Listen to our 25-minute presentation, and explore Biogelx core technology with Dr. Chris Allan, Development Scientist.
At Biogelx, we are helping people who work in translational science, drug discovery and tissue engineering by making in vitro cell cultures more in vivo. We do this through manufacturing and supplying 3D cell culture materials that can be tuned to specific tissues, and synthetic bio-inks that are reproducible and easy to handle. Our portfolio of biomaterials is comprised of synthetic peptide hydrogels that mimic the extracellular-matrix to support cell growth. In addition, the chemical and physical properties of our biomaterials can be precisely tuned to replicate the characteristics of specific tissues so that the cells experience and engage with a realistic 3D environment.
Key Learning Objectives:
The advantages of Biogelx hydrogel technology
Biocompatibility is key: growing a wide range of cell types
Printing with peptide hydrogels: A case study of synthetic bio-ink development
Here to support you: Bio-ink formulation service
Do you want to learn more? Watch our recent webinar on ‘The advantage of peptide hydrogels over other 3D cell culture matrices’. Click on the link below!
Louisa Dean, MSc is a young researcher in the field of Cell Matrix Biology and Regenerative Medicine at the University of Manchester. In her recent project, she investigated novel 3D bioactive biomaterials to evaluate their suitability as substrates for wound-healing acceleration in ex vivo human skin tissue assays. In the following interview, she is sharing her experiences working with BiogelxTM-Collagen.
1. What research does your lab focus on?
Our research focuses on developing new therapies for the management of large-surface and chronic skin wounds, which remain an unresolved clinical challenge – posing a serious burden to patients and public health systems. Hence, with a clear need for effective treatment of these kind of wounds, our project aim was to design and produce novel 3D bioactive biomaterials and evaluate their suitability as substrates for wound-healing acceleration in ex vivo human skin tissue assays. Specifically, we investigated the suitability of synthetic collagen proteins as agents to promote cell spreading in both 2D and 3D (hydrogel substrate) adhesion assays.
2. Why is it so important to use 3D models?
As the critical focus of this research is tissue regeneration, a 3D substrate is needed to act as a porous scaffold for cells to colonize, ideally mimicking the extracellular matrix (ECM) that surrounds cells in their natural context – replicating an in vivo environment.
3. What are the features you think are key for a 3D matrix?
Personally, I think two fundamental properties for 3D substrates are biocompatibility and reproducibility. Another important feature is that the substrate is adaptable to various applications (e.g. mimic various tissues), with mechanical properties that are tuneable – without concerns for how changing the crosslinking chemistry of the matrix would influence the functionalization of the gels with cell-adhesive components.
4. Why did you choose BiogelxTM-Collagen?
Ease of use, easily adjustable range of stiffnesses to produce gels that suitably mimic the desired cell environment for our research application and functionality of gels to incorporate specific components (in this case a collagen biomimetic peptide sequence) into the 3D matrix.
5. How easy was it to use the gel? How easy was to repeat your research results?
Once you are familiar with preparing pre-gel solutions at a desired stiffness from the hydrogel powder, and decide the best gel format for your application, I found that my results (i.e. the level of attachment and spreading by cells) were quite consistent across my experiments.
6. What cell types do you use in your research? Please share your experience in growing cells in our gel(s).
This project began using HT1080 cells, a fibrosarcoma cell line, then from this we moved onto primary HFF cells, human foreskin fibroblasts – chosen for high levels of integrin expression and adherence properties. Each cell type behaved differently across various hydrogel stiffnesses and types (unfuctionalised vs. GFOGER collagen), but both responded well to the gel environment and demonstrated good attachment during culture.
7. What are the next steps in your research?
At the moment we have some promising preliminary results using collagen-functionalised hydrogels (which incorporates the GFOGER peptide into the matrix) with our primary fibroblast cells. So, the plan for the future is to investigate a panel of collagen proteins for these hydrogels – with the aim of applying these functionalised gels to ex vivowound closure assays using human skin.
Cell culture techniques have been widely used for decades in cell biology and preclinical biomedical research. These tools are crucial for understanding the biology of cells, and they have also become a key component of drug screening and toxicity testing in pharmacology research.
Traditional cell cultures are two-dimensional; based on a monolayer system which involves cell growth on a flat plastic surface. Still, substantial evidence suggests that cells cultured in 2D are not representative of cells residing in the complex microenvironment of a tissue. Cells are forced to adapt and generate artificial responses resulting in flattened morphology and poor replication of the complicated cellular signals between cells and their matrix. As a result, data generated by 2D cell culture methods could be misleading, and this discrepancy is thought to make these systems relatively poor models of in vivo response. 3D cell culture models have started to substitute traditional 2D systems. Such 3D systems offer a better alternative by providing similar characteristics to those experienced by cells in their natural matrix within tissues, thus replicating the function, behavior and morphology of cells in vivo. Furthermore, 3D culture models can act as an intermediate assessment stage before turning to in vivo techniques. In the context of drug screening assay, a three-dimensional model allows researchers to accurately evaluate how the cells will react to the drugs in a faster, more ethical and more cost-effective manner than in vivo studies using a living organism. Ultimately, employing such 3D techniques, offers the opportunity to improve the success rate for predicting drug responses in in vitro drug discovery.
