanced therapy medicinal products” (ATMPs), offer new ways to treat diseases and raise hopes for more efficient cures of patients worldwide. To ensure these patients are protected, endotoxin testing is part of the mandatory safety testing of these products, just as it is required for “classic” parenterals. If you are an ATMP manufacturer coming from an academic or research background, you might not be familiar with endotoxin testing requirements.
A number of guidelines for ATMPs are available from different regulatory agencies. For example, the European Pharmacopoeia (EP) offers information on both raw materials for ATMPs (chapter 5.2.12) and gene transfer medicinal products (chapter 5.14). In addition, both the European Medical Agency (EMA) and the US Food and Drug Administration (FDA) have published several (draft) guidelines on the topic (EMA “Guideline on human cell-based medicinal products”, EMA Draft “Guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials“, FDA “Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) – Guidance for Industry”).
All of these regulatory documents and guidances point out the importance of endotoxin testing. While ATMP manufacturers are usually aware of the mandatory end-product release testing, it is worth noting that regulatory bodies specifically state that additional testing should be done throughout the manufacturing process of cell and gene therapy products.
As endotoxin has a variety of biological effects that not only affects the patient, but also a cell culture, endotoxin assessment must be included in raw material and manufacturing process qualifications. This starts with testing incoming raw materials, where a low endotoxin level should be ensured. Likewise, testing of RNA and DNA vectors, plasmids or artificial chromosome DNA should include tests for endotoxin levels, among other specifications. In addition to the raw materials, various materials are needed for collection, selection, culture or even genetic or phenotypic modification of cells. For these as well, a low endotoxin level should be ensured.
After having established the quality of raw material and accessories, in-process control endotoxin tests ensure that the in-process material is of sufficient quality to ensure manufacture of an acceptable final product.
In the end, endotoxin testing is done on the finished product which has to meet the acceptance criteria before being given to the patient. Typically, products must demonstrate less than 5 endotoxin units (EU) per kilogram of patient body weight. In the case of intrathecal injection, the specified endotoxin limit is more stringent, with ≤0.2 EU/kg of patient body weight.
For details on how endotoxin tests have to be performed (e.g. what validation tests are needed, which controls have to be used, etc.), the regulatory documents point to the established “Bacterial Endotoxins Test” chapters (EP chapter 2.6.14 and USP chapter <85>). For additional information, you should also take into consideration EP chapter 5.1.10 and USP chapter <1085>.
Last but not least, there are two specific topics that have repeatedly come up in conversations with ATMP manufacturers starting endotoxin testing. First, it is important that you use accessories that are demonstrated to be suitable for endotoxin testing. This means the endotoxin content needs to be lower than your assay detection limit – a generic “pyrogen-free” or “endotoxin-free” labelled item might not have a low enough level of endotoxin. In addition, you need to make sure that the accessory doesn’t interfere with the assay, i.e. it doesn’t release interfering substances, nor does endotoxin adhere to it irreversibly. Second, you need to validate your sample storage. I’ve had several users telling me that they would freeze samples for some time, and then send them collectively for endotoxin testing. While a sample storage is generally permitted, you need to prove that you can indeed store the sample under the chosen conditions (storage time, temperature and container) without the endotoxin content being affected. Otherwise, you risk a false negative or low endotoxin result that impacts patient safety.
Should you have any questions about endotoxin testing for your cell or gene therapy product, don’t hesitate to contact our Scientific Support Team. We are happy to discuss them with you!
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Transfection of Human Hepatocytes using NucleofectorTMTechnology
Primary human hepatocytes (PHH) are the gold standard in vitro tool for toxicology and drug discovery studies. While PHH users face the challenge of an efficient transfection, a good quality starting material is their first choice to ensure high viability and functionality of hepatocytes. A very gentle handling during thawing process procedure and transfection is crucial.
To address the needs for long-term culture of human primary hepatocytes which were transfected using the NucleofectorTM Technology, conditions preserving cell functionality over time were identified and described in this white paper about transfection of primary human hepatocytes.
Cryopreserved human primary hepatocytes were transfected with program DS-150 by using 5μg CleanCap® mCherry mRNA.
As functionality over longer times is as important as an efficient transfection, sustained and very efficient mRNA expression of up to 85% was observed after transfection with this specific mCherry mRNA. Similar to the non-transfected cells transfected hepatocytes formed complex branched bile canaliculi network within 4 days of culture. In this white paper about transfection of primary human hepatocytes, it was shown that Cholyl-lysyl-fluorescein (CLF staining, Corning) was used for visualization of the bile canaliculi formation (Figure 5B). Cells were incubated for 1 hour at 37°C in medium containing 4µM CLF followed by three washing steps with 500µL medium, prior to examination by fluorescence microscopy.
An overlay of the mCherry transfection efficiency (visualized in red by fluorescence) and CLF bile canaliculi staining is also shown.
In addition to the 7 days of gene expression the cells form a dense monolayer with functional bile canaliculi and an intact morphology.
One aspect may draw your attention: the same program only shows 20% efficiency with the pmaxGFPTM Control Vector or pmaxGFPTM Positive Control. As nucleofection is a cell-specific method rather than substrate-specific, we learned that it is possible to use a “smoother” program for mRNA (keeping in mind that this substrate needs to be delivered only to the cytoplasm). So, for primary hepatocytes a so called “high efficient program for DNA” and a “high functionality program" for mRNA was developed.
DNA transfection for short term:
DNA transfection with the program EX-147 was tested with the pmaxGFPTM Positive Control but also with a ß-galactosidase expressing plasmid. High efficiencies could be seen after 24h and the cells started to show an intact bile canaliculi formation over the following days. However, the albumin secretion might decrease in a donor dependent manner after 24h. Therefore, it might be an efficient condition for short term experiments where high transfection efficiencies are crucial.
Written by Siham and Elke
Scientific Support Specialists, Lonza Pharma-Bioscience Solutions at Lonza
Currently available recombinant alternatives to horseshoe crab blood lysates:
Are they comparable for the detection of environmental bacterial endotoxins?
A Review
The most commonly used method for detecting endotoxin in biological drugs are the Limulus amebocyte Lysate (LAL) based kinetic assays, however, this has not always been the case. Originally, the rabbit was used to test drugs for absence of fever-inducing substances. This Rabbit Pyrogen Test (RPT) only fell out of routine use once the LAL-based bacterial endotoxin test (BET) was developed and proven to be a suitable replacement that was practically superior and much more sensitive. A new method is now available which is based on the same reaction principle as LAL but is produced without the need for animal-derived raw material. This new method is based on recombinant production of Factor C, the same protein that is used in the LAL assay. Therefore, it is important to evaluate the use of these recombinant technologies which may expand and potentially improve the quality of endotoxin testing. These recombinant technologies may also bring additional ethical and ecological benefits by using non-animal materials.
Peer-reviewed literature was evaluated comparing results obtained with recombinant reagents to those with LAL and also TAL (Tachypleus amebocyte Lysate). In particular, the authors sought to establish if results using rFC were equivalent to or better than those with LAL/TAL. A critical review of available data, with special consideration of studies in which naturally contaminated samples were tested side by side, was performed to determine if results using rFC were equivalent to or better than those with LAL/TAL.
Comparing results of rFC and LAL tests requires careful study design in order to reach accurate conclusions. Studies of this nature are inherently difficult to conduct because the variation in results within any type of endotoxin testing is relatively high due to the very low (sub-picogram) level tested and the tremendous sensitivity of the assay. As a consequence, large dilutions of standards and samples are necessary to obtain results in range of the assay and minor differences in procedure (temperature, mixing time and operators) result in significant variation even when the same LAL method is used on the same samples at the same time in the same laboratory. Therefore, this review put more weight on those laboratories that controlled for these well-known variables.
In the publications cited, different types of sample material (from airborne samples to biological products) were analysed with both rFC and LAL methods. The analyte was often a real microbial contamination, however, endotoxin preparations like CSE, RSE or NOE were also used in some cases. In all publications listed, there was a good correlation between recombinant Factor C based methods and LAL.
The authors expect that further studies are likely to arrive at the same conclusion that rFC and LAL produce comparable results, as long as the study accounts for the specificity of LAL with respect to beta glucans. Although the false-positive pathway activation of LAL can be mitigated with glucan blocking buffers, these buffers may not block all of the glucan interference or all types of glucans, which may account for some data in which LAL has detected higher concentrations of endotoxin than rFC in natural waters, for example.
Feel free to learn more about recombinant Factor C and its specificity to endotoxin on our website.
Written by SST
Reference
Jay Bolden, Chris Knutsen, Jack Levin, Catherine Milne, Tina Morris, Ned Mozier, Ingo Spreitzer, Friedrich von Wintzingerode. Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review. PDA J Pharm Sci Technol. Sep-Oct 2020;74(5):602-611
WinKQCL™ Tips and Tricks
WinKQCLTM Software is the premier endotoxin detection analysis software on the market today and beyond the basic functionality of calculating endotoxin results there are a few “tricks” that you may not be familiar with when utilizing the software for your day-to-day testing. Here are a few that you may want to employ in the future:
- First, once the template is setup you can print the plate layout to the actual size of the 96-well plate. This allows the analyst to place the printout under the plate to be used as a guide when adding samples.
- Second, the analyst can manage the final endotoxin result label by selecting the “ET per” dropdown. From there, different endotoxin labels can be selected based on your sample type: EU/ml, EU/μg, EU/mg, EU/unit, EU/mEq and EU/device. Depending on the label selected additional text boxes open for values necessary for that label. For example, when calculating “EU/device” the analyst will also need to enter the number of devices and total volume in which the devices are submerged.
- Lastly, templates can be configured to the exact analyst specifications using the Custom Layout option. When clicking on this option, samples, standards and blanks can be placed into any open well on the template. This is an important option if the analyst would like to move the blank to a different location on the plate, for instance.
Accurate Cell Counting Using a Hemocytometer
Many applications and experiments that involve primary cells and cell lines rely on accurate cell counting. It is always important to determine and know, for example, how many cells you have when you begin a Nucleofection experiment or start a culture.
One of the most common, universal ways of doing this is using a Hemocytometer. It’s a rather basic method that doesn’t require any expensive equipment and works very well when done correctly. There might be some of you that haven’t used a hemocytometer before or perhaps don’t use one very often. This blog post is for you and I think you will find it very straightforward.
The hemocytometer is a device originally used to count blood cells (as the name suggests). It is now used to count other cells and many types of microscopic particles. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a grid of perpendicular lines. The device is carefully crafted so that the area bounded by the lines is known, as well as the depth of the chamber. Therefore, it is possible to count the number of cells in a specific volume of fluid and calculate the concentration of cells in the fluid overall.
When a liquid sample containing cells is placed on the chamber, it is covered with a cover slip, and capillary action completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting.
All hemocytometers consist of 2 chambers, each of which is divided into 9 squares with the dimension of 1 x 1 mm. A cover glass is supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm2 x 0.1 mm or 0.1 mm3, or 10-4 cm3. Since 1 cm3 is equivalent to 1 ml, the cell concentration per ml will be the average count per square x 104. Any dilutions have to be taken into account for the calculation of the cell concentration.
The cell suspension should be diluted so that each such square has between
20 – 50 cells (2 – 5 x 105 cells/ml).
