人皮肤成纤维细胞分离培养的研究

Study of primary cultures method of human foreskin fibroblast

目的:探讨人包皮成纤维(human foreskin fibroblast,HFF)细胞分离培养方法,通过分析HFF细胞体外培养条件,建立稳定的HFF细胞系。方法:用0.25%dispaseⅡ酶消化包皮环切术后皮肤组织,分离表皮和真皮,真皮组织用0.1%Ⅰ型胶原酶消化,收集细胞于含20%胎牛血清的DMEM培养液中,置CO2培养箱孵育,待细胞长成单层后消化传代。结果:消化法得到的人皮肤成纤维细胞24h内贴壁,刚开始贴壁时细胞多数呈圆形,HFF细胞随贴壁时间延长,逐渐伸展成梭形或多边形。传代后的成纤维细胞12h开始贴壁,24h内完全贴壁。HFF细胞采用SABC法染色细胞浆呈棕色,前10代细胞活力均在98%以上。结论:该方法可获得高产量、高活力的HFF细胞,是一种可靠的HFF细胞培养方法。

Objective： To explore the primary cultures method of human foreskin fibroblast （HFF）, and build steady HFF cell lines. Methods： Foreskin was digested with 0.25 % Ⅱ dispase and separated into epidermis and dermis, dermis was digested with 0. 1 % collagenase Ⅰ , the HFF cells were collected into DMEM medium which contains 20% fetal bovine serum, and then placed into CO2 incubator, HFF cells was digested for passage when they formed cell monolayer. Results： HFF cells adhere within 24 h, cells were round at the beginning, and gradually extended into spindle or polygonal. Fibroblasts after the passage begin to adhesion after 12 h, and fully adhere within 24 h. With SABC method, the cytoplasma of HFF cell show brown indicated it was HFF cell. The vigor of HFF cell of 1 to 10 generations all more than 90%. Conclusion： It is an effective method to cultivate HFF cells with high productivity and survival rate.