CLONAL GROWTH OF NORMAL HUMAN EPIDERMAL-KERATINOCYTES IN A DEFINED MEDIUM
CLONAL GROWTH OF NORMAL HUMAN EPIDERMAL-KERATINOCYTES IN A DEFINED MEDIUM
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DOI：
10.1002/jcp.1041100217
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发表时间：
1982-01-01
影响因子：
5.6
通讯作者：
HAM, RG
中科院分区：
文献类型：
--
作者：
TSAO, MC;WALTHALL, BJ;HAM, RG
正常人表皮角质形成细胞（HK）的集落形成是在不含故意添加的未定义的补充剂的培养基中实现的。“定义”一词用于描述这种培养基，尽管其成分中的微量污染物可能有助于其支持的增殖的可能性尚未完全排除。成分确定的培养基由基础培养基MCDB 152组成，其补充有5 ng/ml表皮生长因子（EGF）、10 μ g/ml转铁蛋白、5 μ g/ml胰岛素、1.4 × 10 - 6胰岛素和1.5 × 10 - 6胰岛素。10-6 M氢化可的松，1.0 ×10-5 M乙醇胺，1.0 ×10-5 M磷酸乙醇胺，和2.0 ×10-9孕酮。MCDB 152不同于先前开发用于用少量透析血清增殖HK的MCDB 151（Peehl和Ham，1980 b），仅通过添加来自人成纤维细胞培养基MCDB 104的微量元素混合物（McKeehan等人，1977年）。转铁蛋白是确定培养基中最不确定的组分，其大部分要求可通过在过滤灭菌后向最终培养基中添加新鲜溶解和灭菌的硫酸亚铁来替代。胰岛素和EGF显然是最佳增殖所必需的，氢化可的松是温和有益的。乙醇胺或磷酸乙醇胺必须存在于用于HK增殖的限定培养基中。与我们以前使用的部分确定的系统相比，在确定的培养基中对EGF的需求更大，对氢化可的松的需求更少。在具有低钙浓度（0.03mM）并且不利于角质形成细胞分化的成分确定的培养基中获得非常大的扁平上皮细胞集落。钙浓度越高，生长越少，分化越多。成分确定的培养基对来自角质形成细胞和成纤维细胞的混合接种物的角质形成细胞生长具有高度选择性。
Colony formation by normal human epidermal keratinocytes (HK) was achieved in a medium that contains no deliberately-added undefined supplements. The term defined is used to describe this medium, although the possibility that trace contaminants in its components could be contributing to the multiplication that it supports cannot yet be ruled out completely. The defined medium consists of a basal medium, MCDB 152, supplemented with 5 ng/ml epidermal growth factor (EGF), 10 .mu.g/ml transferrin, 5 .mu.g/ml insulin, 1.4 .times. 10-6 M hydrocortisone, 1.0 .times. 10-5 M ethanolamine, 1.0 .times. 10-5 M phosphoethanolamine, and 2.0 .times. 10-9 M progesterone. MCDB 152 differs from MCDB 151, previously developed for multiplication of HK with small amounts of dialyzed serum (Peehl and Ham, 1980b), only by addition of the trace element mixture from human fibroblast medium MCDB 104 (McKeehan et al.,1977). Most of the requirement for transferrin, which is the least defined component of the defined medium, can be replaced by adding freshly dissolved and sterilized ferrous sulfate to the final medium after it has been filter sterilized. Insulin and EGF are clearly needed for optimal multiplication and hydrocortisone is mildly beneficial. Either ethanolamine or phoshoethanolamine must be present in the defined medium for HK multiplication. There is a greater need for EGF and less for hydrocortisone in the defined medium than in previous partially defined systems that we have worked with. Very large colonies of flattened epithelial cells are obtained in the defined medium, which has a low calcium concentration (0.03 mM) and does not favor keratinocyte differentiation. Less growth and more differentiation are obtained with higher calcium concentrations. The defined medium is highly selective for keratinocyte growth from a mixed inoculum of keratinocytes and fibroblasts.