3D culture models can be divided into two broad categories, scaffold-free and scaffold-based methods. For 3D cell culture experiments, scaffold-based systems provide a high degree of reproducibility, and provide a physical stability not achieved with scaffold-free methods, and which is required for routine assays. Scaffold materials like hydrogels allow the encapsulation of cells and have the potential to better mimic the mechanics, composition, and structural cues of native tissues over polymeric scaffolds. Animal-derived hydrogels such as those based on ECM proteins like collagen or Matrigel (a mixture of ECM proteins derived from a mouse sarcoma) possess an inherent bioactivity and provide biocompatibility. However, the ability to control the properties of such hydrogels is very limited, and they often provide low batch-to-batch reproducibility.These materials can also be difficult to handle which is a major limitation for their use in automated drug screening assays. You can read more hereabout the limitations of these systems and how to overcome them! On the other hand, synthetic hydrogels like Biogelx technology (based on simple peptides) can be consistently manufactured and can be designed to provide both the optimal biophysical and mechanical environment plus the biomimetic cues for specific cell types in a controlled and realistic manner.
In summary, it is now well established that 3D cell culture models can provide more relevant results over their mor traditional 2D counterparts. However, when it comes to developing a 3D cell culture model, it is important to consider your ultimate requirements and thus which of the available 3D culture technologies is best suited. On the related links below, you can learn more about the benefits of 3D cell culture and all the things to consider if you are thinking about switching to a 3D model or have recently started using these systems:
For researchers who wish to culture and maintain cells in a laboratory, the conditions must be precise in order to best mimic the native tissue from which the cells were isolated. For human cells, the physiological temperature of 37°C has be maintained; a humidified atmosphere with 5% CO2 and the correct pH are all important. The cells require the correct nutrition; media supplemented with serum provides the correct vitamins and minerals, amino acids, carbohydrates, hormones and growth factors. Further, this serum must have the appropriate solute concentration in order to maintain the osmotic balance of the cell.
Cell culture usually involves the expansion of cells to larger numbers to measure cell response to certain conditions or to possible therapeutics. In some cases, engineered cells are expanded for the manufacture of biotechnological products by the cells themselves. While the rudimentary practice of maintaining tissue in a salt solution had been explored since the 19thcentury, it was not until the 1940s and 1950s that the cell culture methods that we recognize today were established. During this time, the cultivation of viruses in mammalian cell culture permitted the development of vaccines. For example, the polio vaccine was originally produced through the culture of rhesus monkey kidney cells; a technique that led not only to the Nobel Prize, but also to the sparing of millions of lives from disease and mortality.
Cell culture is typically performed with a monolayer of cells on flat plastic dishes. This 2D culture environment can be convenient. It permits easy observation and evaluation of the cells through microscopy, simple access to cells for the introduction of therapeutic or diagnostic molecules, and it is comparatively inexpensive. Additionally, because the 2D culture technique has existed for so long, it has become an accepted standard by which researchers can compare their cells to those in a large body of existing findings. However, in vitro cell culture on a two-dimensional surface frequently results in the loss of cell functionality, phenotype and morphology, particularly for specialized cells. For this reason, 2D culture is a poor representation of an in vivo environment. Data gathered from 2D cell culture may also not be as accurate as researchers wish. A three-dimensional microenvironment provides a more similar mimic to native conditions within the body. For the evaluation of potential therapeutic drugs and molecules for the treatment of disease, toxicity of the drug is a primary reason for the failure of clinical trials. 3D culture models can act as an intermediate assessment stage before in vivo techniques. A 3D environment allows researchers to accurately evaluate how the cells will react to the drugs in a faster, more ethical and more cost effective manner than in vivos tudies using a living organism.
The extracellular matrix (ECM) is a network of proteins and proteoglycans that provide the cells with the necessary biochemical signals and the mechanical support needed for survival and function. The two primary regions of the ECM include the basement membrane and the interstitial matrix. The basement membrane provides the sheets of ECM material upon which and against which the cells lie. The interstitial matrix fills the interstitial or intercellular space between cells and cell types. Collagen is the most abundant protein in the ECM, providing the bulk of the structural support. There are a number of families and types of collagen based upon the morphology of the assembled protein. As the name suggests, elastin proteins permit the ECM network to have a degree of elasticity, deforming and rebounding in shape in response to mechanical stress. Similarly, hyaluronic acid is an ECM polysaccharide that provides tissues with the capacity to oppose physical compression. Laminins and fibronectins are glycoproteins which promote cell adhesion by providing motifs that are recognized and bound by cell surface integrins. Other components of the ECM include proteoglycans like chrondroitin sulfate, karatan sulfate and heparin sulfate which confer structural and biochemical properties required by cells. Based on a negative charge, the proteoglycans attract Na+ ions which in turn creates a solute concentration that induces osmosis, bringing water into the ECM for hydration. Cell survival and function are influenced by soluble factors and the mechanical properties of the ECM.
