Remnant Effects of Culture Density on Cell Chirality After Reseeding

Proper muscle function requires specific orientation of myotubes. Cell chirality, a mechanical behavior of cells, may participate in myogenesis and give rise to left-right (LR) orientation of muscle tissue. Thus, it is essential to understand the factors effecting the cell chirality. Here, using C2C12 cells as a model system, we report that prior culture condition with high/low density can create remnant effects on cell chirality after reseeding. C2C12 myoblasts were first conditioned by a series of subcultures with plating density at 2200 cells/cm² (low density) or 22 000 cells/cm² (high density). After reseeding on micropatterned stripes fabricated on glass or polydimethylsiloxane (PDMS) substrates, we found that the cells after low-density cultures exhibited a reduced cell aspect ratio and intercellular alignment, leading to an attenuated chiral orientation only appearing on glass substrate. In contrast, chiral orientation was observed in cells after high-density culture on both substrates. By comparing it to the original cells without being subcultured with high/low density, we found that the series of low-density cultures disorganized the formation of actin rings in single cells, which is an essential structure for cell chirality. Moreover, by using high-density culture supplemented with inhibitors of actin polymerization, the effect of low-density cultures was recaptured, suggesting that the series of subcultures with high/low density may be an in vitro aging process that modifies the actin cytoskeleton, causing a remnant attenuation of cell chirality even after trypsin digestion and reseeding. Together, our result suggests a mechanistic insight of how cytoskeletal structures "memorize" the previous experience through modification of the actin filament, opening up new possibilities for morphogenesis and mechanobiology.