RAFT^TM 3D Cell Culture System for Tissue Modeling

1. 1. Mix a single cell type with liquid collagen matrix
2. 2. Apply absorber for 15 minutes

1. 1. Mix together multiple cell types with liquid collagen matrix
2. 2. Apply absorber for 15 minutes

1. 1. Mix a single cell type with liquid collagen matrix
2. 2. Apply absorber for 15 minutes
3. 3. Layer second cell type on top of organoid disc

1. 1. Mix cells with liquid collagen matrix in 96-well format
2. 2. Apply absorber for 15 minutes or  form cells to spheroids using ultra-low attachment plates
3. 3. Transfer 96 well formatted tumoroid or spheroid to 24-well plate
4. 4. Create a liquid cell-free matrix of choice in the plate surrounding the 96-well format tumoroid
5. 5. Apply 24-well absorber to create surrounding matrix.

Various studies have shown that in vitro skin micro-tissues comprising of a fibroblast layer containing dermis topped by a stratified layer of keratinocytes can be formed on polycarbonate transwells using air-liquid interface cultures. The total duration to obtain the full thickness skin models can vary from 28–30 days from seeding the fibroblasts, followed by keratinocytes and differentiation.

The RAFT^TM 3D Cell Culture System could provide an advantage of shortening the culture period to 22–24 days. Our air-liquid interface format provides a scaffold for differentiating keratinocytes, embedding fibroblasts and possibly additional skin cell types.

Normal Human Skin Tissue. Image courtesy of Wikimedia.

Dermal fibroblasts are cells within the dermis layer of skin which are responsible for generating connective tissue and allowing the skin to recover from injury.³ Using organelles (particularly the rough endoplasmic reticulum), dermal fibroblasts generate and maintain the connective tissue which unites separate cell layers. Furthermore, these dermal fibroblasts produce the protein molecules including laminin and fibronectin which comprise the extracellular matrix. By creating the extracellular matrix between the dermis and epidermis, fibroblasts allow the epithelial cells of the epidermis to affix the matrix, thereby allowing the epidermal cells to effectively join together to form the top layer of the skin.

Epidermal-dermal skin substitutes are currently the most utilized tissue-engineered skin model that closely resembles the structure of native human skin. The presence of both keratinocytes and fibroblasts within the epidermal-dermal skin substitutes leads to the production of a variety of growth factors and cytokines which expedite wound healing, highlighting the importance of epithelial-mesenchymal interactions. These epidermal-dermal skin substitutes have been utilized for wound healing assays, toxicological research, co-culture studies, tissue engineering research. 

Primary Human Keratinocytes and Fibroblasts Cultured in RAFT^TM System

In our recent white paper, we describe the procedure in constructing full thickness (FT) skin models using the RAFT^TM 3D Culture System with primary Human Neonatal Epidermal Keratinocytes (NHEK) and primary Human Neonatal Dermal Fibroblasts (NHDF). The RAFT^TM Skin Model consists of a compressed collagen-type-I-based hydrogel that closely mimics the human dermis. This layer is topped with differentiated epidermal keratinocytes in an ALI mimicking the epidermis. We show analyses of the FT skin model to resemble the native skin by immunohistochemistry and immunofluorescence validated using key markers for epidermis and dermis.

Related Products

• Human Neonatal Epidermal Keratinocytes (NHEK) and KGM-Gold^TM Keratinocyte Growth Medium
• Human Neonatal Dermal Fibroblasts (NHDF) and FGM-2 BulletKit^TM Medium
• Buy a RAFT^TM Trial Kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well

References

1. Richard LE and Ellen AR.  Molecular biology of keratinocyte differentiation. Environmental Health Perspectives (1989); 80: 109-116 2.
2. Skin Structure and Function 
3. Darling, David (10 September 2011). "Hypodermis". Encyclopedia of Science.