As I mentioned above, accuracy is important and obtaining accurate cell counts with a hemocytometer depends on the following:
- Accurate mixing of the sample (if bubbles are introduced into the chamber, the chamber will need to be emptied, cleaned, and filled again)
- Number of chambers counted; by performing a redundant test on a second chamber, you can compare the results
- Number of chambers counted; by performing a redundant test on a second chamber, you can compare the results
In a blog format such as this it is difficult to give visual guidelines so if you need to see an example, luckily three are many videos on YouTube these days. It seems you can find everything there and this includes cell counting tutorials using a hemocytometer. So, I would encourage you to search there for some video lessons.
For our purposes, here is a brief overview of counting with a hemocytomer.
- If necessary, trypsinize cells
- Pipet 10 μl of well resuspended cell suspension into Neubauer counting chamber
- Count cells in 1 quadrant. Alternatively as a general rule 2 – 4 quadrants are being counted.
- To determine cell number per ml, multiply cell number by factor of dilution and 104 (disregard trypan blue positive dead cells). If you count 2 – 4 quadrants you need to divide by the number of quadrants
If you are new to using a Hemocytometer, I hope this serves as a helpful introductory primer. If you are more experienced then hopefully this is a good refresher or simply just a handy reference that you can refer back to if needed.
As always if you have questions, please reach out to Scientific Support. We can be reached via email in the United States and Canada at scientific.support@lonza.com and in the EU/Rest of the World at scientific.support.eu@lonza.com
Written by Sean
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
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Setting Up a Primary Cell or Endotoxin Product Reserve
As you might imagine, we get a lot of questions in Scientific Support and not all of them are always directly about science or research. After receiving an initial question about our primary cells for example, a very common question is “how do I/we set up a reserve for a lot of cells?”
This is an excellent question and one that I thought could be addressed in a blog post.
So, how does one set up a reserve then? There are several steps involved and I will break it down for you here.
For our primary cell products, it usually begins with Scientific Support providing the donor characteristics (height, weight, gender, etc.) for the lots we have in inventory and then the customer will choose which lots they are interested in.
From there, Scientific Support will refer the customer to their Lonza Sales Representative. The Sales Representative will confirm inventory for the lot(s) in question with our Sales Support Group and when the amount of vials for the reserve is agreed upon, the Sales Representative will formally submit the Reserve Request Form.
This form is emailed to the customer and then submitted back to our Reserve Coordinator (all necessary email addresses for correspondence will be provided during the process of setting up the reserve).
At that point, the quantity agreed upon is set aside for eight weeks at no charge. This is the testing period which gives the researchers time to test the lot to make sure it will suit their needs. If the lot is acceptable, the customer can then pay a 500.00 fee to reserve the entire lot for 6 months or submit a purchase order for the entire reserve.
If you have more specific questions about placing an order against an established reserve, you can contact your Lonza Sales Representative (link to contact us) or our Sales Support team at
- For America: ussales.support@lonza.com
- For Europe and ROW: LBSSalesSupportEU.Verviers@lonza.com
We also have a reserve process for our endotoxin product which is a little different. Most of the reserve requests for these products are for larger bulk orders and they have an eight week lead time. We also require a signed reserve agreement stating the intent to buy a certain quantity of the reserve within one year.
At the end of the reserve period, if the number agreed upon has not been purchased, we will require a Purchase Order for the remainder of the reserve.
For non-bulk orders, the process is the same except that there is no lead time. A minimum order number maybe required however. You can direct these specific questions again to your Sales Representative or to our Sales Support Team at the email address above.
I hope this helps elucidate the process for you. I would also like to thank Amanda Collin, Lonza Reserve Coordinator for her assistance in explaining the process.
Written by Sean
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
As you might imagine, we get a lot of questions in Scientific Support and not all of them are always directly about science or research. After receiving an initial question about our primary cells for example, a very common question is “how do I/we set up a reserve for a lot of cells?”
This is an excellent question and one that I thought could be addressed in a blog post.
So, how does one set up a reserve then? There are several steps involved and I will break it down for you here.
For our primary cell products, it usually begins with Scientific Support providing the donor characteristics (height, weight, gender, etc.) for the lots we have in inventory and then the customer will choose which lots they are interested in.
From there, Scientific Support will refer the customer to their Lonza Sales Representative. The Sales Representative will confirm inventory for the lot(s) in question with our Sales Support Group and when the amount of vials for the reserve is agreed upon, the Sales Representative will formally submit the Reserve Request Form.
This form is emailed to the customer and then submitted back to our Reserve Coordinator (all necessary email addresses for correspondence will be provided during the process of setting up the reserve).
At that point, the quantity agreed upon is set aside for eight weeks at no charge. This is the testing period which gives the researchers time to test the lot to make sure it will suit their needs. If the lot is acceptable, the customer can then pay a 500.00 fee to reserve the entire lot for 6 months or submit a purchase order for the entire reserve.
If you have more specific questions about placing an order against an established reserve, you can contact your Lonza Sales Representative (link to contact us) or our Sales Support team at
- For America: ussales.support@lonza.com
- For Europe and ROW: LBSSalesSupportEU.Verviers@lonza.com
We also have a reserve process for our endotoxin product which is a little different. Most of the reserve requests for these products are for larger bulk orders and they have an eight week lead time. We also require a signed reserve agreement stating the intent to buy a certain quantity of the reserve within one year.
At the end of year, if the number agreed upon has not been purchased, we will require a Purchase Order for the remainder and ship the rest of the reserve or cancel it and make those products available to other customer.
For non-bulk orders, the process is the same except that there is no lead time. A minimum order number maybe required however. You can direct these specific questions again to your Sales Representative or to our Sales Support Team at the email address above.
I hope this helps elucidate the process for you. I would also like to thank Amanda Collin, Lonza Reserve Coordinator for her assistance in explaining the process.
Written by Sean Fuerst
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
EP publishes Chapter 2.6.32 introducing recombinant Factor C
On 01 July 2020, the EDQM published European Pharmacopoeia (EP) Supplement 10.3 which now contains the new chapter 2.6.32 Test for bacterial endotoxins using recombinant factor C (rFC). This is a milestone for the EP, as it now introduces the recombinant Factor C assay as a compendial assay for endotoxin detection.
Chapter 2.6.32 closely follows the existing chapter 2.6.14 Bacterial Endotoxins, covering equipment, reagents and general calculations before describing the technique and detailing the requirements for preparatory and routine testing.
The rFC test is described as a quantitative, endpoint fluorescent assay, employing recombinant Factor C based on the genetic sequence from the horseshoe crab (all species). Lonza’s PyroGeneTM Assay is a prime example of this and has been well established as such for over 15 years.
Like the classic LAL assays, users have to first demonstrate the linearity of the standard curve in an initial qualification (IQ) assay. This is followed by the typical product-specific validation, where inhibition/enhancement (I/E) testing is carried out to determine the best sample preparation and ensure the sample doesn’t interfere with the test. For interference testing, the EP specifically points out that due to the absence of Factor G in the rFC reagent, reactivity to glucans will not occur.
The setup and requirements for preparatory and routine assays are identical to the quantitative photometric LAL assays:
- Standard curve: at least three standards, additional standards for each additional log range, at least triplicates (IQ) / duplicates (I/E and routine); correlation coefficient ≥ |0.980|
- Negative control: water for BET, at least triplicates (IQ) / duplicates (I/E and routine); no detectable endotoxin
- Positive Product Control (PPC): concentration at or near the middle of the standard curve, at least duplicates; recovery 50-200%
With so much in common, labs are able to easily transfer from their current LAL method to an rFC assay and are now backed up in this decision by a major regulatory agency. Moreover, users of Lonza’s WinKQCLTM Software will find that the PyroGeneTM rFC assay is already available as an option, making the switch on the software side as easy as clicking a button.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Lonza and Sanquin Partner for Commercialization of MAT
Lonza Bioscience and Sanquin Reagents B.V. have entered into a strategic partnership for the commercialization of the Monocyte Activation Test (MAT). This partnership expands Lonza’s extensive portfolio of endotoxin testing products, and enables drug developers and quality control (QC) laboratories to explore the full potential of the sustainable MAT method to bring safe pharmaceuticals to the market. The MAT Assay completes our QC Testing portolio as the Rabbit Pyrogen Test (RPT) is no longer permitted in the EU.
The cryopreserved pooled human Peripheral Blood Mononuclear Cells (PBMCs, pooled from 4 healthy human donors) are produced by Sanquin specifically for use with the MAT. They will now be sold as PyroCellTM MAT Assay and include an optimized MAT Culture Medium Supplement. The cells are developed in line with the requirements of the European Pharmacopoeia chapter 2.6.30, following an optimized donor selection, cell isolation and cell quality testing process. The cells are available on demand and the large number of vials from one batch guarantees a long term test consistency. In addition, Lonza will distribute the Sanquin-branded PeliKine compact Human IL-6 ELISA Kits that have been validated for use with the MAT assay to detect the released cytokine IL-6.
Overcoming Challenges in Cryopreservation
How can freezing damage cells?
Water freezes as a pure substance. Thus, solutes remaining in the liquid state can be damaging to cells through chemical and osmotic effects (1). The process of freezing also causes mechanical destruction as crystal formation distorts cell shape by squashing, piercing, and teasing apart the cells.
Cryoprotecting agents (CPAs) lower the melting temperature by forming chemical bonds with water and increasing the total concentration of solutes in the system. This reduces the amount of crystal formation and increases the pocket size of unfrozen liquid. Thus, the cells are less likely to be mechanically destroyed by crystals because they have more space in the pockets. Most CPAs are penetrating, meaning they actually enter the cells. This reduces the likelihood of cell dehydration. CPAs are also non-toxic and do not precipitate out of solution. Examples include glycerol, dimethyl sulfoxide, ethanediol, and propanediol. (1)
With a shift in regulatory demands of the Cell and Gene Therapy Market has come a shift in cryopreservation techniques (2). Traditional methods often included the use of serum and DMSO. Freezing medium is now being manufactured under cGMP conditions and formulated serum-free and of non-animal origin. Lonza offers a cryoprotectant media with such characteristics: TheraPEAKTM ProFreezeTM Freezing Medium Chemically Defined. Researchers are also using <10% DMSO in the freeze cocktail.
Others are developing more advanced techniques of cooling. Previous methods of vitrification (solidification to a glassy state at a high viscocity without the formation of ice crystals) required the use of extremely high concentrations of CPAs that often caused issues with cell toxicity (1). A team of Japanese researchers at Shinshu University developed a way to vitrify the cells without the use of CPAs (3). This process of ultrarapid cooling, utilizes inkjet cell printing to cool at a rate of 10,000 degrees Celsius/second, causing near-vitrification of the cells.
Of note, most labs are still using the tried and true method of CPAs, such as DMSO. The key to maintaining high viability using the traditional method is by increasing cell concentration. This will reduce the DMSO volume so that the cellular material is more widely spaced in the crystalline matrix (1).
Tips for freezing (2):
- Only freeze the highest quality cells. The cryopreservation process is tough on the cells and only the strongest will survive.
