Process for immortalizing human skin cell lines and a serum-free medium

摘要

The method of immortalizing human skin cells comprises: a).obtaining a human skin tissue sample and preparing the sample for culturing in vitro; b).obtaining keratinocytes and/or melanocytes from the prepared skin sample and seeding the keratinocytes and/or melanocytes into a serum-free growth medium, onto culture plates provided with a coating comprising fibronectin, type I collagen and BSA which facilitates cell attachment and growth; c).changing the medium as necessary to optimize confluent growth of the cultured cells while maintaining the coating on the culture plates; d).transferring the keratinocytes or melanocytes into a serum free growth medium suitable for selecting between keratinocytes and melanocytes onto similarly precoated culture plates; e).infecting the keratinocytes or melanocytes with a retroviral construct; f).transferring the resultant immortalized keratinocytes or melanocytes to a serum free proliferation medium suitable for proliferating immortalized keratinocytes or melanocytes onto similarly precoated culture plates and g).transferring the resultant proliferated keratinocytes or melanocytes to a serum-free differentiation medium which contains high calcium onto similarly precoated culture dishes. An immortalized keratinocyte or melanocyte cell line obtained by the above process that retains the ability to differentiate and to express one or more polypeptides selected form cytochrome p-450 3A5, superoxide dismutase, collagenase type I TNFa or glutathione S-transferase in the same pattern as normal differentiated keratinocytes or melanocytes even after high passage in tissue culture is disclosed. A serum free culture medium comprising amino acids or salts thereof, inorganic salts, vitamins, adenine, ethanolamine, glucose, HEPES, phenol red, putrescine 2 HCl, thiamine HCl, thymidine, epidermal growth factor, insulin, hydrocortisone, phospoethanolamine and bovine pituitary extract is characterized in that it contains transferring and thioctic acid in an amount sufficient for isolating and producing human keratinocytes and melanocyte cell lines as defined above.