Clonal growth of normal human epidermal keratinocytes in a defined medium

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Abstract
Colony formation by normal human epidermal keratinocytes (HK) has been 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 μg/ml transferrin, 5 μg/ml insulin, 1.4 × 10−6 M hydrocortisone, 1.0 × 10−5 M ethanolamine, 1.0 × 10−5 M phosphoethanolamine, and 2.0 × 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 phosphoethanolamine 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.