Cells behave differently in a 3D environment


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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 to be maintained; a humidified atmosphere with 5% COand 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 vivo studies 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 since 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 electrospun 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 nanomaterials, 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 continue 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.

 

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