There is growing evidence to suggest that the prevailing physical microenvironment and mechanical stress regulate cellular functions, including adhesion, proliferation, and differentiation. Moreover, the physical microenvironment determines the stem-cell lineage depending on stiffness of the substrate relative to biological tissues as well as the stress relaxation properties of the viscoelastic substrates used for cell culture. However, there is little known regarding the biological effects of a fluid substrate, where viscoelastic stress is essentially absent. Here, we demonstrate the regulation of myogenic differentiation on fluid substrates by using a liquid-liquid interface as a scaffold. C2C12 myoblast cells were cultured using water-perfluorocarbon (PFC) interfaces as the fluid microenvironment. We found that, for controlled in vitro culture at water-PFC interfaces, expression of myogenin, myogenic regulatory factors (MRF) family gene, is remarkably attenuated even when myogenic differentiation was induced by reducing levels of growth factors, although MyoD was expressed at the usual level (MyoD up-regulates myogenin under an elastic and/or viscoelastic environment). These results strongly suggest that this unique regulation of myogenic differentiation can be attributed to the fluid microenvironment of the interfacial culture medium. This interfacial culture system represents a powerful tool for investigation of the mechanisms by which physical properties regulate cellular adhesion and proliferation as well as their differentiation. Furthermore, we successfully transferred the cells cultured at such interfaces using Langmuir-Blodgett (LB) techniques. The combination of the interfacial culture system with the LB approach enables investigation of the effects of mechanical compression on cell functions.
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