Why Optimize ECM?

Each different cell type in a multicellular organism has its own unique microenvironment which is composed of several factors influencing the cell’s proliferative and differentiation status. In general, these factors fall into one of two categories: soluble cues (growth factors, metabolites, dissolved gases) and insoluble cues (the composition, architecture, and elasticity of the extracellular matrix (ECM) and cell-cell interactions)^1,2. Since proliferation and differentiation of cells in culture has become increasingly important for basic research³, drug discovery⁴ and tissue engineering/regenerative medicine⁵, the incorporation of these cues for in vitro cultivation of cells is crucial for recreating a cell’s natural niche. Recently, emphasis has been placed on finding the appropriate mixture of the soluble cues in liquid media for culturing stem cells with minimal attention to the insoluble triggers ^6-9. Insoluble cues such as the glycoproteins within the ECM however play significant roles in specifying cell fate by affecting the same signaling pathways used by soluble cues such as growth factors³. Recent data in mammalian cell and human mesenchymal stem cell differentiation show that the impact of insoluble cues; however, is far more profound than previously thought. In particular, the proper composition of the ECM, its presentation to the cell, and its appropriate stiffness significantly improve the biological relevance of an in vitro cell culture since the cell acts and looks far more like its in vivo counterpart in the presence of the appropriate soluble medium.

One example in mammalian cell culture that illustrates the utility of providing the appropriate ECM composition and presentation is in vitro mammary epithelial cell differentiation. Mammary epithelial cells have two requirements for in vitro differentiation into bilayered acini capable of producing milk proteins: the ECM must be properly presented to the cell and it should be laminin-rich. Mammary scp2 epithelial cells do not produce b-casein in the presence of prolactin when the cells are simply grown as two-dimensional monolayers; the monolayer must first be overlaid with a laminin-rich basement membrane extract¹⁰. Indeed, purified laminin by itself can substitute for the basement membrane extract, underscoring the additional importance of matrix composition¹⁰.

Another example in mammalian cell culture is branching morphogenesis during early kidney development. One of the first steps in early kidney development is the extensive branching morphogenesis of a structure known as the ureteric bud (UB) within surrounding mesenchymal tissue. To recapitulate UB branching in-vitro, three-dimensional culture with the appropriate soluble and insoluble cues plays a crucial role¹¹. Simple hydrogels such as alginate-based gels and Puramatrix did not work, underscoring the need for a surrounding matrix fortified with additional ECM proteins and growth factors¹¹.

Matrix stiffness, which matches that of a specific target cell’s in vivo microenvironment, acts as a potent signal to differentiate mesenchymal stem cells (MSCs) into the target cell. More importantly, recent data illustrates that specific matrix stiffness allows MSCs to more fully differentiate into the matching target cell type than it can with soluble inducers alone. In non-inductive media, matrix stiffness by itself is a strong inducer of MSC differentiation (measured in terms of morphology and tissue-specific gene and protein expression profiles) when MSCs are plated on matrices of different elasticities12. In tissue-specific inductive media; however, the levels of various tissue specific protein marker levels were comparable to in vivo levels only when the appropriate stiffness was matched to the correct inductive medium¹². For example, in myogenic inductive media, the MyoD1 muscle marker protein was expressed at approximately half the level of that in C2C12 muscle cells when MSCs were plated on matrices of different stiffness. Only when the matrix stiffness matches that of muscle (11 kPa), are MyoD1 levels in differentiating MSCs and C2C12 cells comparable¹².

In sum, the emerging picture for the future of cell culture is the imperative to optimize both the soluble medium and the insoluble cues. Since both sets of cues affect the same signaling pathways through crosstalk and the outcome of mixing these cues in one culture system cannot be predicted a priori ^1,8,9,13, it stands to reason that both sets of cues must be co-optimized for best results. Co-optimization of the many combinations of both soluble and insoluble cues lends itself to high-throughput approaches where several groups have begun work ^1,8,9,14,15. It is logical that such screening approaches will be crucial as ideal cell culture systems are developed for differentiating adult and embryonic stem cells for future applications in tissue engineering and regenerative medicine. In the future, since the purpose of cell culture optimization in vitro is to grow cells for in vivo therapies, much effort needs to be placed on developing hydrogels which can bridge both needs. The best hydrogel will not only be able to supplant well-performing, animal-based hydrogels such as Matrigel, but it will also act as an in vivo animal-free cellular delivery vehicle which is biocompatible, biodegradable, injectable, and flexible enough to allow its stiffness to match that of the target cell type for reasons described here.