An improved method for culture of epidermal keratinocytes from newborn mouse skin
An improved method for culture of epidermal keratinocytes from newborn mouse skin
复制标题
DOI：
10.1023/a:1016385109922
复制
发表时间：
2001-01-01
期刊：
影响因子：
--
通讯作者：
Larjava, Hannu
中科院分区：
文献类型：
--
作者：
Hakkinen, Lari;Koivisto, Leeni;Larjava, Hannu
从转基因小鼠品系中分离和长期培养表皮角质形成细胞是非常必要的，但大多数技术都不成功。在这份报告中，我们详细描述了一种从新生小鼠皮肤中分离假设的角质形成细胞干细胞并在培养中长期保持它们的简化方法。用酶法分离新生小鼠皮肤角质形成细胞，建立细胞培养体系。为了选择可能的角质形成干细胞进行培养，这些细胞被允许在由I型胶原和纤维连接蛋白组成的复合基质上附着10分钟。去掉未贴壁的细胞，将贴壁的细胞培养在含有低钙浓度、9%胎牛血清、新生小鼠皮肤成纤维细胞的条件培养液和EGF的特定培养液中。传代培养时，将细胞接种在组织培养塑料上。分离的细胞呈典型的基底层角质形成细胞形态，表达上皮细胞特有的整合素αvbeta6。αvbeta6整合素的表达水平与人皮肤角质形成细胞相当。在器官培养模型中，角质形成细胞也能够分化形成表皮。通过使用所描述的方案，从冷冻的股票中的角质形成细胞已经传代了26次，而细胞活力、增殖率和形态都没有改变。
Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca2+ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin alphavbeta6. The expression level of alphavbeta6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.