Developing a hydrogel culture system to promote satellite cell self-renewal

Adult skeletal muscle is maintained and repaired following damage by satellite cells (SCs). SCs in culture differentiate quickly and lose the ability to incorporate into muscle, suggesting interactions between the niche and satellite cells are important for regulating SC fate. Polyethylene glycol (PEG) norbornene hydrogels were used to replicate both the stiffness and molecular composition of the native SC environment. Initial cultures of SCs on PEG hydrogels yielded inconsistent results which we believed were caused by non-SC contamination influencing SC binding. To test this, I developed a new technique to isolate SCs using magnetic beads. Initial SC isolations from whole muscle yielded ~5000 cells per mouse. After I optimized this procedure, magnetic beads were capable of isolating ~40,000-100,000 SCs per mouse. When seeded onto uncoated PEG hydrogels, magnetically isolated SCs did not attach. When magnetically isolated SCs were seeded onto PEG hydrogels coated with extra-cellular matrix proteins, SC attachment efficiency was low and variable between experiments, suggesting other factors are necessary for SC adhesion in vivo.