- Control the rate of freezing. The cells should be cooled to 4°C before adding the CPA, and then they should be frozen immediately thereafter. Freezing too quickly causes ice formation and freezing too slowly can cause cell dehydration and shrinkage. Cooling rate must be optimized to the cell type.
- Cells must be stored below -130°C to protect viability.
- Thaw cells quickly to reduce cell toxicity. Be sure to dilute out the DMSO and add buffer close to melting point while cell metabolism is still suppressed.
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References:
(1) David E Pegg. Principles of Cryopreservation. Methods Mol Biol. 2007;368:39-57.
(2) Lauren Tanabe. Overcoming Cryopreservation Challenges. Biocompare.com June 2020
(3) Shinshu University. A new way to 'freeze' cells promises to transform the common cell-freezing practice. ScienceDaily.com April 2019
Rapid Screening of Potential COVID-19 Treatments Using AI Software
SARS-CoV-2 or COVID-19 has dramatically affected the world in way that hasn’t been seen in over 100 years. Everyone in the world is currently feeling it’s impact and the scientific community is working around the clock to find answers. Despite our efforts, we are still without a vaccine or reliable treatment for this disease.
Working to find possible treatments, researchers at Recursion along with colleagues at Utah State University used high throughput screening for rapid identification of potential treatments for COVID-19. They infected normal human renal cortical epithelial cells (HRCE), normal bronchial epithelial cells (NHBE), and Caco-2 cells with SARS-CoV-2. Vero cells were also infected as a control. Cells were then analyzed using a phenotypical software analysis tool that the researchers refer to as a “deep learning-enabled drug discovery platform.”
A number of drugs and compounds were evaluated using their software platform that provides rapid image analysis to quickly determine potential treatments. The phenotypic evaluation is accomplished by an algorithm that can analyze cytological structures of cells with poor morphology thus enabling quick assessment of the efficacy of treatments.
The only antiviral in their assay that showed efficacy is remdesivir. None of the other antiviral drugs tested including chloroquine and hydroxychloroquine showed any benefit in the human models that were screened. The researchers did observe some small benefit to the cells using beta-blockers, mTOR/PI3K inhibitors and Vitamin D equivalents. There was some increase of the viral phenotype using a beta-agonist.
As we start to cautiously reopen our world again, hopefully this type of approach can narrow the number of potential candidates for treatment and contribute to faster recovery of patients.
For more information, please check out the full article for rapid identification of potential treatments for COVID-19.
Please note that the research article described in this blog is currently a preprint, which means it has yet to be peer reviewed.
Written by
Joseph
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Using Mesenchymal Stem Cells for Therapeutic Applications
Stem cells have promising use in cell therapy applications because they have the potential to generate an entirely new organ. Mesenchymal stem cells (MSCs), despite various challenges due to their heterogeneity, have become increasingly popular for therapeutic use.
Mesenchymal stem cells can be defined as stem cells, stromal cells, or medicinal signaling cells. They are classified as advanced therapeutic medicinal products in the United States and Europe. In the U.S. this means that they are cell therapy products, but Europe further differentiates between somatic and tissue engineered products. These differences in classification dictate the regulatory processes in which cell manufacturing must be compliant.
The following article will summarize the therapeutic properties, quality attributes, and expansion methods of MSCs, as well as some major challenges these cells create when used in cell therapy applications.
Therapeutic properties of MSCs
MSCs can be isolated from bone marrow, adipose tissue, or umbilical cord blood. They can also be found in other adult, fetal, and perinatal tissues. These cells are heterogeneous and polyclonal with 3 subpopulations:
- Type I : spindle shaped, fibroblast-like
- Type II : large, flat, epithelial-like, senescent
- Type III : small, round, self-renewing
The minimal criteria for MSCs in cell therapy manufacturing are as follows:
- Show plastic adherence
- Can differentiatie into cartilage, bone, and fat tissue in vitro
- Express CD73, CD90, and CD105, but not CD11b, CD14, CD19, CD34, CD45, or HLA-DR
- Migrate to injury sites, secrete chemoattractants (cytokines, chemokines, growth factors, extracellular vesicles) that recruit stem cells to repair the damage
Quality Attributes of MSCs
The heterogeneity of these cells can pose challenges when implementing manufacturing guidelines. Thus, the establishment of Quality by Design (QbD) principles and Critical Process Parameters (CPPs) is essential.
Critical Quality Attributes of MSCs include the following:
Potency
Measured via:
- Percent viability
- In vitro functional assays such as MSC differentiation capacity (RNA/protein analysis and secretome analysis (ELISA, mass spectrometry)
Sterility, purity
- Purity guaranteed using cell-specific sorting
- Sterility tests
Expansion of MSCs in vitro (CPPs critical process parameter)
Seeding density — important to consider because of the variability of starting material and source yield (i.e. bone marrow aspirate vs. adipose tissue, etc.). Therapeutic applications require at least 1 x 108 cells per dose.
Culture age — expansion process should not allow the phenotypic changes of the MSCs to alter their original purpose for therapy (i.e. transformation, cytokine secretion, etc.)
Culture medium — Component considerations include, but are not limited to glucose, glutamine, fetal calf serum, human serum, and human platelet lysate. Glucose is the main carbon source for MSCs and can be provided at concentrations of 1 g/L- 4.5 g/L. Glutamine is a second carbon source (provide at 2-4 mΜ). It is recommended to use a more stable form of glutamine such as GlutaMAX to prevent unwanted byproducts such as ammonia. Purified serum proteins and lysates can be used in place of whole serum to reduce the amount of undefined components provided in the media. Serum-free media is the most optimal and GMP-compliant. However, the main issue is that cells cultured in serum-free media senesce more quickly.
Culture vessel conditions — Oxygen supply (21%) in tissue culture flasks is much higher than under physiological conditions (5-7%). This can increase Reactive Oxygen Species (ROS) and thus, should be adjusted accordingly. Temperature and pH must be optimized for each MSC subtype. Vessel material for culture is typically polypropylene, but the cells can also grow on glass or dextran. Cells cultured in serum-free conditions typically require additional coating with adhesion-promoting factors such as fibronectin or vitronectin.
Cell detachment — Standard use of non-specific, proteolytic methods such as trypsin can be damaging to cell markers and reduce viability. Enzymatic methods are more specific to the cell type. The use of dissolvable growth surfaces or thermosensitive surfaces are also an option, but can cause cell aggregation because they act on the cell surface rather than the cells themselves.
Freezing methods — freeze slowly, thaw quickly for allogeneic MSCs
MSC manufacturing for clinical trials
Surprisingly only about 20% of cell therapy centers reported the use of bioreactors, about 80% use T-flasks or cell factories
Advantages of bioreactor use include:
- Automation of expansion and harvest, reduces operation error and contamination risk, fluctuation in culture conditions, more batch to batch consistency
- Traceability – control and monitoring of CPPs
- Examples of bioreactors include fixed (macrocarriers) and fluidized bed, stirred tank (microcarriers), wave, wall-rotating, vertical wheel
- Use of microcarriers allow for higher expansion factor
Major challenges in MSC production for CT
The biggest challenge in using MSCs for cell therapy is being able to develop a process that mimics the natural MSC niche while allowing scale up that doesn’t compromise MSC properties. Due to their heterogeneic properties, it is difficult to standardize these cells. The International Society for Cell and Gene Therapy (ISCT) has published standards for harmonization of potency assays in attempt to overcome this issue.
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References
Jan Barekzai, Florian Petry, Jan Zitzmann, Peter Czermak and Denise Salzig (November 5th 2019). Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products, New Advances on Fermentation Processes, Rosa María Martínez-Espinosa, IntechOpen, DOI: 10.5772/intechopen.90029.
From LAL to rFC Assay
Bacterial endotoxins can cause fever and vomiting and even be fatal. Therefore, testing for bacterial endotoxins is mandatory for injectable drugs. The Limulus Amebocyte Lysate (LAL) assays are currently the most commonly used methods for detecting endotoxins. As the name suggests, it is derived from amebocytes, which are specialized immune cells in the horseshoe crab. These cells contain granules that will naturally form a clot when they encounter endotoxins. This mechanism was exploited and the LAL Assay introduced to the market more than 50 years ago.
Horseshoe crabs have long been harvested as bait for commercial fisheries. To avoid negative effects on the horseshoe crab population, fishing quotas are imposed in the United States since 1998 by the ASMFC, and non-profit organizations like the ERDG have ongoing help programs to protect the population. Due to these efforts, the stock of the atlantic horseshoe crab was found to be good or neutral in 2019. But when looking to Asia, no regulations exist and thus the horseshoe crab populations in this region are highly endangered by fishing, but also by destruction and pollution of ecosystems. In addition, there is a growing need for endotoxin tests and the available ressource “amebocyte lysate” might become scarce.
The industry has recognized the need to change its approach and widely adopt more sustainable, animal-free endotoxin testing methods that do not affect horseshoe crabs or the wider ecosystem in which they play a key role.
Although such change can take time and require significant investment in the short term, these initial costs can be surpassed by the long-term benefits. When the traditional qualitative Rabbit Pyrogen Test (RPT) was replaced by the LAL assay, this change ultimately benefited the industry by providing an ethically more acceptable assay with higher sensitivity that can be run with relative ease and speed.
One sustainable and animal-free endotoxin test method is the PyroGeneTM Assay, based on the recombinant Factor C (see also Blog Article “Adopting rFC for the lab”). Lonza has early recognized the need for an alternative and developed this assay 15 years ago. Companies have started to adopt this method and in September 2018, Eli Lilly’s new drug, EmgalityTM (galcanezumab), was the first drug approved by the US FDA that uses the rFC assay instead of an LAL-based method.
In 2019, several pharmacopoeias (USP, EP, JP and CP) have published draft chapters or announced plans for draft chapters to include rFC in the compendia (see also Blog Article "Recombinant Factor C adopted into Ph. Eur.").
With all those changes in the regulatory authorities towards acceptance of the recombinant FactorC we expect another boost for adoption of PyroGene in labs for release testing.
Your SST and Allen Burgenson
CRISPR/Cas9 based Genome Engineering of NK Cells Using Nucleofector™ Technology
During checking for CRISPR in literature we stumbled over the following interesting topic:
CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy
Since NK cells become more and more popular for their role in cancer therapy we decided to make you aware of this article focusing on technical points such as their transfection as well as assay readouts for genome editing experiments.
The authors (Lambert et al. 2020) bring light to the research of NK cells done in the past and how this cell type can be used with the help of the CRISPR/Cas9 technology from a huge number of research teams working on different topics.
Since mid of 1970s NK cells were often described as a tumor killing cell type by acting as part of innate immunity. Following several studies, it is now known that this special cell type is playing an important role not only by destroying tumor cells but also when the human body seems to be challenged with bacterial and viral infection and also in the regulation of immune responses.
