Cells Require the Right Physical Environment

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Three-dimensional cell culture models are favored over two-dimensional culture techniques for many in vitro culture applications. Within the body, the extracellular matrix (ECM) provides a network of proteins and proteoglycans that determine structure and signaling for cells in their natural environment. A 3D microenvironment more closely mimics native tissue and provides better conditions for the cells compared to traditional culture on a 2D surface such as polystyrene. 3D cell culture excels in replicating tissue-specific conditions in vitro and has applications in therapeutic fields such as tissue engineering, drug discovery and toxicity testing using organ models.

Cellular function is determined by the influence of microenvironmental cues including both biochemical and physical signals.  Growth factors, elastic modulus, stress, and strain of the local environment have been demonstrated to impact cell behavior.  In vitro culture of cells is often associated with the loss of cell-specific morphology, phenotype, and function.  In order to address this shortcoming in translational discovery, researchers are developing and generating improved materials to mimic in vivo conditions in vitro3D cell culture involves scaffolds to support the cells. The sub-macro scale morphology of the scaffolds may be micropores, microfibers, or nanofibers.  Nano-scale materials provide the advantage of increased surface area for cell adhesion and interaction with the culture substrate.  The cells receive signals from all sides rather than on one dimension; these interactions are necessary for cell survivorship and functionality.

3D culture scaffolds often take the form of hydrogels.  In some cases, the materials can be 3D-printed.  Hydrogels are composed of up to 99% water with a porous polymeric network providing structure.  Mechanical properties of the hydrogels are comparable to that of soft tissues and in some cases can be customized in stiffness to a particular tissue type.

Commonly used 3D cell culture materials are frequently animal-derived.  Examples include Matrigel™, collagen and decellularized tissues. Naturally-derived materials typically provide a superior mimic to a native environment, however, they can also trigger an immune response. Matrigel is an undefined mixture secreted by mouse sarcoma cells that is used as a cell culture substrate.  Matrigel provides a network of ECM proteins and growth factors that promote cell adhesion and survival.  However, animal-derived products such as Matrigel are not fully characterized and pose questions about reproducibility and batch-to-batch consistency.  Similarly, collagen is the primary structural protein in the body, so it is a natural fit for cell culture conditions.  However, collagen used for cell culture is isolated from rats, pigs or cows, and raises comparable concerns as Matrigel.

Synthetically produced cell culture matrices include polyethylene glycol (PEG), polycaprolactone (PCL) and polylactic acid (PLA).  Synthetic polymers are typically not biodegradable; they are also not biocompatible in the sense that they do not provide the correct signals to the cells, and thus, they have low cell adhesion.  Cell-adhesive molecules may be introduced to the system or tethered to the monomer subunits. Synthetic polymers do confer the advantage of thorough characterization and ease of scaled-up production.

An improvement on the animal-derived materials and some purely synthetic polymers are peptide-based cell culture scaffolds such as PuraMatrix™ and the Biogelx products. These materials are synthesized in a lab and are composed of relatively short, well characterized peptide sequences with high purity and batch-to-batch consistency. Biogelx products offer the added benefit of tunable stiffness. Increasing the peptide concentration in the hydrogel increases the elastic modulus of the microenvironment, offering a further layer of customization to the in vitroculture format.

Stem cells show great promise in the field of tissue engineering.  Stem cells are capable of long-term expansion and can be directed to differentiate into theoretically any cell in the human body.  2D culture on top of a thin Matrigel film remains one of the accepted and widely implemented culture methods for stems cells.  Large-scale expansion of stem cells may likely be performed in a 3D culture format in the future.  Current 3D models have had the challenge of imprecise cell agglomeration at high densities.  Also, high-volume suspension-based 3D culture of stem cells subjects the cells to harmful sheer forces from media flow.  Stem cells are particularly sensitive to the mechano-elastic microenvironment.  Appropriate elastic modulus provides stem cells with the signals needed to maintain their stemness and multi or pluripotency. Stiffness of a cell culture substrate has also been used to influence mesenchymal stem cell differentiation, including with the above-mentioned materials.

Culture of cancer cells is typically aimed to screen potential therapeutics or to determine oncogenic mechanisms that result in disease.  Most preclinical therapeutics that show promise in 2D culture fail at the next stage when they are tested in a complex organism such as a mouse.  3D tumor models such as spheroids allows researchers to generate more of both cell-to-matrix and cell-to-cell interactions that accurately simulate the organization of a tumor microenvironment. This is particularly important with a disease such as cancer that involves dysregulation of cell signal transduction.

Modeling and maintaining cellular function in a laboratory environment outside of the human body is a significant challenge to research and translational therapeutics.  Development of materials that can customized in architecture and biochemistry provide a close mimic to the native microenvironment and are desired for in vitro culture models. A 3D cell culture system allows researchers to optimize growth and survival environments over traditional cell culture performed on a two-dimensional plastic surface.  A number of academic labs and companies are addressing the need for these materials and are demonstrating their efficacy for tissue engineering and drug discovery applications.


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