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The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca 2+  concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca 2+  elevation regulated the phosphorylation of MLC and improved both cell expansion and cell–cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca 2+  enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca 2+  promoted MLC phosphorylation and formation of cell–cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca 2+  was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca 2+  could facilitate the fabrication of many types of cell graft.

ROCK inhibitor combined with Ca2+ controls the myosin II activation and optimizes human nasal epithelial cell sheets