This review outlines features of the cells in great detail (such as their function in physiological and pathological conditions) and also points out their potential utilization in treatment of cancer or tumor patients. However, to overcome some limitations e.g. long-term presence at tumor site in vivo it might be helpful to genetic engineer these cells by the help of the CRISPR/ Cas9 technology. This book chapter outlines an overview about methods, protocols and results done in the past and a new approach of specific genomic modification by introducing Cas9 RNPs into the cells. The authors hand over a precise protocol about the transfection of primary human NK cells by using the 4D NucleofectorTM Device. In their model, they genetically engineered the NK cells by knocking out the CXCR4 gene. The engineered cells showed reduced CXCR4 expression and thus reduced migration upon specific chemokine stimulation. All CRISPR materials (crRNA/ tracrRNA, Cas9 protein) are used from IdT (integrated DNA technology) a company which referred on their website to start with the cell type –specific optimized protocol using the NucleofectorTM Technology. In this case, the optimized protocol for human NK cells is not yet available but they come up with the primary solution P3 and program CM-137 as their favorite conditions for the 4D NucleofectorTM Device. With this set up they tested the efficacy of the CRISPR/Cas9 genome editing tool in human NK cells and propose this protocol as a valuable approach for developing NK cells based therapies. Additionally, this technology enables to learn more about this fascinating cell type in research.
Please have a look at the chapter and the detailed description of the protocol. I am sure we are not the only one who find this interesting?”
Written by
Gabriel und Elke
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References
Lambert M, Leijonhufvud C, Segerberg F, Melenhorst JJ, Carlsten M. CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy. Methods Mol Biol. 2020;2121:213–239
CRISPR/Cas9 based Genome Engineering of NK Cells Using Nucleofector™ Technology
During checking for CRISPR in literature we stumbled over the following interesting topic:
CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy
Since NK cells become more and more popular for their role in cancer therapy we decided to make you aware of this article focusing on technical points such as their transfection as well as assay readouts for genome editing experiments.
The authors (Lambert et al. 2020) bring light to the research of NK cells done in the past and how this cell type can be used with the help of the CRISPR/Cas9 technology from a huge number of research teams working on different topics.
Since mid of 1970s NK cells were often described as a tumor killing cell type by acting as part of innate immunity. Following several studies, it is now known that this special cell type is playing an important role not only by destroying tumor cells but also when the human body seems to be challenged with bacterial and viral infection and also in the regulation of immune responses.
This review outlines features of the cells in great detail (such as their function in physiological and pathological conditions) and also points out their potential utilization in treatment of cancer or tumor patients. However, to overcome some limitations e.g. long-term presence at tumor site in vivo it might be helpful to genetic engineer these cells by the help of the CRISPR/ Cas9 technology. This book chapter outlines an overview about methods, protocols and results done in the past and a new approach of specific genomic modification by introducing Cas9 RNPs into the cells. The authors hand over a precise protocol about the transfection of primary human NK cells by using the 4D-NucleofectorTM Device. In their model, they genetically engineered the NK cells by knocking out the CXCR4 gene. The engineered cells showed reduced CXCR4 expression and thus reduced migration upon specific chemokine stimulation. All CRISPR materials (crRNA/ tracrRNA, Cas9 protein) are used from IdT (integrated DNA technology) a company which referred on their website to start with the cell type –specific optimized protocol using the NucleofectorTM Technology. In this case, the optimized protocol for human NK cells is not yet available but they come up with the primary solution P3 and program CM-137 as their favorite conditions for the 4D-NucleofectorTM Device. With this set up they tested the efficacy of the CRISPR/Cas9 genome editing tool in human NK cells and propose this protocol as a valuable approach for developing NK cells based therapies. Additionally, this technology enables to learn more about this fascinating cell type in research.
Please have a look at the chapter and the detailed description of the protocol. I am sure we are not the only one who find this interesting?”
Written by
Gabriel and Elke
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References
Lambert M, Leijonhufvud C, Segerberg F, Melenhorst JJ, Carlsten M. CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy. Methods Mol Biol. 2020;2121:213–239
Video Showing the Division in CD34+ Human Cord Blood Hematopoietic Stem/Progenitor Cells
Hematopoietic Stem/Progenitor Cells
Hematopoietic Stem Cells (HSC) are cells isolated primarily from bone marrow and umbilical cord blood. They can self-renew and differentiate into many different types of blood cells [1].
These characteristics raised the interest of researchers in using them as potential cellular treatment for a variety of diseases over 50 years ago. Nowadays, transplant of HSC is routinely used as therapy for several cancers, blood or immune disorders and metabolic diseases [2], [3]. Moreover, research progress could widen the therapeutic window of HSC transplants to treat brain disorders such as Alzheimer or Dementia, or neurodegenerative diseases such as Multiple Sclerosis, amongst others [2]–[7].
In vitro Challenges
In vitro cultures of HSC still represent a challenge in research because their fragility prevents them from replicating and also because, in order to differentiate, samples of HSC must be cultured with a highly specific combination of cytokines [8], [9]. Additionally, given their similarity to many other blood or bone marrow cells, purifying HSC is a challenge, and the fact that only 1:10000-15000 bone marrow cells or 1:100000 blood cells is known to be a stem cell [1] does not ease the task.
To address the identification problem, cell surface protein markers [10] can be used. One of the most commonly used markers for HSC recognition is CD34, a transmembrane phosphoglycoprotein encoded by the gene with the same name [11]. CD34+ HSC isolated from umbilical cord blood present the advantage to efficiently proliferate without losing their ability to differentiate into the several blood cell types[12].
Observation of a Mitosis in an Umbilical Cord Blood Isolated CD34+ HSC Sample
A sample of human cord blood CD34+ stem/progenitor cells in X-VIVOTM 15 serum-free hematopoietic cell medium supplemented with recombinant human thrombopoietin (25ng/mL), Rlt3 ligand (25ng/mL) and stem cell factor (13ng/mL). The culture was also coated with fibronectin.
Live cell imaging was performed with Nanolive’s 3D Cell Explorer for 15 hours (3 images/min) and cell division was captured. To date and to our knowledge, this live cell movie featuring CD34+ HSC is the first one ever obtained.
One can distinguish characteristic steps of mitosis such as chromatin condensation inside the nucleus that lead to chromosome formation, the alignment of the chromosomes in the metaphase plate and the migration of sister chromatids to opposite ends of the cells. Eventually, two daughter cells were obtained as a result of the contractile ring effect during cytokinesis.
Considering that any stress would trigger programmed cell death in such a sensitive cell type, the mitosis captured acquires further significance. Indeed, it is a sign of not just the homogeneity of the cell culture, but also of the fact that both the medium and freezing procedures of the sample were optimal and that Nanolive’s technology was completely non-invasive, which can also be reaffirmed by observing healthy behavior in most of the cells surrounding the dividing cell.
Your Scientific Support Team in cooperation with Nanolive
References:
- Bujko K, Kucia M, Ratajczak J, Ratajczak MZ. Hematopoietic Stem and Progenitor Cells (HSPCs). Adv Exp Med Biol. 2019;1201:49-77
- “Hematopoietic Stem Cells: What Diseases Can these Stem Cells Treat?” [Online]
- Y. M. Hawsawi, F. Al-Zahrani, C. H. Mavromatis, M. A. Baghdadi, S. Saggu, and A. A. A. Oyouni, “Stem Cell Applications for Treatment of Cancer and Autoimmune Diseases: Its Promises, Obstacles, and Future Perspectives” Technol. Cancer Res. Treat., vol. 17, p. 1533033818806910, 2018.
- A. Tyndall and J. M. van Laar, “Stem cells in the treatment of inflammatory arthritis” Best Pract. Res. Clin. Rheumatol., vol. 24, no. 4, pp. 565–574, Aug. 2010.
- T. Franceschetti and C. De Bari, “The potential role of adult stem cells in the management of the rheumatic diseases” Ther. Adv. Musculoskelet. Dis., vol. 9, no. 7, p. 165, 2017.
- C. A. Rush, H. L. Atkins, and M. S. Freedman, “Autologous Hematopoietic Stem Cell Transplantation in the Treatment of Multiple Sclerosis” Cold Spring Harb. Perspect. Med., vol. 9, no. 3, p. a029082, Mar. 2019.
- K.A. Kwak, “Current Perspectives regarding Stem Cell-Based Therapy for Alzheimer's Disease” Stem Cells Int, 2018; 2018: 6392986
- S. J. Morrison, D. E. Wright, S. H. Cheshier, and I. L. Weissman, “Hematopoietic stem cells: challenges to expectations” Curr. Opin. Immunol., vol. 9, no. 2, pp. 216–221, Apr. 1997.
- M. A. Walasek, R. van Os, G. de Haan, "Hematopoietic stem cell expansion: challenges and opportunities" Ann N Y Acad Sci. 2012 Aug;1266:138-50
- I. L. Weissman and J. A. Shizuru, “The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases” Blood, vol. 112, no. 9, pp. 3543–53, Nov. 2008.
- D. L. Simmons, A. B. Satterthwaite, D. G. Tenen, and B. Seed, “Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells” J. Immunol., vol. 148, no. 1, pp. 267–71, Jan. 1992.
- D. K. Kim, Y. Fujiki, T. Fukushima, H. Ema, A. Shibuya, and H. Nakauchi, “Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells” Stem Cells, vol. 17, no. 5, pp. 286–94, 1999.
Tolerogenic Dendritic Cell-Based Therapy for Multiple Sclerosis
Dendritic cells are central players of the immune response and they have the unique ability to activate naïve T-cells. They can either induce immunity or establish tolerance by interacting with multiple cells of the immune system. Like this, dendritic cells are crucial in maintaining the balance between attacking pathogens or foreign molecules and sparing the body’s own tissue.
In autoimmune diseases, this tolerance against self-tissue has broken down. Worldwide, it is estimated that approximately 1 in 15 individuals suffers from autoimmune disease. Existing therapies against autoimmune diseases mostly include treatment with non-specific immunomodulatory drugs. Those systematically suppress the function of many immune effector cells and therefore often cause serious and sometimes life-threating side effects. There is an urgent need of more disease-specific treatments and for treatments that have a curative effect and help to re-establish the tolerance against self-antigens.
Tolerogenic DCs (tolDCs) are essential in the maintenance of central and peripheral tolerance by induction of clonal T-cell deletion and anergy, inhibition of memory and effector T-cell responses and generation and activation of regulatory T-cells. TolDC therefore are promising candidates for the re-establishing of a permanent antigen-specific tolerance.
In the last years, researchers have developed many protocols for the in vitro generation of potent and stable tolerogenic dendritic cells from monocytes. TolDC cells can be induced from immature monocyte derived dendritic cells i. e. by:
- Pharmacological agents: using immunosuppressive drugs like corticosteroids, rapamycin, aspirin or Vitamin D3
- Cytokines: incubation with TGF-beta or IL-10 or a combination of both
- Genetic engineering: recombinant expression of apoptosis inducing elements, such as FasL PD-L1 and TRAIL or suppression of NF-Kappa etc
For a review see i.e. in Domogalla et al Front. Immunol. 2017
In the publication of Zubizarreta et al in PNAS the authors describe a phase 1b clinical trial to investigate the safety and feasibility of tolDC therapy for the CNS-related autoimmune disorders Multiple Sclerosis and Neuromyelitis optica with peptide-loaded tolerogenic dendritic cells.
