Quickly transform your single-cell-type 2D culture model to 3D in 2 quick steps.
- 1. Mix a single cell type with liquid collagen matrix
- 2. Apply absorber for 15 minutes
The RAFTTM 3D Cell Culture System offers a new way to develop organoids, tumoroids, and microtissue. The RAFTTM 3D Culture System is designed to enable self-assembly of cells into a dense natural protein scaffold.
Develop microtissues and organoids from cells and cell lines in less than 1 hour.
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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 RAFTTM 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.
The skin provides a vitally important protective separation between the internal and the external environments1. There are three structural layers to the skin: the epidermis, the dermis and subcutis. The epidermis is the outer layer, serving as the physical and chemical barrier between the interior body and the exterior environment. The dermis is a deeper layer providing the structural support of the skin. Subcutis is made up of loose connective tissue and fat, which can be up to 3 cm thick on the abdomen.
Epidermis is a stratified squamous epithelium consisting of several cell types. The most abundant cell type of epithelial layer of the skin is the keratinocytes which synthesize the protein keratin2. Protein bridges called desmosomes connect the keratinocytes, which are in a constant state of transition from the deeper layers to the superficial. The epidermis varies in thickness based on the tissue of origin. The four separate layers of the epidermis are stratum basale (basal or germinativum cell layer), stratum spinosum (spinous or prickle cell layer), stratum granulosum (granular cell layer) and stratum corneum. These layers are formed by the differing stages of keratinocyte maturation.
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.
In our recent white paper, we describe the procedure in constructing full thickness (FT) skin models using the RAFTTM 3D Culture System with primary Human Neonatal Epidermal Keratinocytes (NHEK) and primary Human Neonatal Dermal Fibroblasts (NHDF). The RAFTTM 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.
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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.
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.
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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.
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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.
RAFTTM System allows for the creation of tissue-like structures. The 3D matrix of the type 1 collagen-based RAFTTM 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 RAFTTM System. Our results show that the RAFTTM 3D System represents a more robust model for the long-term maintenance of liver-specific functions.
Recent data (Gieseck et al.) established in the RAFTTM 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.
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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.
RAFTTM 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 RAFTTM System with additional optimized protocols that allow for applying standard histological, biochemical and imaging techniques to 3D cultures.
Applying standard microscopy techniques can be challenging on 3D cultures. Due to the translucent properties of RAFTTM 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 RAFTTM 3D System for several days prior to fixation and immunocytochemical staining. The protocol demonstrated an efficient permeability of the antibodies through the RAFTTM Matrix to visualize these 3D cultures.
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 VialightTM Assays could seamlessly assess viability and proliferation of HCT116 colon cancer cell line and human dermal fibroblasts cultured in RAFTTM 3D Cultures.
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 NucleofectorTM Technology and RAFTTM 3D Culture System attempt to bridge these gaps. The NucleofectorTM System has been an established method to accomplish efficient transfection in hard-to-transfect cell types. When coupled with the RAFTTM 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 NucleofectorTM Transfection Technology, the RAFTTM System used this efficient approach to create 3D cultures which maintained transfected substrate for over 4 days.
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Buy a RAFTTM trial kit today in one of these formats - 24-well, 24-well cell culture insert, 96-well
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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 RAFTTM 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 RAFTTM 3D Cell Culture System.
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Daniels et. al (2015) demonstrates that the RAFTTM 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.
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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 RAFTTM 3D Cell Culture System, spatially defined organotypic blood-brain-barrier models mimicking epithelial and endothelial tissues can be made simply.
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 RAFTTM System.
Blood-Brain Barrier Model in RAFTTMCultures. Figure A shows primary human astrocytes in RAFTTM Cultures. Figure B shows co-culture of astrocytes with brain endothelial cells hCMEC/D3 and transport of glucose-coated gold nanoparticles in primary human astrocytes and/or brain endothelial cells (hCMEC/D3). Data courtesy of Gromnicova et al (2013) PlosOne 8(12)
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.
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Classic 2D cell culture systems are often not suitable for mimicking the complex structures of human cornea, skin or blood brain barrier. With the RAFTTM 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 RAFTTM System provides an ideal solution for researchers to develop an in vitro corneal model within rat and bovine type 1 collagen.
A 3D in vitro human cornea model containing biomimetic corneal limbal crypts established in RAFTTM System. H&E stained paraffin embedded section shows that the HLE (human limbal epithelial) cells formed a healthy, 3-4 cell multi-layered epithelium on the flat surface of the HLF (human limbal fibroblasts) embedded in collagen. Data Courtesy of Levis et al (2013) Biomaterials.
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.
RAFTTM 3D Culture System – Translating LSCD Research into Clinical Applications
RAFTTM 3D Culture System uses high density collagen scaffolds which are very robust and transparent. Our customers have leveraged this capability of RAFTTM 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, RAFTTM 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 RAFTTM 3D Cell Culture System.
Data Courtesy of Julie Daniels and the team. Figure shows subjective assessment of RAFTTM TE and HAM transparency. Macroscopic images of text through either RAFTTM TEs (A) or HAM (B) were captured for qualitative comparison. As the image demonstrates, RAFTTM TEs showed comparable transparency at certain collagen concentrations which is an important criteria to serve as an alternative to HAM treatment. Read the full paper.
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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 RAFTTM 3D System and compared to a conventional 2D system. The data from this study shows that islets embedded in the RAFTTM 3D System maintain their tissue integrity better than in monolayer and suspension cultures. "Overall the use of RAFTTM 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)
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The RAFTTM 3D Cell Culture Kit is a simple-to-use 3D cell culture system that enables the production of robust, reproducible 3D cultures in less than an hour. The RAFTTM Kit offers a complete solution with the necessary reagents and consumables to develop human or animal organoids for disease modelling, toxicity studies, and cancer cell migration.