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

A major challenge of engineering larger macroscale tissues <i>in vitro</i> is the limited diffusion of nutrients and oxygen to the interior. For skeletal muscle, this limitation results in millimeter scale outcomes to avoid necrosis. One method to address this constraint may be to vascularize <i>in vitro</i>-grown muscle tissue, to support nutrient (culture media) flow into the interior of the structure. In this exploratory study, we examine culture conditions that enable myogenic development and endothelial cell survival within tissue engineered 3D muscles. Myoblasts (C2C12s), endothelial cells (HUVECs), and endothelial support cells (C3H 10T1/2s) were seeded into Matrigel-fibrin hydrogels and cast into 3D printed frames to form 3D <i>in vitro</i> skeletal muscle tissues. Our preliminary results suggest that the simultaneous optimization of culture media formulation and cell concentrations is necessary for 3D cultured muscles to exhibit robust myosin heavy chain expression and GFP expression from GFP-transfected endothelial cells. The ability to form differentiated 3D muscles containing endothelial cells is a key step toward achieving vascularized 3D muscle tissues, which have potential use as tissue for implantation in a medical setting, as well as for future foods such as cultivated meats.