A Chemically Defined Common Medium for Culture of C2C12 Skeletal Muscle and Human Induced Pluripotent Stem Cell Derived Spinal Spheroids.

INTRODUCTION: Multicellular platforms and linked multi organ on chip devices are powerful tools for drug discovery, and basic mechanistic studies. Often, a critical constraint is defining a culture medium optimal for all cells present in the system. In this study, we focused on the key cells of the neuromuscular junction i.e., skeletal muscle and motor neurons. METHODS: Formulation of a chemically defined medium for the co-culture of C2C12 skeletal muscle cells and human induced pluripotent stem cell (hiPSC) derived spinal spheroids (SpS) was optimized. C2C12 cells in 10 experimental media conditions and 2 topographies were evaluated over a 14-day maturation period to determine the ideal medium formulation for skeletal muscle tissue development. RESULTS: During early maturation, overexpression of genes for myogenesis and myopathy was observed for several media conditions, corresponding to muscle delamination and death. Together, we identified 3 media formulations that allowed for more controlled differentiation, healthier muscle tissue, and long-term culture duration. This evidence was then used to select media formulations to culture SpS and subsequently assessed axonal growth. As axonal growth in SpS cultures was comparable in all selected media conditions, our data suggest that the neuronal basal medium with no added supplements is the ideal medium formulation for both cell types. CONCLUSIONS: Optimization using both topographical cues and culture media formulations provides a comprehensive analyses of culture conditions that are vital to future applications for in vitro NMJ models.