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

It has been shown that an estimated 70% of cancer cells can cluster together and form higher-order structures that are referred to as tumoroids or spheroids, which then proliferate to make larger, more complex and multi-layered structures. The cells in the center of the tumoroid are exposed to a hypoxic environment and the core can become necrotic, which more closely resembles the inner tumor mass in vivo. This special behavior of cancer cells cannot easily be mimicked in a classical 2D cell culture environment. This provides a compelling argument for the adoption of tumoroid-enabling 3D cell culture techniques for oncology research. Please select your area of interest from the navigation above to learn more.

The Benefits of RAFT^TM 3D Cancer Models

• Cancer cell lines from breast, liver and lung cancer have shown tumoroid structure formation in RAFT^TM 3D System
• The cancer cells are being studied in a biologically relevant collagen environment. Therefore the cells can interact with the matrix through matrix-metallo-protease activity and integrin/DDR cell surface receptors and signaling cascades
• The cultures are amenable to various assays allowing the generation of dose-response curves. Assays that have been shown to be compatible with the RAFT^TM System are: Cell proliferation assays, Mitochondrial function assays, Cell signaling assays, and Protein phosphorylation assays  

Cancer Tumoroids and Immune Cells

Oncoimmunology is an emerging area and there is growing emphasis on utilizing better models to understand this application. To properly study the interaction between immune cells and target tumor cells, an appropriate in vitro model system mimicking tumor microenvironment must be established. However, much of the data published to date used cancer cells plated as a two-dimensional (2D) monolayer. A growing amount of data has shown that cells cultured in this manner lack the cell:cell and cell:matrix communication, metabolic gradients, and polarity demonstrated in vivo.

The ability to perform matrix infiltration studies is also eliminated with the use of 2D cell culture. By embedding cancer cells into a three-dimensional (3D) matrix and allowing the formation of tumoroids, the shortcomings of using 2D cultured cells can be overcome as communication networks and cellular gradients observed within in vivo tumors are re-established. 

Related Products

• Natural Killer Cells
• Buy a RAFT^TM Trial Kit today in one of these formats -  24-well, 24-well cell culture insert, 96-well

Angiogenesis and Cancer

The properties and behavior of cancer cells and tumoroids are strongly influenced by the surrounding extracellular matrix. Therefore, each meaningful oncology model should contain a representative extracellular matrix to mimic tumor microenvironment more closely. Tumor progression is mediated by micro-environmental conditions that include cell-cell and cell-extracellular matrix (ECM) interactions. Tumor metastasis is influenced by the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumor microenvironment.

Related Products

• HUVECs and EGM-2^TM Endothelial Cell Growth Medium
• Buy a RAFT^TM Trial Kit today in one of these formats -  24-well, 24-well cell culture insert, 96-well

Breast Cancer Model in RAFT^TM System

Relevant Downloads and More White Papers

• Engineering a vascularised 3D in vitro model of cancer progression
• Parallel Preparation of RAFT^TM 3D Cell Cultures on the Tecan Freedom EVO^® - Increasing Throughput for a Dose Response Experiment
• The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

Hepatocytes are the primary cell type of the liver and function to provide the majority of the detoxification in the body. It is widely accepted that hepatocytes lose their function rapidly when cultured on a planar 2D surface. There is a desire within drug discovery and academic research to create a liver model that maintains higher levels of functionality for a longer period of time, ideally for many weeks.This would enable chronic exposure experiments to be carried out with some confidence that the results would bear similarities to results from primary cells and humans. The liver model is also an area where co-culturing of different cell types has proven to be of the most use. In addition to the parenchymal hepatocytes, researchers have been adding the non-parenchymal stellate and kupffer cells which clearly aid in supporting culture longevity and hepatocyte function.

