Biocompatibility vs. Consistency? You can have both.

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Three-dimensional cell culture is now clearly established as a better alternative to traditional 2D models to recreate the complexities that cells encounter in real‐life tissues.

In the body, cells are present in an extracellular matrix (ECM) consisting of a complex 3D architecture, and they adapt to their surrounding environment by responding to physical and chemical cues, which have critical implications for cellular function. In 2D cell culture, the inability of cells to achieve a realistic structural organization prevents the models from effectively mimicking properties such as in-vivo-like cellular morphology, viability, proliferation, differentiation, response to stimuli, metabolism, and general functionality. Consequently, 3D scaffolds are becoming more relevant for researchers.

Balancing Act: Biocompatibility vs. Consistency - 1

In terms of 3D scaffolds, hydrogels are the most widely used. Gels utilized as scaffolds for 3D culture must be biocompatible and should incorporate biological cues to mediate tissue formation and aid in guiding cell adhesion and proliferation. Both natural and synthetic 3D scaffolds have undeniable advantages over 2D surfaces. However, there are several differences worth considering.

Biomaterials of natural origin are often based on extracellular matrix (ECM) components such as collagen, hyaluronic acid and fibrin. They also include other naturally derived materials like alginate or silk. Natural materials might provide essential cues and contain adhesion sites that mediate biocompatibility, but they usually lack the mechanical tunability of synthetic scaffolds, which is often important for researchers wishing to better tailor their 3D models. A more important limitation is based on their intrinsic ‘natural’ origin which increases lot-to-lot variability and makes these materials very inconsistent and not as reproducible as it is essential for reliable research studies and drug testing.

Animal-sourced gels have been used extensively as substrates for 3D cell culture in cancer research. The best known commercially available is Matrigel, a natural extracellular matrix material reconstituted from a mouse sarcoma and composed of proteins such as laminin and collagen, plus an unknown mixture of growth factors and enzymes. However, Matrigel has disadvantages, such as its exact constituents are not clearly defined, and it suffers from batch‐to‐batch variation. Matrigel can also present handling difficulties when dispensed as a chilled liquid. These limitations are not ideal for 3D cultures which are intended for routine predictive drug testing. Additionally, a chemically defined scaffold would be more suitable as it has been suggested that tumor‐derived Matrigel and similar ECM combinations may result in the development of cell adhesion‐mediated drug resistance within the tumor microenvironment.

Collagen Type I is another animal-derived gel matrix extensively used for 3D cell culture. When working with collagen hydrogels, volume changes due to swelling of the matrix can occur during culture, which can lead to difficulties with reproducibility. Furthermore, such animal-derived matrices may contain remaining growth factors and viruses which can again increase batch-to-batch variability.

In addition, collagen hydrogels currently being used as 3D models have the major drawback of presenting very low rigidity, which does not mimic the naturally stiff environment of some tissues or solid cancers. To overcome these issues, other materials allow the formation of scaffolds that are mechanically stronger or matrices that match the desired stiffness found in the tissue in vivo. The stiffness of the matrix affects cell growth and morphology.  In cancer research, extensive experimental evidence has shown that mechanical stimuli from the tumor microenvironment play a key role in affecting numerous kinds of cell behavior, both in normal and in pathological conditions.

Balancing Act: Biocompatibility vs. Consistency - 2

Some of these challenges can be addressed with synthetic materials. These scaffolds have advantages such as a defined chemical composition and tunable mechanical properties that have been shown to modulate cell differentiation.  For example, Biogelx hydrogels (a scaffold framework of simple self-assembled peptides with high water content) are mechanically tunable (0.5 – 100 kPa) and their peptide nanofibers, unlike soft collagen, can offer similar mechanical properties to a range of native tissues. These materials have a dynamic nature in that the scaffold can reorganize and cells can migrate through the material by displacing the nanofibers. This is important for instance to regulate hypoxic conditions in 3D tissue culture models to mimic oxygen levels found in native tumors, and it is controllable via the 3D matrix and cell density. Moreover, due to their synthetic origin, these materials provide reproducibility and consistency, with no batch-to-batch variations. The hydrogels can be designed to support specific types of cell growth and function. Biogelx synthetic hydrogels are peptide-based, thus fully biocompatible. Furthermore, the hydrogels can be functionalized with short biomimetic peptide sequences that are key to several ECM proteins, providing the essential cues and adhesion sites to enhance biocompatibility. GFOGER for collagen, IKVAV and YIGSR for laminin, and RDG for fibronectin are some of the available options.

Balancing Act: Biocompatibility vs. Consistency - 3

In summary, there are now more options than ever before for growing cells in 3D culture, and innovative materials and methods are becoming available to support the generation of new platforms that are suitable to sustain 3D cell growth and tissue formation in vitro. This needs to be done in a reliable and reproducible manner following clearly defined protocols, which will help to encourage the scientific community to adopt these new approaches while recognizing the limitations of conventional 2D cell culture.


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