A 3D environment provides more surface area for cell contact with the matrix, as well as for more cell-to-cell interactions. A delicate balance is required for cell-to-cell interactions in culture. If too few cells are present in a given area or volume, the cells will not survive and replicate. Conversely, cells that are grown too densely will undergo selective pressures that can change the phenotype of the cell. For example, stem cells that are grown over confluence will begin to differentiate and no longer behave as stem cells. Gap junctions are the spaces between cells. Cells communicate with one another through the exchange of small molecules, ions, and even electrical signals. A 3D cell culture environment allows a cell to interact with the network of cells in the surrounding space, rather than with only the limited number of cells on a flat surface.
The mechanical properties of the ECM dictate cell gene expression and cell differentiation, as well as cell migration. The ECM of soft brain tissue versus stiff bone, and every tissue type in between will vary the way that cells interact with their microenvironment and with each other. Focal adhesions are the structures of aggregated protein within a cell that function as the mechanical connection between the cell and the ECM. Through ligand binding, focal adhesions sense the amount of force required to deform the matrix and transduces this signal to the cell. It is the dynamic process of focal adhesion assembly that permits a cell to migrate along and throughout its substrate. Complex tissues are organized into a hierarchy; certain cells are positioned together and work together for a particular function, these cells are then separated from neighboring cell types by a layer of ECM. The roles of the cells are compartmentalized and complementary with one another, and spatial organization is paramount to function.
2D culture plates are typically made of polystyrene; sometimes the plates can be coated with a thin film of physiologically relevant material such as collagen or fibronectin in order to increase cell attachment, survival, and phenotype maintenance. Some of the first 3D cell culture materials were electro-spun fibers that formed a substrate for cell adhesion. Hydrogels are frequently employed as scaffolds for 3D cell culture. Hydrogels are porous polymeric networks which provide structure, and are composed of up to 99% water. Cells can be encapsulated within the hydrogels or the cells can be seeded into pre-formed hydrogels. Hydrogels can be formed from isolated components of the natural ECM, such as collagen, hyaluronic acid and fibrin. Alginate is a polysaccharide derived from algae that can be cross-linked with ions to form a hydrogel. There are also a number of synthetic hydrogel materials including polyethylene glycol and polyacrylamide. Peptide-based hydrogels are becoming an increasingly popular choice. Some short peptide sequences can assemble into nanofibers, which in turn form the architecture of the gel. Many of these peptide hydrogels can be customized in stiffness in order to better match native tissue. Additionally, many of the above materials can be functionalized by adding cell-adhesive molecules to the monomeric subunits. Non-hydrogel scaffolds also exist. Cell culture scaffolds may be composed of micropores, microfibers, nanopores and nanofibers. Materials with morphology on a nano-scale provide a greater substrate surface area for cell adhesion and interaction. Micropores and microfibers are not too dissimilar to 2D culture, as the cells may not be as ensconced and encapsulated in the construct as with nano materials, which deliver signals to all sides of the cell. Scaffold-free 3D cell culture methods such as hanging drop techniques also exist, as well as suspension and agitation-based approaches for non-adherent cells.
3D culture environments are limited by diffusion; oxygen and nutrients must be able to reach all of the cells, and waste products must not remain trapped in the hydrogel construct. Within the body, a network of veins and capillaries would be supplying the tissues. In a lab, bioreactors and microfluidic devices can be employed to maintain cell survivorship in a 3D culture system. These systems permit nutrient and gas exchange as well as waste removal from the culture environment through constant flow and monitoring of conditions.
The microenvironment in which cells are grown influences cell morphology, gene expression and cell function. A cell culture system must accurately mimic the native environment of the cell with all of the correct biochemical and architectural signals. Cells require mechanical support, receiving signals from all three dimensions so that they can interact with both the substrate and with each other. Highly specialized cells can lose their phenotype in culture conditions outside of the body. Stem cells have been demonstrated to display greater differentiation potential when grown in a 3D environment rather than 2D. Researchers commonly use spheroid aggregates of encapsulated cells in order to mimic a tumor microenvironment when studying disease mechanisms and treatment of cancer. All of these applications rely on 3D cell culture because the cells behave differently in a 3D environment compared to a 2D flat plastic dish. Development and refinement of 3D cell culture systems continues to be a priority for researchers. Accurately modeling native tissue environments has implications for stem cell technologies such as tissue engineering and regenerative medicine, as well as cell therapy and drug discovery. Additionally, biotechnological processes that rely on the expansion of large numbers of cells while maintaining phenotype will benefit from 3D cell culture systems.