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

The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca²⁺ 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 Ca²⁺ 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, Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca²⁺ could facilitate the fabrication of many types of cell graft.