Modified cell-electrospinning for 3D myogenesis of C2C12s in aligned fibrin microfiber bundles

Electrospinning methods can generate scaffolds with alignment cues to guide the development of myogenic precursors into 3D skeletal muscle grafts. However, cells seeded onto these scaffolds adhere to the exterior resulting in regions of acellularity within the scaffold interior. To overcome this limitation, we modified an aqueous solution-electrospinning method to encapsulate C2C12s and electrospin them into fibrin/polyethylene oxide (PEO) microfiber bundles. We demonstrated that loading C2C12s as cellular aggregates (80-90 μm in diameter) and modifying several other electrospinning parameters dramatically increased cell viability following exposure to the 4.5 kV electric field. C2C12-seeded fibrin/PEO microfiber bundles were cultured for up to seven days. Uninduced and myogenically induced C2C12s proliferated, elongated and became multinucleated. Myogenic induction increased the number of myotube-associated nuclei (36.4 ± 12% vs. 6.2 ± 1.9%), myotube length (122.4 ± 10.9 μm vs. 59.9 ± 8.3 μm), and myotube diameter (16.76 ± 2.06 μm vs. 12.49 ± 0.93 μm). The data presented in this study demonstrates for the first time that cells can be loaded inside the aligned fibrin hydrogel 3D construct during aqueous solution electrospinning while retaining their potential for de novo tissue formation.