Optimization of Culture Media and Cell Ratios for 3D In Vitro Skeletal Muscle Tissues with Endothelial Cells

A major challenge of engineering larger macroscale tissues in vitro is the limited diffusion of nutrients and oxygento the interior. For skeletal muscle, this limitation results in millimeterscale outcomes to avoid necrosis. One method to address this constraintmay be to vascularize in vitro-grown muscle tissue,to support nutrient (culture media) flow into the interior of thestructure. In this exploratory study, we examine culture conditionsthat enable myogenic development and endothelial cell survival withintissue engineered 3D muscles. Myoblasts (C2C12s), endothelial cells(HUVECs), and endothelial support cells (C3H 10T1/2s) were seededinto Matrigel-fibrin hydrogels and cast into 3D printed frames toform 3D in vitro skeletal muscle tissues. Our preliminaryresults suggest that the simultaneous optimization of culture mediaformulation and cell concentrations is necessary for 3D cultured musclesto exhibit robust myosin heavy chain expression and GFP expressionfrom GFP-transfected endothelial cells. The ability to form differentiated3D muscles containing endothelial cells is a key step toward achievingvascularized 3D muscle tissues, which have potential use as tissuefor implantation in a medical setting, as well as for future foodssuch as cultivated meats.

Automated summary

A major challenge of engineering larger macroscale tissues in vitro is the limited diffusion of nutrients and oxygen to the interior. Vascularizing in vitro-grown muscle tissue may address this constraint by supporting nutrient flow into the structure. This study explores the culture conditions necessary for the development of 3D muscles containing both myoblasts and endothelial cells.