Hydrogels, a type of biomaterial, are an invaluable part of biomedical research as they are highly hydrated and properties such as elasticity, porosity, and ligand density can be tuned to desired values. Recently, culture substrate stiffness was found to be an important regulator of muscle stem cell self-renewal. Polyethylene glycol (PEG), a synthetic polymer, can be fabricated into hydrogels that match the softness of skeletal muscle tissue, thereby providing a culture surface that is optimal for maintaining muscle stem cell self-renewal potential ex vivo. In this Chapter, we describe a method to produce flat PEG hydrogels across a range of stiffnesses, including a formulation that matches the bulk stiffness of healthy skeletal muscle (12 kPa), while maintaining a constant ligand density. Since PEG is inert to protein adsorption, the steps required to surface functionalize the hydrogel with an adhesive interface (e.g., laminin) are also described.
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