AIM: To explore the method for rapidly acquiring sufficient amount of keratinocytes and dermal fibroblasts in the best growth status as the seed cells for tissue-engineered skin. METHODS: Skin biopsy samples were treated with 3 methods, namely with trypsin(2.5 g/L), thermolysin(35 g/L) or dispase(9.0 × 103 U/L) respectively(method A, B and C, respectively) . The dissociation of the epidermis from the dermis was assessed, and the keratinocytes and dermal fibroblasts were cultured. The cells were counted and plated, and the cloning and seeding efficiency were examined. The proliferation ability of the culture cells was investigated by means of MTT assay and BrdU analysis. Cells obtained by the above methods were used to construct a tissue-engineered skin graft.RESULTS: Under A, B and C conditions, the adhesion rate of the eratinocytes and fibroblasts were (8.7±0.2)%, (12.1 ±0.2)%, (15.9 ±0.4)%, and (20.2±2.4)%, (30.5±1.8)%, (38.3±1.9)% repectively, showing significant differences between them( t = 3.3506, t0. 05/2.18 =2.101, P＜0.05). The cloning rate of the keratinocytes was (0.110±0.025) %, (0.260% ±0.026) % and (0.370 ±0.021)%, respectively,with significant difference between them ( t = 2. 2875, t0.05/2, 18 = 2. 101, P＜0.05 ) . Dispase yielded the best separation, cell recovery, as well as cloning and seeding efficiency. Under different conditions, the cell proliferation, adhesion rate and cloning rate were all higher than those with other digestion solution. Trypsin treatment was the worst in terms of separation of the dermis from the epidermis, and the cells thus obtained possessed poorer proliferation ability than other groups. The cells obtained by all the methods can be used to construct a skin graft. CONCLUSION: Keratinocytes and dermal fibroblasts in the best growth status can be rapidly obtained from a skin biopsy with dispase treatment to be used as the seed cells for tissue-engineered skin. Appropriate digestion method, incubation time and temperature are crucial for retaining the activity of the primary cells.%目的:探讨如何以简便快捷的方法获得大量最佳生长状态的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞.方法:用2.5 g/L胰酶、35 g/L热溶素和9.0×103U/L Dispase分别在A,B,C不同条件下处理皮肤标本,观察表皮与真皮的分离情况,并分别培养角朊细胞和真皮成纤维细胞,观察比较细胞的贴壁率和克隆形成率,通过MTT和BrdU检测观察细胞的生长状态,并分别用不同方法获得的细胞构建组织工程皮肤.结果:A,B,C 3种方法角朊细胞的贴壁率(%)分别为:8.7±0.2,12.1±0.2,15.9±0.4;成纤维细胞的贴壁率(%)分别为:20.2±2.4,30.5±1.8,38.3±1.9.组间比较差异有统计学意义(t=3.3506,t0.05/2.18=2.101,P＜0.05).角朊细胞克隆形成率(%)分别为:0.110±0.025,0.260±0.026,0.370±0.021.组间比较差异有统计学意义(t=2.2875,t0.05/2.18=2.101,P＜0.05).在各种条件下,Dispase处理组获得的细胞数量、细胞贴壁率和克隆形成率均优于其他消化液处理组,细胞纯度高,生长状态良好;用其他方法获得的细胞生长状态相似,都可成功构建组织工程皮肤.结论:使用9.0×103U/L Dispase可以简便快捷的获得大量生长状态良好的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞,合适的消化方法、时间和温度可以最大限度保持原代细胞的活力.
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