(A) Schematic diagram illustrating the positions of the tissues shown in B (explant tissue, central part of the migrating cells and leading edge of the migrating cells). 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 cellCcell 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 Morin hydrate that combining ROCK inhibition with elevation of Ca2+ could facilitate the fabrication of many types of cell graft. Subject terms: Regeneration, Post-translational modifications, Translational research Introduction Previous studies have attempted to optimize the fabrication of epithelial cell grafts by supplementing the culture medium with agents such as cholera toxin1, epidermal growth factor2, amphotericin B3, insulin and hydrocortisone4. As a result of advances in cell culture techniques, many types of epithelial cell can be expanded in vitro to form cell sheets, which have been used for regeneration of the cornea5, esophagus6C8 and middle ear9. The selection of agents for optimization of cell culture is based on cell behaviors such as proliferation and differentiation. A key modulator of cell behavior is the non-muscle myosin light chain II (MLC), which is regulated by phosphorylation and has been implicated in cell migration, proliferation and differentiation10C12. Thus, controlling the level of phosphorylated MLC is considered important for the successful fabrication of cell grafts. Rho signaling induced by Rho-associated kinase (ROCK) results in the phosphorylation and activation of MLC and hence muscle contraction13C15. Y-27632 is a ROCK inhibitor (ROCKi) that was originally developed as a muscle relaxant16 but subsequently shown to enhance the proliferation of many cell types including human embryonic stem cells and keratinocytes17,18. Additionally, Y-27632 has been co-injected with cultured corneal endothelial cells into the anterior chamber of the eye to regenerate the corneal endothelium in patients with bullous keratopathy19. Currently, a ROCKi is considered almost a necessity for cell expansion because of its anti-apoptotic and proliferative effects17. Ionized calcium (Ca2+) induces calmodulin-mediated signaling that regulates the activity of MLC20,21. Ca2+ is also recognized as a differentiation-inducing factor for epithelial cells and is essential for cellCcell adhesion via cadherin22C25. Culture medium containing low levels of Ca2+ prevents the differentiation of epithelial cells and promotes cell expansion26,27. Remarkably, Zhang et al. reported that the combination of a ROCKi with low-Ca2+ medium resulted in a 1012-fold expansion in epithelial cell number28. Therefore, addition of a ROCKi and alteration of the extracellular Ca2+ concentration are two Morin hydrate methods that can be used to regulate the MLC activity, proliferation and differentiation of epithelial cells. We recently succeeded in treating patients Morin hydrate with severe middle ear disease by surgically transplanting nasal mucosal cell Morin hydrate sheets into the middle ear9. The cell sheets were fabricated using a two-step process involving explant culture Mouse monoclonal to NFKB p65 (proliferation) and cell sheet culture (differentiation) in keratinocyte culture medium (KCM)29,30. The transplant surgery required up to 9 nasal mucosal cell sheets per patient, which would necessitate a highly efficient culture protocol to generate enough autologous cell sheets from a single specimen of resected nasal mucosa. Analyzing the effects of various agents on cell behavior during the two stages of culture would provide important information regarding how to optimize cell proliferation and differentiation during the fabrication of cell sheets. In this study, we examined the effects of co-administering different concentrations of Y-27632 and Ca2+ on cell proliferation and differentiation during explant culture and cell sheet culture. Furthermore, we analyzed the expressions of various proteins, particularly MLC and its phosphorylated forms, to better understand the effects of these agents on cell phenotype and behavior. We also investigated the wound healing potential of nasal mucosal cell sheets fabricated using optimized levels of Y-27632 and Ca2+. Results ROCK inhibition and Ca2+ are important for explant culture.