Autologous tolerogenic dendritic cells have been generated from patients PBMCS under GMP compliant conditions using the Lonza X-VIVOTM Medium. In the protocol, GM-CSF and IL-4 have been used for differentiation and addition of Dexamethasone for the induction of the regulatory phenotype. TolDC from MS patients were loaded with seven myelin peptides and DC from NMOSD patients were stimulated with peptides from AQP4 patients
As primary endpoint, the safety of CNS-peptide loaded tolDC therapy was tested and as a secondary endpoint, signs of efficacy and markers of immune tolerance in treated patients with MS and NMOSD were determined. More information on the clinical trial can be found in the National Clinical Trial No NCT02283671
The researchers showed, that i. v. administration of peptide-loaded tolDC is safe and feasible. Immunological assessment confirmed the induction of Tr1 cells producing high levels of IL-10. This is the basis for future trials to determine if the observed increase of regulatory T-cells and IL-10 production can modify the disease course in MS or NMODS.
Currently, two further phase I clinical trials for tolerogenic dendritic cell-based treatment for multiple sclerosis are running (Trial registration numbers NCT02618902 and NCT02903537) described in Willekens et al, BMJ Open 2019. In the two harmonized trials two different routes of cell administration (intradermal vs intranodal) are compared. In this study, VitD3-treated tolDC loaded with myelin-peptides are prepared from leukapheresis from MS patients.
Results from phase I clinical trials using tolDC also in other autoimmune disease (review for example) also showed promising results with regards to safety of administration. Future phase II trials will need to investigate the efficacy of these patient-tailored treatments. I am excited to see, if tolerogenic dendritic cells with become a new tool for treatment of autoimmune diseases.
Written by SST
Using HLA-Typed Cells in Research
Writing today’s article did not start with an interesting publication I read, or with a fancy technology, I heard of. It started with a question. I was asking myself, why we at Lonza Scientific Support are asked more frequently for HLA-data for our primary cell types.
I know that HLA-matching is important when it comes to transplantation – of organs, tissue or bone marrow. However, HLA types are also connected with many diseases or autoimmune disorders. Like this it can be a critical factor when it comes to assay development in research.
The major histocompatibility complex (MHC) is a set of genes that code for cell surface proteins essential for the immune system to recognize foreign molecules. The human MHC is called Human Leukocyte Antigen (HLA).
MHC proteins supports self versus non-self recognition in coordination with T Cell Receptor proteins. It could be so easy, but it is not: The MHC genes display the greatest degree of polymorphism in the human genome.
On chromosome 6 there are 6 different HLA genes:
- HLA-A, HLA-B and HLA-C belong to MHC class I, which can be found on all nucleated cells in the body.
- HLA-DR, HLA-DQ and HLA-DP belong to class II and can be found in addition on certain immune cells.
Each of them coming with a huge allele diversity. HLA-B for example has more than 3000 different allele subtypes. And now take into consideration that each individual has 2 alleles - donor differences reaches a new dimension.
HLA status has a high impact on research areas like tumor immunology. When considering cancer immunotherapies, like adoptive T-cell transfer, it must be proven upfront that the T-cells will not attack healthy tissue or will have any other cytotoxic effects and cross-reactions.
HLA typed cells and tissues can also be used in exploring disease mechanisms for example in autoimmune disorders. The HLA status can predict diseases like Arthritis, Type I diabetes (HLA-DRB1*04:01) or Multiple Sclerosis (HLA-DRB1*15:01). It can have an impact on disease progression as well, like in case of HIV.
In drug development the HLA type plays a role in immunogenicity, hypersensitivity or even non-response of the patient to a specific drug due to the patient’s HLA status.
How to handle this situation? Well, test and focus is a good way.
And the fact that HLA A2*01 is the most prevalent MHC class I allele family in humans however helps to focus. Most Therapies are developed for patients with this HLA status.
At the moment we have HLA data available for many lots of fresh bone marrow and bone marrow cells, as well as different blood cell types and liver cells. More to come! Just get in touch with us at scientific.support@lonza.com to discuss your needs.
Written by Isabella
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Models for Safety Testing of Biopharmaceuticals
If you want to protect yourself from an infection, you get a vaccination, e.g. non infectious viral proteins. Your immune system reacts with several mechanism and also produces antibodies to attack foreign proteins.
The same can happen, if you get multiple doses of biopharmaceuticals (BP) such as monoclonal antibodies conjugated with specific drugs. The immune system might generate an unwanted immune response. The ADA (anti-drug-antibodies) can potentially decrease the efficacy of the drug, modify the clearance or induce hypersensitive reactions. Due to the safety issues associated with immunogenicity, it is of great importance to reduce the risk for immunogenicity in the clinic.
The scientist from Novo Nordisk A/S published a novel approach in PLOS ONE about the developend a new assay for early drug testing during the development. This novel assay uses CD4+-enriched T cells and irradiated PBMCs comprising the APC population, and the effects of the biopharmaceuticals are tested by cell prolifation and cytokine secretion (IL-2 Elispot).
Advantages to other immune cell assays:
- Controlled number of specific T cells
- Ctonger response of enriched specific T cell fraction
- no generation of DCs needed - hence allowing high througput analysis, cytokine contribution from non-specific cells can be limited
- 2 independent read outs of the immune response and activation
The results they present in “Quantitative analysis of the CD4+ T cell response to therapeutic antibodies in healthy donors using a novel T cell:PBMC assay”, show that the novel test can evaluate the immunogenicity potential of biopharmaceuticals in a very early stage of drug development. They also show that in vitro immunogenicity tests of several known BPs are also documented in clinical trials.
The selection of the right drug candidates with low immunogenicity potential is crucial to increase the patients safety.
Written by Peter
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Short Guideline for an Efficient Transfection Using NucleofectionTM Technology of Human Dendritic Cells
As part of the immune system, dendritic cells (DC) play a pivotal role in catalyzing the adaptive immune response. Once they capture and process local antigens, they migrate to draining lymph nodes in order to prime T lymphocytes. For their essential function, they are more and more exploited in tumor immunotherapies.
Since DC are often used in vitro screens, the need for a reliable method for their transfection became a key step. For this purpose, we try to present here a short guideline for the transfection of human DC cells (monocyte-derived) in suspension, using the 4D-NucleofectorTM Device.
In the case of immature DCs after thawing the cells you can culture them in T75 flasks at 2.5 E+05 cells per flask with the corresponding culture medium (RPMI1640 + 10% FCS + 2% Ultraglutamin + 1% Na-Pyruvat + 1% PS + 25 ng/ml IL-4 + 50 ng/ml GM-CSF) for 5 days. Change medium on day 2 and 5. For the mature phenotype, change the medium on day 5 to differentiation medium (RPMI1640 + 10% FCS + 2% Ultraglutamine + 1% Na-Pyruvat + 1% PS + 25 ng/ml IL-4 + 50 ng/ml GM-CSF + 10 ng/ml IL-1β + 20 ng/ml TNF-α + 10ng/ml IL-6 + 500 ng/ml PGE2) and incubate for 24 hours.
For the transfection using NucleofectionTM Technology, we recommend to use per sample 2 x 106 (for cuvettes) and 4 x 105 (for strips). To collect the cells, centrifuge them at 90xg for 10 minutes at room temperature. Resuspend the cell pellet carefully in room temperature nucleofection solution P3 (mixed with the supplement). Prepare mastermixes by dividing cell suspension according to number of substrates. Add required amount of substrates to each aliquot (max. 10% of final sample volume). For the DCs it is important when using mRNA as substrate to pipette it directly into the cuvette, then add the cell suspension and immediately transfect the sample. This step is recommended because the cells, once in contact with the mRNA, will start to degrade it, resulting in suboptimal transfection efficiencies.
For the transfection, we would recommend depending on the substrate either CB-150 program for mRNA or EH-104 for DNA.
In order to have a better viability after the transfection, we suggest to include a recovery step immediately after applying the pulse: add culture or differentiation medium to cuvette (500 µl) or 80 µl to each well of the strip and allow to stand at room temperature for 10 min.
Mix cells by gently pipetting up and down two to three times. Plate desired amount of cells in culture system of your choice. Immature and mature DCs should be kept in their respective medium after nucleofection until nucleofection results will be analyzed.
The maturation of immature DCs can also be started directly after transfection using NucleofectionTM Technology. In this case, differentiation medium should be added for the recovery step and the cells will be transferred into differentiation medium.
Since the results might be donor dependent, we would recommend to check the transfection efficiency at different time points and in the first experiment to use the pmaxGFP plasmid provided in the kit, as a positive control. Based on the results of the initial set of transfections, we encourage you to contact the Scientific Support Team for further optimizations.
We wish you happy transfections!
Written by Elke & Gabriel
Scientific Support Specialists, Lonza Pharma-Bioscience Solutions at Lonza
Reprogramming of Human Erythroblasts into iPSC for Sickle Cell Disease Research
Sickle Cell Disease (SCD) is a severe monogenetic blood disorder that is caused by mutations in the HBB gene, thus affecting hemoglobin and leading to anemia and organ failure.
Millions of people are affected worldwide and therefore, iPS derived from SCD patients should be considered as useful tools to gain further insides into disease pathogenesis, drug discovery and eventually therapy.
By using Lonza’s NucleofectorTM Technology, researchers from Brazil were able to reprogram erythroblasts from SCD patients and age and sex matched healthy individuals to allow for SCD studies (Martins et al., 2018 and Paredes et al., 2019).
By delivering episomal vectors to PBMC derived erythroblasts, they generated three iPS clones with homozygous SCD and three healthy iPS clones as control. All clones showed a pluripotent stem cell morphology, expressed a variety of pluripotency markers and differentiation potential was confirmed by RT-PCR and staining markers for all three germ layers in a EB formation assay. By passage 15, all iPSC displayed a normal karyotype and showed loss of episomal vectors.
These results show that reprogramming of human somatic cells into iPS by using the NucleofectorTM Technology is a well-established and well-documented method giving researchers many opportunities for gaining further insights in disease mechanisms.
Written by SST
CAR-T Therapy Overview
What is CAR-T therapy and how does it work?
Normal human T cells can destroy cancer cells, but unfortunately, some cancer cells can evade the normal immune response. What if we could modify T Cells to recognize those cancer cells and destroy the cancer using our body’s own defenses rather than using radiation or chemicals? This is precisely what CAR-T Therapy aims to accomplish.
CAR stands for Chimeric Antigen Receptors. A gene to code for these receptors is inserted into T Cells via transfection. Lonza’s NucleofectionTM technology has been cited for many studies using CAR-T cells. Here are a couple of citations for Lonza’s 4D-NucleofectorTM LV Unit with CAR-T cells.
- T cell therapies for hematologic malignancies
- Biomanufacturing for clinically advanced cell therapies
After the cells are modified, these additional receptors enable the transfected T cells to recognize and destroy the cancerous cells.
Challenges
Unfortunately, these modified T cells can sometimes have very severe side effects. The most prominent side effect is Cytokine Release Syndrome (CRS). However, this can often be mitigated through medication. There can also be neurological side effects as well, such as confusion. You can read more about side effects on Dana-Farber Cancer Institute’s blog.
The treatment is not always effective, and Riddell et.al. goes into some detail regarding the challenges for the efficacy of the treatment.
It can also be time consuming, expensive, and sometime it is not possible to use the patient’s cells for the media.
The future of CAR-T Therapy
Allogeneic treatments could potentially offer a solution in which healthy donor T cells are used for treatments. You can learn more about allogeneic CAR-T and their eas of access.
