Microfluidic regulation of porous poly(L-lactide-co-ε-caprolactone) microcarrier to refine myoblast differentiation potential

With the growing interest in cell-based regenerative therapies for skeletal muscle injuries, there is a critical need for a microcarrier strategy specifically designed for superior functioning of myoblasts. Monodisperse porous poly(L-lactide-co-ε-caprolactone) (PLCL) microcarriers with precisely controlled diameters were thus developed using microfluidics to explore their effect on differentiation performance of mouse skeletal myoblasts (C2C12 cells). A capillary microfluidic device was designed with varying parameters to fabricate four representative sets of microspheres with diameters of 177 μm, 270 μm, 355 μm, and 472 μm. C2C12 cells were cultured on these microspheres, and their adhesion and viability were assessed. Myogenic differentiation ehaviour of these cells was evaluated by specific gene expression and myosin heavy chain (MHC) staining. Inflammatory response of macrophages to these microcarriers was assessed to qualify their suitability for muscle repair. Notably, microcarriers of 472 μm exhibited the optimal performance in promoting myogenic differentiation of C2C12 cells. The expressions of myogenic differentiation markers MYOG, Desmin, and MYH2 were significantly enhanced on the 472 μm group, showing respective increases of 2.41-fold, 2.15-fold, and 6.87-fold compared to those of the 177 μm group. Moreover, the formation of long myotubes distributed in a global domain was observed on the 472 μm group with a fusion index nearly three times as high as that on the 177 μm group. These microcarriers could effectively inhibit the expression of pro-inflammatory factors IL-6, TNF-α and IL-1β by macrophages in a high M1 polarization environment. The results offer a microfluidic based solution for in vitro engineering of injectable constructs for muscle repair and regeneration.