Primary Human and Animal Hepatocytes Cultured in RAFT^TM System

RAFT^TM System allows for the creation of tissue-like structures. The 3D matrix of the type 1 collagen-based RAFT^TM Culture provides a more natural cell culture environment and therefore a potentially superior model for in vitro screening. In a recent study, we compare cell viability and cell morphology of rat and human hepatocytes, and the maintenance of Cytochrome P450 (CYP) activity in human hepatocytes grown in the traditional Sandwich Model with that of cells cultured in the 3D RAFT^TM System. Our results show that the RAFT^TM 3D System represents a more robust model for the long-term maintenance of liver-specific functions.

iPSC-derived Hepatocytes Cultured in RAFT^TM System

Recent data (Gieseck et al.) established in the RAFT^TM 3D Cell Culture System has demonstrated that human iPS-derived hepatocytes show enhanced levels of maturation markers and cytochrome P450 3A4 activity levels when compared to cells maintained in a 2D culture.

Related Products

• Hepatocyte Culture and Maintenance Medium
• Human or Animal Hepatocytes
• Buy a RAFT^TM Trial Kit Today in one of these formats - 24-well, 24-well cell culture insert, 96-well

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

3D culture is transforming cell biology research and tissue engineering applications. These advanced tools are allowing researchers to develop higher-order structures with cells in vitro. This, in turn, allows cells to grow and interact in an environment more closely mimicking in vivo. However, such revolution in research is also coupled to new challenges. It can be very difficult to apply standard cell analysis techniques on 3D cultures which have long been established in 2D environment. The tight structure of some spheroids, which form a necrotic core, the lack of transparency of many plastic materials used in 3D methods or the dense fiber network of some 3D hydrogels can interfere with basic techniques such as imaging, transfection, cytotoxicity assays or biotherapeutic applications.

RAFT^TM 3D Culture System is attempting to address such challenges with 3D methods enabling researchers to work with a 3D system that is well supported with protocols to conduct downstream assays. Lonza continues to develop and support the RAFT^TM System with additional optimized protocols that allow for applying standard histological, biochemical and imaging techniques to 3D cultures. 

Visualization of RAFT^TM 3D Cultures with Standard Microscopy Techniques

Applying standard microscopy techniques can be challenging on 3D cultures. Due to the translucent properties of RAFT^TM Scaffolds, immunofluorescently stained 3D cultures can be visualized with subcellular resolution under a standard fluorescence microscope. The breast cancer cell line MCF7 and human dermal fibroblasts were cultured in RAFT^TM 3D System for several days prior to fixation and immunocytochemical staining. The protocol demonstrated an efficient permeability of the antibodies through the RAFT^TM Matrix to visualize these 3D cultures.

Optimization of Cell-Based Assays Made Simple

The assessment of cell viability in 3D cultures can be equally challenging because most cell based assays have been optimized for traditional 2D culture. It is generally recommended that the assays are optimized by researchers to make them work for 3D method in use. The higher density of the cells and the abundant presence of extracellular matrix molecules in certain 3D methods add to the complexity of using these assays. Lonza attempts to make this optimization step easier. With slight modifications to standard 2D protocol, Lonza’s Vialight^TM Assays could seamlessly assess viability and proliferation of HCT116 colon cancer cell line and human dermal fibroblasts cultured in RAFT^TM 3D Cultures. 

Transfecting 3D Cultures Efficiently

Transfection of cells in 2D culture is already challenging if it comes to hard-to-transfect cell types. Given the complexity of 3D cultures, standard 2D transfection techniques pose bigger challenges. Lonza’s Nucleofector^TM Technology and RAFT^TM 3D Culture System attempt to bridge these gaps. The Nucleofector^TM System has been an established method to accomplish efficient transfection in hard-to-transfect cell types. When coupled with the RAFT^TM System, achieving high transfection efficiency becomes seamless in 3D environment. In a recent technical note, we show a more successful approach where cells can be transfected in 2D prior to transfer into 3D method. The prerequisite for this is achieving high transfection efficiencies while keeping cells viable so that a large number of cells transferred into 3D express the gene of interest and stay viable over a longer period. In combination with Lonza’s Nucleofector^TM Transfection Technology, the RAFT^TM System used this efficient approach to create 3D cultures which maintained transfected substrate for over 4 days.