Reprogramming of the patients T Cells without removing them from the blood would be even more efficient and could potentially side step the rejection of the implanted cells. You can read more about in-situ CAR-T therapy.
Written by Joseph
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Recombinant Factor C Adopted into Ph. Eur.
The European Pharmacopoeia (Ph. Eur.) Commission has officially adopted general chapter 2.6.32 Test for Bacterial Endotoxins using Recombinant Factor C!
The Importance of Data Integrity in the Endotoxin Test
Data Integrity can be broken down into a simple definition:
- The maintenance of…
- The assurance of…
- The accuracy and consistency of…
- You can learn more about Kevin’s book
- Learn more about Data Integrity
- We also offer a technical tip at our QC InsiderTM Toolbox
- The FDA provides a guidance document on "Data integrity and compliance with drug CGMP questions and answers guidance for industry"
Cancer Invasion Regulates Vascular Complexity in a 3D Biomimetic Model
Organoid cultures are remarkable in that they recapitulate accurate tissue growth within 3D cultures. When it comes to cancer organoids though, it is difficult to assess and quantify cancer invasiveness, especially for solid epithelial cancers. The Lonza RAFTTM System has been used to generate dense, cell-seeded collagen scaffolds. These can be tissue-engineered to produce cancer masses, which can then be nested into stromal compartments. Because of the two compartments, 3D tumouroid models allow for quantitation and morphology of cancer invasion and even predict metastatic potential.
Exciting New Developments in the Production of Viral Vaccines
According to World Health Organization's report on "Progress and Challenges with Achieving Universal Immunization Coverage", vaccination is one of the public’s most cost-effective forms of biomedical treatment. However, in 2018, almost 20 million children across the world were left undervaccinated. Thus, it is imperative to develop more cost effective techniques for virus production, making it more affordable to individuals on global level.
Vero cells are the most widely accepted cell type for vaccine production. However, their growth is anchorage-dependent. Until recently, culture at high densities was only made possible through the use of microcarriers, which are glass or plastic beads used as supporting matrices. The main downsides to culturing cells in adherent formats are that these often require additional time and labor-intensive steps compared to suspension applications.
Scientists at the National Research Council of Canada were able to adapt the Vero cell line CCL-81 to suspension culture without the use of microcarriers and effectively propagate vesicular stomatitis virus (VSV) in small and large scale applications (1). They found that the suspension cultured cells achieved a higher viral titer compared to those cultured in adherence format. In addition to this, the suspension cells generated a higher titer with increasing cell concentration, especially in bioreactors compared to small-scale shaker flasks. Cultures in which the fed-batch method was emplyed had an improved yield as well.
An exciting new avenue at the forefront of clinical development includes the use of vaccinations to treat primary or metastatic tumors. Scientists at the University Medical Center Groningen were able to effectively establish a GMP-compliant manufacturing process to produce a vaccine targeting human papillomavirus (HPV) induced tumors. High-risk HPVs can integrate early proteins E6 and E7 into their host’s genome, transforming native cells to a malignant form. The vaccine, named Vvax001, aims to target these proteins.
Vvax001 is based on recombinant Semliki Forest Virus (rSFV). rSFV was chosen as the system for treatment for the following reasons:
- RNA from this virus does not integrate
- rSFV infection is cytolytic via apoptosis
- rSFV activates both the innate and adaptvie immune systems to target viral proteins
- There is no immune response directed at the viral vector itself
Briefly, the Vero cells were subjected to a series of electroporations with viral RNA encoding replicase, the E6 and E7 fusion proteins, capsid and spike proteins. The virus was then purified using anion and cation exchange chromatography, then analyzed for contaminants such as mycoplasma and other viruses. Stability testing confirmed a shelf life of at least 6 months. Researchers were able to achieve a mean viral titer of 4.0 x 108 infectious particles/mL after purification and the mean recovery was 19%.
It is apparent that the vaccine field is rapidly expanding and there is promise of improving techniques to reduce time and cost for production. Ultimately, it is our goal to make these vaccines more available to people across the globe, even those in seemingly unreachable areas so that we can prevent the spread of major biomedical diseases.
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Happy Holidays - Best Wishes for 2020
Dear valued Customer, Researcher, Scientist, and Colleague,
In June 2019, we launched with great success the Lonza Bioscience blog and also share the content on our Lonza Bioresearch Solutions LinkedIn page and Lonza QC Testing Solutions LinkedIn page. The content helps to support our customers, the references and publications show what you can achieve with the Lonza Bioresearch and Endotoxin products.
A Big Picture Approach to Recombinant Protein Production
An increasing number of blockbuster drugs are recombinant mammalian proteins (1). In fact, the FDA has approved over 100 for therapeutic use. Production of recombinant proteins in mammalian hosts allows scientists to generate proteins with the post-translational modifications that contribute to the overall function of the protein. In other words, these proteins more closely mimic in vivo functions compared to those produced in other hosts such as bacteria, yeast, or insect cells. Let us take a brief look into the main elements of protein production in mammalian hosts to get a better picture of what it entails.
Host Cell Lines
CHO
These are hardy, reliable cells that allow for adaptability to serum-free conditions and growth at very high densities(1). Many human pathogens do not replicate in CHO, which is advantageous from a regulatory standpoint. It is also worth noting that CHO lines facilitate glycosylation of recombinant proteins, which is important for overall function(2).
HEK293
Proteins produced in this line are actually a closer match to in vivo human proteins. Scientists have also developed the HKB11 cell line (hybrid between HEK293S cell line and human B cell line) which is easy to transfect and secretes very large quantities of protein.
Methods of Delivery (1)
Transient transfection
Plasmid DNA is expressed using reagent. There is no need for clonal selection because the gene/s are not incorporated into the genome of the host. Downside is large quantities of plasmid are required for effective delivery.
Stable transfection
Gene of interest is expressed using editing technology such as CRISPR. Requires a clonal selection step.
Recombinase Mediated Cassette Exchange
Procedure in which a preexisting gene cassette is exchanged for an analogous cassette carrying the gene of interested. For protein production, the recombinant target is directed to a genomic “hotspot” to increase yield.
Viral Delivery
Allows for delivery of nucleic acid at large volume, produces high yield of secreted protein without the need for clonal selection.
Clonal Selection (3)
Methotrexate (MTX)
Inhibits dihydrofolate reductase (DHFR). Used in combination with DHFR deficient lines in which the DHFR clonal selection gene is supplied in the expression cassette containing the gene of interest.
Glutamine Synthetase (GS)
Methionine sulfoximine (MSX) inhibits GS. Used in combination with GS-deficient cell lines in which the GS clonal selection gene is supplied in the expression cassette containing the gene of interest.
Benefit to this approach: The drug concentration can be increased to “fine tune” and generate the strongest cell clones that produce the most protein.
Expansion methods (4)
Batch
All nutrients are supplied in the initial base medium
Fed-batch
Nutrients are added as they are depleted
Perfusion
Medium is circulated through a growing culture, while waste is removed, additional nutrients are supplied, and the product is harvested.
Written by Angela Dover
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
References:
New USP Chapter <1085> Guidelines on the Endotoxins Test
After the withdrawal of FDA’s 1987 guideline on the LAL test, there were gaps in the regulatory documents regarding certain aspects of endotoxin testing like RSE/CSE standardization. On 01 December 2019, USP therefore published General Chapter <1085> Guidelines on the Endotoxins Test.
In vitro Exercise Models Used to Study Skeletal Muscle Responses
As an avid weight-lifter I have always been fascinated by the ability of our muscle cells to adapt and respond to various stimuli. As a scientist, it also sparks curiosity because it is also something that can be directly observed without a microscope. We see an increase in muscle size (hypertrophy), muscle definition, and muscle response as direct results of the exercise we do.
However, there are definitely many factors and processes controlling these responses going on underneath the surface that we cannot directly observe. These can be difficult to study and observe in vivo and I began to wonder if the effects of exercise could be studied in vitro. It’s a question I had not asked myself before and again, as a scientist, that made me even more curious and I began my search for answers.
That’s when I came across the paper that I present you with today. The authors developed a unique approach to study actual muscle contractility by using EPS (electric pulse stimulation). That itself is not new as it has been previously used to study contractility in cultured myotubes but with mixed results. These authors have now applied it to contractile human and mouse hybrid myotubes and the results are very encouraging.
As research in this field continues, hopefully in vitro models can be used to more effectively study muscle cell responses to stimuli and the multi-faceted array of cell signaling events that occur.
Written by Sean
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Lonza’s Northern European Endotoxin Seminar Held in Manchester, UK
After a very successful start the European Seminar Series moved on to the UK where a workshop was being offered in Manchester.
The Workshop was opened by an exciting talk from Lonza’s Alan Baines who talked about the evolution of endotoxin testing over his 43 year’s experience in the endotoxin testing market. Ruth Noé spoke about common assay issues, a topic which is always well received during Lonza customer training sessions. LER continued to be a hot topic for discussion on the first day , which was part of the session covering assay interference. David Graham’s (SNBTS) talk about endotoxin testing considerations for ATMPs brought a new perspective to the clinical impact of endotoxin testing. Later that day alternative methods were presented. The Monocyte Activation Test (MAT) introduced by Eelo Gitz from Sanquin (shown on the photo on the left) got a lot of attention as it is quite different from the LAL Assay by detecting Pyrogens in general. The readout for the MAT uses and ELISA to detect the immune response from the monocytes, stimulated by Pyrogens present in the sample. There was also much discussion about the regulatory updates for rFC testing led by Lonza’s Allen Burgensen.
Data Integrity and Automation were the focus of day 2. The customer presentation about Laboratory 2.0 futureproofing (Christine Robson (FujiFilm ) was the opening talk followed by Sinead Cowman explaining how MODA can help with keeping data intact and traceable. The talk by Bernice Fairley of Fujifilm on their approach to showing robustness in their testing routines by OOS/OOT management procedures was met with many nods of agreement around the room. Nisha Gill’s (Oxford BioMedica) talk about business considerations for assay automation certainly sparked some discussion at the open table session including data integrity which closed the meeting.
The participants highly appreciated the opportunity that Lonza has given to discuss current trends in the field of endotoxin testing
Written by SST
Endotoxin Seminar for Pharmaceutical Industry Held in Cologne
For the past few years Lonza has been organizing an Endotoxin Testing Summit at the west coast of the US. The summit focuses on recent developments in the area of endotoxin detection.
Confidence for Adopting the rFC Method in Your Lab
The article, written by Allen Burgenson, Lonza’s Global Subject Matter Expert for Testing Solutions, explores the history of LAL and the regulatory status of the recombinant Factor C assay to emphasize the acceptance of the assay by authorities.
