On The Origin of Shear Stress Induced Myogenesis Using PMMA Based Lab-on-Chip.

One of the central themes in cell and tissue engineering is to develop an understanding as to how biophysical cues can influence cell functionality changes. The flow induced shear stress is regarded as one such biophysical cue to influence physiological changes in shear-sensitive tissues, in vivo. The origin of such phenomena is, however, poorly understood. While addressing such an issue, the present work demonstrates the intriguing synergistic effect of shear stress and spatial constraints in inducing aligned growth and differentiation of myoblast cells to myotubes. In a planned set of in vitro experiments, the regulation of laminar flow regime within a narrow window was obtained in a PMMA-based Lab-on-Chip (LOC) device, wherein the murine muscle cells (C2C12), chosen for their phenotypical differentiation stages, were cultured under graded shear conditions. The two factors of shear stress and spatial allowance were decoupled by another two sets of experiments. This aspect has been conclusively established using a PMMA device having a fixed width microchannel with varying shear and an identical amount of shear with different width of channels. On the basis of the extensive analysis of biochemical assays (WST-1, picogreen) together with gene expression using qRT-PCR and cell morphological changes (fluorescence/confocal microscopy), extensive differentiation of the myoblasts into myotubes is found to be dependent on both shear stress and spatial allocation with a maximum at an optimal shear of ca. 16 mPa. Quantitatively, the mRNA expression of myogenic biomarkers, i.e., myogenin, MyoD, and neogenin, exhibited 10- to 50-fold changes at ca. 16 mPa shear flow, compared to that under static conditions. Also, myotube aspect ratio and myotube density are modulated with shear stress and are in commensurate with gene expression changes. The flow cytometry analysis further confirmed that the cell cycle arrest at the G1/G0 phase triggers the onset of myogenesis. Taken together, the present study unambiguously establishes qualitative and quantitative biophysical basis for the origin of myogenesis toward the critical shear stress of murine myoblasts in a microfludic device, in vitro.