Department of Dermatology, The Third Affiliated Hospital of Soochow University, 185 Juqian Road, Changzhou, 213003, China.
Department of Dermatology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, 233000, China.
Cell Tissue Bank. 2020 Jun;21(2):257-264. doi: 10.1007/s10561-020-09818-3. Epub 2020 Feb 27.
To characterize the tolerance of different types of human epidermal cells to trypsinization in vitro and develop a new method to separate and purify melanocytes according to their tolerance to trypsinization. Epidermal cells were obtained by separating the epidermis from human foreskins. Some of those cells were used for routine culture, and then were subjected to differential trypsin digestion. The remaining epidermal cells were resuspended in a 0.25% trypsin solution and then were neutralized by the addition of bovine serum at different time points. Immunofluorescence staining of HMB45, K15 and vimentin was used to identify melanocytes, keratinocytes and fibroblasts, respectively. We found that Keratinocytes, melanocytes and fibroblasts are primary cells obtained from conventional cultures of human skin. Purified keratinocytes and melanocytes can be obtained by conventional differential trypsin digestion, but fibroblasts in the melanocyte population quickly gain a survival advantage after passage. With longer trypsin digestion times, the number of adherent cells decreased, the time required for cell attachment increased, and the proportion of melanocytes increased. There were no obvious keratinocytes in cell populations obtained after 12 h of trypsinization of epidermal cells, and more short spindle-shaped melanocytes appeared, all of which were HMB45-positive. In conclusion, the tolerance of human epidermal melanocytes to trypsinization in vitro was better than epidermal keratinocytes, and that property can be used to purify melanocytes and was better than traditional differential trypsin digestion. The morphology of cells that survived the long-term trypsin digestion changed and they had good proliferative activity, but seemed to be more immature.
为了研究不同类型的人表皮细胞对胰蛋白酶消化的耐受性，并根据其对胰蛋白酶消化的耐受性开发一种新的方法来分离和纯化黑素细胞。表皮细胞通过分离人包皮获得。部分细胞用于常规培养，然后进行差异胰蛋白酶消化。剩余的表皮细胞悬浮在 0.25%胰蛋白酶溶液中，然后在不同时间点加入牛血清中和。使用 HMB45、K15 和波形蛋白的免疫荧光染色分别鉴定黑素细胞、角蛋白细胞和成纤维细胞。我们发现角蛋白细胞、黑素细胞和成纤维细胞是从常规培养的人皮肤中获得的原代细胞。通过常规的差异胰蛋白酶消化可以获得纯化的角蛋白细胞和黑素细胞，但在传代后黑素细胞群体中的成纤维细胞很快获得生存优势。随着胰蛋白酶消化时间的延长，贴壁细胞的数量减少，细胞贴壁所需的时间增加，黑素细胞的比例增加。在表皮细胞 12 小时胰蛋白酶消化后获得的细胞群体中没有明显的角蛋白细胞，并且出现了更多的短梭形黑素细胞，均为 HMB45 阳性。总之，人表皮黑素细胞对体外胰蛋白酶消化的耐受性优于表皮角质细胞，这种特性可用于纯化黑素细胞，优于传统的差异胰蛋白酶消化。在长期胰蛋白酶消化中存活下来的细胞形态发生变化，增殖活性良好，但似乎更不成熟。
