Abstract:

Lin Jianhong, Wang Yu, Lu Bin, Liu Zhi, Jiang Leiwei, Wang Jinzhao, Zhang Wei, Li Shijun, Lu Hongguang Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China（the current affiliation of the first author was Department of Dermatology, First People′s Hospital of Shaoyang, Shaoyang 422000, Hunan, China） Corresponding author: Lu Hongguang, Email: hongguanglu@hotmail.com 【Abstract】 Objective To reconstruct tissue-engineered skin by co-culture of human fibroblasts, keratinocytes and melanocytes on human de-epidermized dermis with keratinocyte serum-free medium （K-SFM）. Methods Healthy children′s prepuce tissues were treated with pancreatin and collagenase to prepare epidermal and dermal cell suspensions respectively. After keratinocytes and melanocytes were cultured separately up to passage 3 and fibroblasts up to passage 5, the density of these cells was adjusted to 2.5 × 105/ml for the following experiment. Human de-epidermized dermis containing some components of the basement membrane was prepared. Firstly, fibroblast suspensions were seeded at the bottom of 6-well plates followed by 24-hour culture. Subsequently, the prepared de-epidermized dermis was added into the 6-well plates, then, keratinocyte and melanocyte suspensions were seeded on the surface of the de-epidermized dermis with the melanocyte ∶ keratinocyte ∶ fibroblast ratio being 1 ∶ 4 ∶ 1. After 4 hours of culture, the cell mixtures were divided into two groups: an experimental group cultured with K-SFM, a control group cultured with a mixed medium containing DMEM with 10% fetal bovine serum, K-SFM and M254 at a ratio of 1 ∶ 1 ∶ 1. De-epidermized dermis cultured with K-SFM or the mixed medium alone served as blank control groups. After submerged cultivation for 3 days, the tissue cultures were maintained at an air-liquid interface for another 11 days with the culture medium changed every 3 days. Finally, these cultures were subjected to hematoxylin and eosin staining, periodic acid-Schiff （PAS） staining and immunohistochemical staining for Melan-A, S-100, HMB45, cytokeratin-Pan, P63, K5, K6, K14, Ki67, vimentin, collagen Ⅳ and laminin. Results After 14-day submerged and air-liquid interface culture, a well-structured epidermis developed in both the experimental group and control group with the formation of the stratum corneum. The immunohistochemical study showed positive staining for cytokeratin-Pan, P63, K5, K6, K14, Ki67, laminin in both the experimental group and control group, but positive staining for Melan-A, S-100, HMB45 and collagen Ⅳ in only the experimental group. Conclusion Tissue-engineered skin can be constructed using keratinocytes, melanocytes and fibroblasts co-cultured on de-epidermized dermis with K-SFM in vitro.