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.

中文翻译：

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人表皮细胞对体外胰蛋白酶消化的耐受性。

表征不同类型人表皮细胞对胰蛋白酶作用的体外耐受性，并开发一种根据其对胰蛋白酶作用的耐受性分离和纯化黑素细胞的新方法。通过从人包皮分离表皮获得表皮细胞。其中一些细胞用于常规培养，然后进行差异胰蛋白酶消化。将剩余的表皮细胞重悬于0.25%胰蛋白酶溶液中，然后在不同时间点通过添加牛血清进行中和。HMB45、K15 和波形蛋白的免疫荧光染色分别用于识别黑素细胞、角质形成细胞和成纤维细胞。我们发现角质形成细胞、黑素细胞和成纤维细胞是从人类皮肤的常规培养物中获得的原代细胞。纯化的角质形成细胞和黑色素细胞可以通过常规差异胰蛋白酶消化获得，但黑色素细胞群中的成纤维细胞在传代后很快获得生存优势。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。表皮细胞胰酶消化12 h后获得的细胞群中未见明显的角质形成细胞，出现较多的短梭形黑素细胞，均为HMB45阳性。综上所述，人表皮黑素细胞在体外对胰蛋白酶消化的耐受性优于表皮角质形成细胞，且该特性可用于纯化黑素细胞，且优于传统的差示胰蛋白酶消化。经过长期胰蛋白酶消化后存活下来的细胞形态发生了变化，具有良好的增殖活性，但似乎更加不成熟。