Relevant Downloads

Buy a RAFT^TM trial kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

The development of more complex in vitro airway models is needed for the assessment of novel drugs and chemicals because of the limited biological relevance of animal models to humans as well as ethical considerations¹. Many cell-based assays are usually developed in 2D with limited cellular and functional representation of the native tissue. An optimal co-culture model is needed to truly understand the cellular interactions and mimic the features of airway remodeling in the diseased states. For instance, tissue injury is associated with airway remodeling in several airway diseases including asthma, chronic obstructive pulmonary disease, and fibrosis alveolitis². In the case of epithelial injury, certain airway epithelial-derived mediators can stimulate the proliferation of smooth muscle cells.

With current 2D and 3D methods, it is sometimes challenging to layer and establish 3D co-culture models with multiple cell types to achieve the complexity of an airway model. In order to better understand cellular interactions, we developed a preliminary 3D co culture system with bronchial epithelial and smooth muscle cells from normal and asthmatic donors using the RAFT^TM 3D Cell Culture System. As a next step, we seek to develop a stratified air-liquid interface model using bronchial epithelial cells and smooth muscle cells.

The morphology and the growth pattern of bronchial smooth muscle cells appeared to be influenced by the RAFT^TM 3D Cell Culture System.

Related Application

Daniels et. al (2015) demonstrates that the RAFT^TM System can be used to support the air liquid interface model with human corneal epithelial cells in a co-culture with limbal melanocytes. After one week in submerged culture followed by another week of air-lifting, multi-layering and stratification of the epithelial sheet was observed. Limbal melanocytes served as a feeder layer and supported the formation of thicker epithelial cell sheet.

Related Products

• Human Bronchial Epithelial Cells (NHBE) and BEGM^TM BulletKit^TM Bronchial Epithelial Growth Medium
• B-ALI^TM BulletKit^TM Bronchial Epithelial Air-Liquid Interface Medium
• Human Bronchial Smooth Muscle Cells (BSMC) and SmGM^TM-2 BulletKit^TM Smooth Muscle Growth Medium
• Airway Cells from Asthma, COPD and Cystic Fibrosis Donors
• Buy a RAFT^TM Trial Kit Today in one of these formats - 24-well, 24-well cell culture insert, 96-well

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

¹ Mol Pharm. 2014 Jul 7; 11(7): 2082–2091. doi:  10.1021/mp5000295

² Am J Respir Cell Mol Biol. 2009 Sep;41(3):297-304. doi: 10.1165/rcmb.2008-0358OC

Blood Brain Barrier Models

Many tissues are composed of multiple cell types that are often organized within well-defined layers. Examples of such tissues are the human skin, the cornea or the blood-brain-barrier. Classic 2D cell culture systems are often not suitable for mimicking the complex structure of these tissues. With the RAFT^TM 3D Cell Culture System, spatially defined organotypic blood-brain-barrier models mimicking epithelial and endothelial tissues can be made simply.

Human Brain Endothelium

The blood-brain barrier is formed by microvascular endothelial cells, pericytes and astrocytes. It prevents the entry of most large hydrophilic molecules and many potentially harmful toxins from the blood into the brain. On the other hand, it also prevents the entry of many therapeutic agents into the brain. Considerable efforts are made to develop therapeutics that can cross the blood-brain-barrier and these efforts can be supported by high-value 3D in vitro blood-brain-barrier models. Below see an example of a 3D blood-brain barrier model developed by researchers based on the RAFT^TM System.

Nervous System Injury Repair

Artificial tissues constructed from cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In a new study, the effectiveness of using a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested.

Read the full publication.