In 1973, the FDA decided to regulate LAL because the product is derived from horseshoe crab blood. Later, in 1980, the pharmacopoeia adopted LAL as a compendial method. When Lonza (at that time Cambrex BioScience) developed the rFC product (PyroGeneTM rFC) in 2002, regulatory authorities (FDA, CBER and CDRH) concluded that rFC did not need to be regulated for use in testing pharmaceuticals. Another aspect for the quality of the assay is the quality of the reagents. The rFC reagents are made in the same FDA-licensed facility as our traditional LAL products. Lonza also filed a Master File with FDA/CBER in 2008 which contains the same information about reagents and manufacturing that would be submitted to FDA for a BLA or LAL. This Master File was cross-referenced by several pharmaceutical companies in preparing regulatory filings with FDA, resulting in the first product, Emgality® by Eli Lilly, being approved by FDA using the PyroGeneTM rFC. This clearly shows regulatory acceptance of the rFC assay, which was in fact already stated in the FDA 2012 Q&A Guidance. In our revision to the stimulus article we also presented data about detection of endotoxin from different species and did not find significant differences to LAL based methods. In addition, other labs confirmed that rFC assay was able to detect and quantifiy endotoxin from several different gram-negative species as well as the LAL assays. The data is published in scientific journals and references are given in the article.
In summary, we conclude that the rFC reagents perform as well as LAL reagents in detecting endotoxin of various species in different matrices in both our and our customer’s hands. There was no significant difference between rFC and kinetic chromogenic reagents. The FDA has determined that rFC reagents are acceptable for final product release, provided they are validated against the criteria found in USP <1225>.
Written by Ingo
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
How 3D Spheroids Are Reversing Hyperglycemia
Were you aware that type I diabetes (T1D) is an incurable, insulin-dependent, disease that accounts for roughly 10% of the more than 420 million global cases of diabetes?
The majority of cases occur as a result of the automimmune destruction of pancreatic beta cells and if left untreated can result in major organ damage and death. Diagnosed cases continue to increase at an annual growth rate of 3.92% per year and its incidence varies 50–100-fold around the world, with the highest rates in northern Europe and in individuals of European decent. Living with T1D is a constant balancing act requiring full-time attention to avoid acute, life-threatening hypoglycemia or the long-term damage done by hyperglycemia. Blood sugar levels must be monitored either with finger pricks or a continuous glucose monitor. Insulin doses must then be carefully calculated based upon activity and stress levels, food intake, illness and additional factors. These calculations are rarely perfect resulting in a tremendous emotional and mental burden. Transplantation of the pancreas or isolated islets can reverse insulin dependence, but this approach to therapy is limited to a low volume of cases due to the risks of surgery and immunosuppression, limited availability of donor human pancreas, and cost. Preservation and restoration of beta cell function remains the Holy Grail of research into T1D and this paper by Navarro-Tableros et. al highlights the ability of islet-like structures generated from Lonza human liver cells to reverse hyperglycemia in diabetic mice.
Written by Lori
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Video Protocol for CRISPR/Cas9 Genome Editing
The Journal of Visualized Experiments (JoVe) shows a very helpful application video for genome editing of several different cells are organisms. Farboud et al. explain effective and adaptable techniques and a straightforward protocol for precise genetic manipulation with the CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 RNP (CRISPR associated ribonucleotide protein). The authors show different methods for different species. The video starts with Nucleofection using the 4D-NucleofectorTM X Unit for co-transfection of the CRISPR/Cas9 complexes into human primary T cells and mobilized hematopoietic CD34+ stem cells (HSPCs). It highlights important steps when you mix the complexes and shows useful tips for successful experiments. The group from California shows in the second part of the video the genetic manipulation of entire organisms like the roundworm Caenorhabditis elegans and the crustacea Parhyale hawaiensis with microinjection methods.
Spotlight on a Cell Type: Human Hepatic Stellates
Liver disease is a major worldwide health issue, which has personally affected both friends and family in my own life. It has been estimated more than 50 million people in the world are affected by chronic liver disease. 4.5% to 9.5% of the general population globally suffer from cirrhosis1,2,3.
What are Stellates?
Why should you care?
- Melato M, Sasso F, Zanconati F. Liver cirrhosis and liver cancer. A study of their relationship in 2563 autopsies. Zentralbl Pathol 1993; 139: 25–30.
- Graudal N, Leth P, Marbjerg L, Galloe AM. Characteristics of cirrhosis undiagnosed during life: a comparative analysis of 73 undiagnosed cases and 149 diagnosed cases of cirrhosis, detected in 4929 consecutive autopsies. J Intern Med 1991; 230:165–171.
- Lim YS, Kim WR. The global impact of hepatic fibrosis and end-stage liver disease. Clin Liver Dis 2008; 12: 733–746.
CRISPR in the Classroom
The CRISPR (clustered regularly interspaced short palindromic repeats) – CAS (CRISPR associated protein) system is a prokaryotic adaptive immunity mechanism. Emmanuelle Charpentier and Jennifer A. Doudna adapted this system as a tool for gene editing experiments, for which they became the winners of the Nobel Prize 2020 in Chemistry. This new revolutionary technology has become a valuable tool in cell biology.
USP Chapter 85 Including rFC - Draft for Comment Released
After the European Pharmacopoeia (EP) already took steps towards including the recombinant Factor C (rFC) test as a compendial assay, the United States Pharmacopeia (USP) is now following suit. On 3 September 2019, the US Pharmacopeial Forum published a draft for a revised Chapter <85> that would include recombinant reagents. Unlike the EP, whose draft was for a completely new “rFC chapter” 2.6.32, the USP chose to propose an adaptation of the existing chapter on Bacterial Endotoxin Testing.
The proposed text mentions the recombinant Factor C reagents right alongside the “classical” compendial LAL reagents. Instead of only three LAL tests, four methods for BET would now exist: gel clot, turbidimetric assay, chromogenic assay (both LAL and with recombinant reagents), and the endpoint fluorescent rFC method. Test requirements and specifications (e.g. confirmation of linearity of the standard curve, necessity of performing test for interfering factors, valid correlation coefficient and PPC recovery range) remain unchanged and would equally apply to the recombinant assay.
The draft is still open for comment until 30 November 2019 in the Pharmacopeial Forum. Registration is required, but the subscription is free.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Optimization of Therapeutic Gene Editing in Human Hematopoietic Stem Cells
Sickle cell disease (SCD) is the most common inherited blood disorder in the US and affects million people worldwide. The mutation of just one amino acid in the beta-chain of hemoglobin (Hb) causes the production of an altered Hemoglobin S.
This results in multiple health problems like severe pain due to tissue damage, hypertension, kidney failure, anemia and stroke.
It was 1948 when pediatrics Janet Watson postulated for the first time the importance of fetal hemoglobin (HbF;α2γ2), in sickle cell diseases. Meanwhile, it has been shown that the re-activation of fetal hemoglobin (HbF) expression prevents the polymerization of sickle hemoglobin and therefore might prevent or retard the resulting health issues.
BCL11A erythroid enhancer, is responsible for the repression of fetal hemoglobin and for the activation of beta-hemoglobin. Introduction of point mutations in the core sequences of the BCL11A erythroid enhancer region causes de-repression of HbF in adult cells and reduced expression of the sickle-cell beta-hemoglobin and formation of polymerized sickle-hemoglobin.
Using this approach allows to use CRISPR / Cas9 based NHEJ repair to introduce point mutations into the BCL11A enhancer, without the necessity of co-transfection of a DNA template as it would be required for a repair of the effected beta-globin gene
- in vitro differentiated erythrocytes from SCD did resist sickling
- in vitro differentiated erythrocytes from beta-thalassemia patients showed higher frequency of enucleation, large size and circular shape erythroid cells due to improved globin chain balance.
Written by
Peter
What do Malaria and Bladder Cancer Have in Common?
Chloroquine is one possible answer. Chloroquine has its origin in 1934 when Hans Andersag synthesized it at I.G. Farbenindustrie in Wuppertal-Elberfeld, Germany (by the way, the city where I grew up) as a therapeutic for malaria. Nowadays, many malaria parasites are resistant.
In addition to malaria treatment, Chloroquine is used for therapy of rheumatoid arthritis. In cell culture lab, it helps to improve transfection efficiency and gets more and more into the focus of cancer treatment. Used as sensitizer for radiotherapy and chemotherapy, I wonder about the mechanism behind it? What does Chloroquine do that makes it an “all purpose weapon”?
It was in 1964 that a Japanese group showed that Chloroquine can be used to treat malignant bladder tumors. In 2018, 54 years later, in the paper of Chen et al Chloroquine has been used as a one-time treatment on bladder cancer cell lines, to understand a bit of the mechanism behind it.
They checked the lysosomes and found Chloroquine to induce lysosomal membrane permeability that enhances apoptosis in a time- and dose-dependent manner via activation of Caspase-3. Following the above link, please find also live cell imaging videos of the treated cells recorded by the CytoSMARTTM System.
This is why I am a big fan of live cell imaging. Time courses and focusing on individual cells can sometimes give more insight rather than investigating the average of a cell population at only one or two time points.
Also in 2018 a group from China investigated the effect of Chloroquine on the anti-tumor immune response of macrophages.
It becomes clear that we are not at the point of understanding the whole story of what this drug is doing. It’s hopeful and scary at the same time, isn’t it?
Written by Isabella
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Using Longer Single Guides to Increase Editing Efficiency
CRISPR has quickly become the method of choice for genome editing applications. Its simple concept and seemingly endless potential drive researchers to search for methods to improve their results. Everyone wants higher editing efficiency, less errors, and more specificity. Can that be achieved easily? I was looking for answers when I found the paper that I present you with today.
Endotoxin Testing of Blood-Based Samples
As the proverb says, blood is thicker than water, and in endotoxin testing, you can take this literally. Unlike water, blood-based samples contain many components interfering with endotoxin detection assays, making them one of the most difficult sample types to test. Whole blood is highly interfering, so often serum or plasma is used instead.
Blood proteins like albumin are widely-known interference factors which may mask any endotoxin contained in the sample. To free the endotoxin bound to these proteins, as well as inactivate any proteases that may be present, a heat treatment may be performed. Make sure to include a “hard” spike (i.e. add a known amount of endotoxin before the heat treatment), so you can show the treatment doesn’t lead to an endotoxin loss. Alternatively, you can add a dispersing agent like PYROSPERSETM Dispersing Agent to counteract endotoxin masking.
Another potential interfering factor are anticoagulants (e.g. heparin) that may be present in your blood preparation. They are used to prevent the blood from clotting, but unfortunately, they also interfere with the LAL enzymes. Anticoagulants act as chelators, binding divalent cations that are necessary for the LAL enzymes to function. Adding MgCl2 solution can overcome this problem.
For more tips regarding endotoxin testing of blood-based samples, download our TechTip “Serum/Plasma Testing with PYROGENTTM-5000 and Kinetic-QCLTM Assays” from the QC InsiderTM Portal.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
CRISPR-Based Genetic Screen in Physiological Relevant Primary Cells
I recently joined the webinar from Judd Hultquist to learn more about CRISPR screenings. I wanted to know how genome editing can be used for research and therapeutic approaches in HIV. Large scale RNAi-based screenings can be automated and are very common to identify host dependent factors (HDF), which viruses need for entry, replication and transmission. Many of these screens are done with standard cell lines like HEK293 or CHO cells with a high number of false positive candidates with low or no reproducibility in the natural host of HIV. In this article, Hultquist used the primary T cells for the screen and demonstrates that CRISPR-based genetic screens in physiologically relevant cells can specifically identify non-essential host proteins critical for viral infection.
Applying this approach to other pandemic and epi¬demic viruses will allow robust and unbiased identification of novel therapeutic targets. Here you find the link to the Nature Genetics publication.
