人表皮细胞分离培养的研究

目的探索来源于人的表皮细胞的分离培养方法,为其进一步作为皮肤组织工程中的种子细胞或临床应用奠定前期研究基础。方法取3~9岁健康儿童包皮环切术后包皮,经分离酶处理分离真表皮,再将表皮以胰蛋白酶消化为细胞悬液,分别接种于有血清培养基DMEM和无血清培养基K-SFM中进行细胞培养,观察表皮细胞生长融合情况及克隆形成率。结果表皮细胞在DMEM和K-SFM培养液中均能融合成片,但在K-SFM中的融合成片时间明显短于在DMEM中所需时间;在K-SFM中2周时克隆形成率显著高于在DMEM中的克隆形成率。结论两步酶法分离表皮细胞接种于K-SFM中培养,是一种简便有效的表皮细胞分离培养方法。

Isolation and Culture of Human Epidermal Keratinocytes

Objective To develop a protocol of isolation and culture of human keratinocytes in order to serve as seeding cells in tissue engneering of skin and the treatment of severe burns and other traumatic skin defects or lesions. MethodsForeskin obtained from 3 to 9 year-old boys with circumcision were treated with dispase II. The epidermis was treated with trypsin to make single epidermal cells suspension. Keratinocytess were cultured in medium DMEM supplemented with serum or medium K-SFM. The rate of colony formation was calculated and the growth conditions were evaluated. ResultsThe cells grew well in both DMEM and K-SFM culture media. However, the time needed for colony formation in K-SFM medium was significantly shorter and the efficiency of colony formation was significantly higer than that in DMEM medium. ConclusionsIt is an simple and efficient method to isolate keratinocytes by dispase II and trypsin and to culture them in K-SFM medium.