Relevant Downloads

• A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions
• Glucose-Coated Gold Nanoparticles Transfer across Human Brain Endothelium and Enter Astrocytes In Vitro
• An allogeneic ‘off the shelf’ therapeutic strategy for peripheral nerve tissue engineering using clinical grade human neural stem cells

Related Products

• Normal Human Astrocytes (NHA) and AGM^TM BulletKit^TM Astrocyte Growth Medium
• Human Endothelial Cells and Media
• Buy a RAFT^TM Trial Kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

Classic 2D cell culture systems are often not suitable for mimicking the complex structures of human cornea, skin or blood brain barrier. With the RAFT^TM 3D Cell Culture System, spatially defined organotypic barrier models mimicking epithelial and endothelial tissues can be made simply. Generation of an artificial human cornea in vitro could have applications as a tool for pre-clinical development of novel therapies. Cornea, in vivo, is high in collagen type 1 concentration and the RAFT^TM System provides an ideal solution for researchers to develop an in vitro corneal model within rat and bovine type 1 collagen.

Limbal Stem Cell Deficiency Research (LSCD)

Limbal epithelial stem cells (LESCs) are a population of cells responsible for maintenance and repair of the corneal surface. Injury or loss of these cells can lead to limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed¹. Transplantation of cultured human limbal epithelial cells (hLE) on a carrier known as human amniotic membrane (HAM) can restore vision. However, this treatment has its challenges since clinical graft manufacture using HAM can be costly, unreliable due to supply issues, and inconsistent from donor variability. Research has aimed to develop alternative carrier methods to HAM to increase success rate of the LSCD treatment. In order to serve as a carrier for hLE cells to the cornea, it is important that the alternative method has the right optical and mechanical properties (i.e. material should be as transparent as possible) as well as the capability to expand and carry cells to the cornea.

RAFT^TM 3D Culture System – Translating LSCD Research into Clinical Applications

RAFT^TM 3D Culture System uses high density collagen scaffolds which are very robust and transparent. Our customers have leveraged this capability of RAFT^TM Constructs to understand if they can potentially be utilized as a reliable and robust tissue equivalent (TE) to HAM.

In a study by Julie T. Daniels and her team at University College London, RAFT^TM 3D Constructs were able to support optimal hLE expansion and stratification conditions as well as provide a tunable option to develop a consistent production process for an alternative method to HAM.

Watch our webinar "Overcoming Current Challenges with 3D Cell Culturing Webinar" and learn how Prof. Dr. Julie T. Daniels and her team have developed multi-layer corneal models using the RAFT^TM 3D Cell Culture System. 

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.

Diabetes mellitus (DM) affects millions of people worldwide. Development of novel tissue culture techniques for maintaining pancreatic islets is critical in order to improve access to donor material, better understand the disease group and to support the basis for improved transplantation therapies.¹

Pancreatic islets are clusters of non-proliferating cells with limited viability making them difficult to utilize for long-term studies. Constant access to donor material is also a challenge, in particular, for human-sourced pancreatic islets. Most of the donor pancreas are sought for islet transplantation procedures with limited availability for research use. As a result, there is an unmet need to improve the viability and maintenance of islets to alleviate the accessibility concerns for research. Currently, pancreatic islets are utilized in a variety of research areas with a primary focus on understanding and improving therapies for diabetes.

In a recent study (Szebeni et al., Cytotechnology. 2017 Apr; 69(2): 359–369), pancreatic islets were cultured in the RAFT^TM 3D System and compared to a conventional 2D system. The data from this study shows that islets embedded in the RAFT^TM 3D System maintain their tissue integrity better than in monolayer and suspension cultures. "Overall the use of RAFT^TM provided excellent results in preserving islet spheroid viability, structure integrity and insulin, glucagon production for at least 18 days ex vivo". (Szebeni et al., p. 369)

Relevant Downloads

• Real architecture for 3D Tissue (RAFT^TM) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells. Cytotechnology (2017) 69:359–369 

Related Products:

• Human Pancreatic Islets
• Human Diabetic Cells and Media
• Buy a RAFT^TM Trial Kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well

References:

¹ Szebeni et al, Cytotechnology (2017) 69:359–369

Any questions? You wonder if the RAFT^TM System is applicable to your project? Contact us to get support.