Covalently Functionalized Hydrogels on Oxygen Permeable Membranes for Stable, Scalable, and Xeno Free Growth of Undifferentiated Human Embryonic Stem Cells

Technology

This invention is a novel method for culturing pluripotent stem cells. The tissue culture dish is comprised of an oxygen-permeable substrate, which allows for modulation of oxygen partial pressure at the cell-substrate surface interface. Oxygen enters the substrate through surface facing away from culture media and cells. This modification eliminates the difference between the partial pressure of oxygen in the incubator and the cells in the incubator, enabling precise control over oxygen concentration in the cultured cells.The oxygen permeable substrate is coated with stable nanoscale matrices covalently functionalized to have a desired chemistry. The matrix can modify the surface chemistry of the oxygen-permeable substrate and allow for the facile, controlled, and stable attachment of biological molecules to the surface without adversely affecting the oxygen permeability. Functional groups and their density may be controlled during the coating process using a chemical vapor deposition process to generate the desired functional matrix, such as a hydrogel. This coating can mimic natural stem cell microenvironments and/or developmental processes. The inventors identified that altering the density of the functional biological molecules can either inhibit differentiation and maintain pluripotency or promote directed differentiation.  

Problem Addressed

Pluripotent stem cells are attractive candidates for a variety of cell-based based diagnostics and therapies because they are capable of self-renewal and differentiation into any somatic cell type. In vivo, stem cell maintenance and differentiation processes are regulated by soluble factors, partial pressure of oxygen, cell-substrate surface interactions, among other factors. The individual role of these factors in directing self-renewal or differentiation remains largely unknown. Conventional methods for culturing stem cells in vitro involve growing stem cells on irradiated mouse embryonic fibroblasts (MEFs) or on Matrigel, a commercially available, mouse-derived, extracellular matrix-like substrate. These methods introduce xenogeneic proteins that do not allow for reproducibility of cell-substrate surface interactions since both Matrigel and MEFs vary from lot to lot. Moreover, these plating methods do not offer surface substrate flexibility to promote differentiation when desired. In addition, conventional cell culture methods cannot carefully regulate the partial pressure of oxygen in culture vessels, which has important implications for cellular health. Improving control over partial pressure of oxygen would facilitate the culturing of stem cells in physiologically relevant oxygen conditions. Accordingly, new methods for research and clinical uses that could more accurately model the stem cell microenvironment are needed for better in vitro stem cell maintenance and differentiation. The present technology overcomes the current limitations of stem cell culture practices by introducing precise control over partial pressure of oxygen on covalently functionalized hydrogels to enable stable growth and reliable differentiation of human stem cells.   

Advantages

• Enables physiologically relevant culture of pluripotent stem cells
• Reproducible expansion of human pluripotent stem cells