With improved computer models, automated systems and 96-Well ShuttleTM Device for transfection, the group will publish new data soon. The latest results of the CRISPR screen Judd Hultquist presented was recently shared in the following webinar: High-throughput CRISPR CAS9 genome engineering primary cells.
Written by Peter
Manager Scientific Support, Lonza Pharma- Bioscience Solutions at Lonza
Understanding Alzheimer's Disease Variants
Scientists elucidating the mechanisms of the R47H Trem2 variant discover they cannot be replicated in humans
Currently, 5.7 million Americans are living with Alzheimer’s disease (AD), a number that is expected to rise to approximately 14 million by 2050. This strongly begs the need to understand the underlying factors of this disease so that our society can press towards a cure.
Until now, scientists have studied several AD disease variants including Apo lipoprotein E (ApoE) ε4 allele. However, the R47H Trem2 variant, which has been shown to increase the risk of late onset AD, remains largely uncharacterized. TREM2 (triggering receptor expressed on myeloid cells 2) can play both a detrimental and protective role in the regulation of microgliosis, a cellular response to CNS injury.
Researchers at the Ludwig Maximilian University of Munich and German Center for Neurodegenerative Diseases created two mouse models exhibiting the phenotypes of the R47H Trem2 variant, which resulted in a loss of protein function. Trem2 knockout mouse models with the R47H variant overexpressed or knocked in via CRISPR/Cas technology both exhibited reduced mRNA and protein expression. This was due to aberrant splicing of exon 2, which results in the formation of a premature stop codon. However, this phenotype could not be replicated in human iPSC-derived microglia-like cells, emphasizing the need to develop a model more specific to humans.
To read more, click here.
Written by Angela
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
PDA Technical Report on Low Endotoxin Recovery
If you have attended any conferences about endotoxin testing these past years, chances are you heard a talk about Low Endotoxin Recovery (LER). LER has been a hot, and hotly-contested, topic since it was first presented by Cheng et al. in 2013. In March, the Parenteral Drug Association (PDA) published its Technical Report 82 to help companies with their LER studies.
Most importantly, the report provides guidance on developing robust and scientifically sound LER hold-time studies. While it is not a “cookbook”, it details points like endotoxin source, spiking, storage time/container/temperature, as well as data analysis and interpretation. For those who encounter LER in these studies, the PDA report outlines a number of mitigation strategies, like the addition of dispersants and other sample treatments, or the switch from bacterial endotoxin testing to a biological system.
Other sections are dedicated to the proposed mechanism of LER, the history of LER studies, as well as the definition of terms that have been used somewhat interchangeably in the past. Of special interest for people tasked with looking into LER might also be the appendix – it comprises 12 case studies, and with 80 pages makes up the bulk of the report.
Technical Report 82 is available on the PDA website.
Written by Saskia
Scientific Support Specialist, Lonza Pharma-Bioscience Solutions at Lonza
Tech Tips for Primary Cell Culture: Common Errors
There is no worse feeling than going into the lab to check your cells only to find your culture is not growing or worse yet not even attached to the flask.
Cell culture can involve a huge cost both financially and in time. No one wants to waste time and money, especially if you’re on a tight budget.
I have compiled a list below of some common errors that should be avoided to keep your primary cells healthy.
Centrifuging the cells out of cryopreservation
- For many cell types, the damage to primary cells by centrifugation is much harsher than residual DMSO in the culture media.
- Following the recommended seeding density and recommended volume of media per culture vessel will sufficiently dilute the residual DMSO.
- Changing media the day after seeding cells will remove any residual DMSO.
- Check the Lonza optimized protocol to determine the appropriate thawing and seeding procedure.
Using alternate media for culturing the cells
- For many of our cell types, Lonza has specialty media specifically formulated for optimal culture conditions.
- Cells can become conditioned to specific culture media. Changing the nutrients can lead to a lack of cell attachment when plating and/or slow proliferation.
- In particular, endothelial cells can become dependent on VEGF. Therefore, trying to switch to a VEGF-free media can cause the cells to not adhere or grow very slowly.
- Check the Lonza optimized protocol to determine the appropriate media for our cells.
Re-frozen and late passage cells
- Primary cells are finite and will senescence when passaged too many times.
- Cells should be used within the specified guaranteed number of doublings
- Some cell types may be able to be re-frozen using our protocol that can be downloaded here, but later passage cells may not be able to be recovered from cryopreservation. In addition, Lonza offers no guarantee for any cryopreserved cells.
Subculturing Reagents
- Primary cells often require lower concentration Trypsin/EDTA formulations for optimal proliferation after passaging.
- Using too harsh a trypsin can lead to cell death or slow proliferation after passaging.
- Check the Lonza optimized protocol for the appropriate subculture reagents.
Some of Lonza’s cell culture media are serum free and not effective at neutralizing trypsin activity. Using these media to neutralize trypsin can lead to issues post subculture. Always be sure to use the appropriate neutralizing solution.
Written by Joseph
Gut-Brain Microbiome Potential Influence on Alzheimers Disease
When Alzheimer’s disease (AD) recently took the life of a family member and condolences poured in I discovered that almost everyone I know has family or friends who’ve been touched by this devastating disease. In fact, there are currently 50 (1) million people worldwide living with AD and it’s the fifth-leading cause of death (2). However, the actual cause of or treatment to prevent or cure the disease continue to elude us.
Of late, there has been a revival of interest in the gut-brain axis and how neurotoxins from the gastrointestinal (GI) tract microbiome may contribute to progressive age-related neurodegenerative disease development and progression. A recent paper by a team from Lousiana State University (3) provides potential evidence of this. The authors show there is an AD neocortex affinity for lipopolysaccharide (LPS), which is a neurotoxin GI microbiome-derived compound, resulting in selective impairment of transcription of neuron-specific elements known to be required for the maintenance of neuronal cytoarchitecture, synaptic connections and neuron signaling operations.
In addition, their work also shows that when LPS-treated Lonza HGN cells are exposed to LPS the results mimic those noted in the AD neocortex. This is further evidence that primary culture provides a highly useful experimental platform for further study of LPS effects on AD-like processes and their pathogenic consequences.
To learn more about the details of this study, please click here.
Written by Lori
Scientific Support Manager, Lonza Pharma-Bioscience Solutions at Lonza
- Patterson C. World Alzheimer Report 2018. The state of the art of dementia research: New frontiers. London: Alzheimer’s Disease International
- Causes of death
- Walter J. Lukiw, Lin Cong, Vivian Jabe and Yuhai Zhao, Frontiers in Neuroscience. Microbiome-Derived Lipopolysaccharide (LPS) Selectively Inhibits Neurofilament Light Chain (NF-L) Gene Expression in Human Neuronal-Glial (HNG) Cells in Primary Culture, Frontiers in Neurosci., 05 December 2018
TechTip: Screening Consumables for Bacterial Endotoxin Testing (BET)
Plastic accessories like pipette tips or multiwell plates are widely used for endotoxin testing. The harmonized Pharmacopoeias require that any plastic consumables have to be free of detectable endotoxin and - equally important but often not considered - they must be free of interfering factors. That means this is a global requirement. Lonza offers pre-screened and certified accessories, but if specific items have to be used, we have a guideline for how consumables can be qualified for endotoxin testing.
The most crucial portion of the pipette tip is the part coming into contact with the solution to be dispensed. Therefore, it can be used to draw in and dispense water for BET essentially mimicking how the tip is actually used in the test. Instead of pooling solutions, we suggest rinsing a number of tips with the same water to lower the solution volume and thus allow the tips to be tested at a low endotoxin limit. Another important consumable is the multiwell plate. For the 96-well plates, we want to avoid hot wells and will apply a similar limit as for the tips. We suggest testing the plates also with a procedure that mimics how they are used in BET, for example utilize water for BET in most of the wells and a few wells as positive controls. Evaluation of the plates is based on the reaction times.
When screening items from a new vendor a more extensive protocol may be required, including testing three different lots. In addition, it is important to remember that not all plastic is the same. Different types of plastic have different characteristics and due to production procedures, there will be variability from vendor to vendor and from lot to lot.
The complete article including detailed examples, protocols and calculations can be downloaded free from the QC InsiderTM Toolbox.
Written by SST
Binding Affinity of Endotoxin to Different Types of Plastic
The FDA Guideline "Pyrogen and Endotoxins Testing: Questions and Answers" indicates that the ability to detect endotoxin can be affected by storage and handling of the sample. As endotoxin molecules are known to absorb to surfaces, the type of sample container is quite relevant. Researchers from Sartorius Stedim Biotech therefore measured recovery of Control Standard Endotoxin (CSE) from rigid containers constructed of polystyrene, polycarbonate, polypropylene and polyethylene terephthalate (PETG) at four different time points across 24 hours.
Sample containers were filled with WFI, heated to 37°C and extracted for 60 minutes at ambient temperature. The extraction liquid was pooled and tested using the kinetic turbidimetric LAL assay.
The authors conclude that polycarbonate or polystyrene collection containers are suggested for the most accurate endotoxin testing results when the assay is conducted within 6 hours of the sampling event. Overall polystyrene is suggested as the preferred sample collection container for endotoxin assays due to its relatively high recovery and stability over time. Nevertheless, as there is significant change in recovery of endotoxin across most materials between 1 and 6 hours after inoculation, they recommend to conduct the endotoxin assay as soon as possible after sampling, preferably within 1 hour of sample collection.
This is a nice study about sample container suitability.
Written by SST
Reference:
Aseptic Sampling Best Practices, Endotoxin Binding Affinity, Sartorius Stedim Biotech GmbH
The Horseshoe Crab Experience
For many years, the Atlantic Horseshoe Crab (Limulus polyphemus) was on a path to extinction. Misnamed a “crab” since they are actually related to arthropods, these prehistoric creatures were once only thought of as an angler’s nuisance and were ultimately killed for either eel bait or fertilizer in the United States.
Today the crabs are lauded as their blood is used in an assay that detects endotoxins from Gram-negative bacteria that can be found in vaccines and other injectable drugs. The blue blood is processed into a component of the LAL (Limulus Amebocyte Lysate) Assay and used by pharmaceuticals companies. The crabs are not killed for this blood; on the contrary, they are protected by the U.S. Department of Natural Resources (DNR), the Atlantic States Marine Fisheries Commission (ASMFC) and local environmental agencies.
In the late spring, sensing the tides and using the light from the moon, the crabs come to the shores of east coast beaches in the United States and Yucatan Peninsula to spawn. Unfortunately, not all of the crabs make it back to the ocean and could be left stranded on their backside on the beach – only to desiccate and perish if they are not flipped and returned to the water.
In 1998, the Ecological Research & Development Group (ERDG) launched the “Just Flip ‘em!TM” program in order to help the stragglers get back to the ocean. During the annual Lonza Endotoxin Testing Summit, the attendees travel to Pickering Beach in Delaware to assist with this program. Folks from around the globe that have never seen or touched a horseshoe crab now have the opportunity to learn about them from Glenn Gauvry (ERDG President) and can participate in the crab flipping. 2019 marks the fifth year anniversary of the Lonza Endotoxin Testing Summit.
- You can learn more about the Lonza Endotoxin Testing Summit
- Learn about the Just Flip ‘em!TM program
- Learn about the ERDG program
Written by Travis