Two-Dimensional Differential Adherence and Myoblast Alignment Driven Fabrication of Engineered C2C12 Muscle Organoids

Abstract

Differential adherent surfaces were used to induce attachment of C2C12 myoblasts that later would form engineered muscle organoids. Narrow channels, 60–400 μm wide, were micromachined in a 2% agarose gel using an ArF laser, and subsequently filled with extracellular matrix. Upon addition of 1 mL of a 2×10⁴ cells/mL myoblast suspension, cells selectively adhered to the ECM lined channels in a non-confluent manner and we monitored their growth at various time points. The adherent myoblasts were flourescently imaged with a propidium iodide live/dead assay, which revealed that the cells were alive within the channels. After 72 hours growth, the myoblasts grew, proliferated and differentiated into myotubes. Myoblasts residing in the narrow channels (60–150 μm wide) aligned parallel to the channel, while myoblasts adhered to the wide channel (400 μm) were randomly aligned within the channel. The fully-grown 1 cm long organoids maintained an aspect ratio on the order of 100:1. These results provide the foundation for future three-dimensional tissue growth using differential adherent