Adult muscle stem cells (also called satellite cells) are an anatomically defined population of cells that are essential for muscle regeneration. In aging and dystrophic diseases, muscle stem cells (MuSCs) exhibit functional and molecular heterogeneity; therefore, single-cell assay technologies are critical to illuminate the mechanisms of pathological stem cell dysfunction. Here, we describe the process of generating mechanically tunable hydrogels with micro-scale well features ("microwells") using micro-contact printing for clonal muscle stem cell culture assays. Microcontact printing (μCP) is a simple and versatile method for generating cell culture substrates for micro-scale features for spatially restricting the cultures of single cells and their progeny. We explain how to use photolithography and polydimethylsiloxane casting to generate stamps capable of printing purified extracellular matrix proteins onto soft, hydrated poly(ethylene glycol) hydrogels to generate arrayed microwells in a defined pattern. We summarize methods to analyze the viability, migration, and differentiation of individual MuSC clones within hydrogel microwell cultures.
Keywords: Hydrogel; Laminin; Live-cell imaging; Microcontact printing; Migration; Photolithography; Self-renewal; Stem cell expansion.