A ROCK inhibitor promotes keratinocyte survival and paracrine secretion, enhancing establishment of primary human melanocytes and melanocyte-keratinocyte co-cultures.

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3-4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%-7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y-27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y-27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y-27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y-27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.