人角质形成细胞体外分离与无血清培养的实验研究

Isolation and serum-free culture of human skin primary keratinocyte

目的探讨一种体外人皮肤角质形成细胞分离与原代培养的方法,为角质细胞的多方面研究提供细胞来源。方法采取两步消化法对皮肤进行消化以获取角质形成细胞,采用无血清培养技术进行角质细胞的体外培养,进行细胞形态学观察以及生长曲线的绘制与分析。结果分离酶和胰蛋白酶联合两步消化法能够很好地分离表皮与真皮,活细胞率高,培养后细胞融合时间短,同时又能够避免成纤维细胞的污染,可获取较多高纯度的角质形成细胞。角质细胞无血清培养可以在体外快速稳定增殖,并在多次传代后保持了正常形态。结论两步消化法和体外无血清培养是一种理想的皮肤角质形成细胞分离培养的方法。

[Objective] To establish an optimal method for in vitro culture and amplification of primary keratinocytes from the human skin. [Method] The keratinocytes were isolated from the human skin by two-step combined dissociation. The cultured cells in serum-free media were identified by morphology. Growth curves were plotted and analyzed. [Result] The combination of dispase and trypsin digestion could separate the epidermis from the dermis complete and result in high survival rate of cells and shorted time for confluences without contamination of fibroblast, which can provide a lot of highly pure keratinocytes. The keratinocytes grew steadily and rapidly in serum-free culture, and maintain the normal morphological characteristics after many times of transfer. [ Conclusion ] Two-step combined dissociation and serum-free culture in vitro for human skin primary keratinocytes is an ideal method.