Improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof

Abstract

The present invention relates to improved continuous (immortalized) cell lines, in particular keratinocytes and melanocytes derived from normal human skin tissue. The present invention also relates to novel serum-free media for isolating, producing and maintaining said improved continuous keratinocyte and melanocyte cell lines. The present invention also relates to methods for producing primary melanocytes and keratinocytes under serum-free conditions without any feeder cells.

Description

[0001] The present invention relates to improved continuous (immortalized) cell lines derived from normal human skin tissues, in particular keratinocytes and melanocytes which retain the ability to express differentiation proteins characteristic of differentiated melanocytes or keratinocytes, even in high passages. The present invention also relates to novel serum free media which do not require the use of feeder cells.[0002] The production of immortalized cell lines derived from human skin tissues has been previously described. In general such methods comprise transfection or transformation of human skin cells, e.g., keratinocytes and melanocytes, cultured in vitro with agents which provide for immortalization.[0003] Immortalization refers to the production of cells which are able to be cultured for prolonged time periods in vitro, ideally indefinitely. These cells are also referred to as continuous cell lines. By contrast non-immortalized cells are only capable of growing for a fi...

AI Technical Summary

Benefits of technology

0053] Moreover, as described in greater detail infra, it has been surprisingly discovered that epinephrine is a strong growth factor of normal keratinocytes when used in serum free medium. Specifically, the NR-3 medium described infra contains epinephrine which has been found to enhance the growth of normal keratinocytes (see FIG. 1). This is quite surprising given the fact that epinephrine has previously been reported to inhibit the growth of keratinocytes (Halprin, J. Invest. Dermatol., 81:553-557 (1983)) or to only have a moderate effect on keratinocyte cell growth (Koizumi et al, J. Invest. Dermatol., 96:234-237, 1991).

0054] Also, it has been surprisingly discovered that the continuous coating of the culturing dishes or plates used to culture primary and immortalized keratinocytes and / or melanocytes, in particular with a coating or "cocktail" containing fibronectin, BSA and type I collagen improves both the attachment of keratinocytes and melanocytes to culture plates or culture dishes, as well as enhancing cell growth. The use of such a coating material has not been previously described for use with immortalized keratinocytes and / or melanocytes.

0055] As discussed, the present invention further specifically provides a novel serum-free medium referred to as the NR-3 medium. This medium allows for culturing and isolation of normal keratinocytes and / or melanocytes from human skin under serum-free conditions without the use of a feeder layer. This medium has been found to improve the growth of normal keratinocytes and to allow the establishment of normal keratinocyte cultures without any contact with serum or feeder cells.

0056] The exact composition of the NR-3 medium is described in Table 1. This medium contains various amino acids, inorganic salts as trace elements, vitamins, growth factors and other substituents. For example, this medium contains as growth factors epidermal growth factor (EGF recombinant), insulin, hydrocortisone, transferrin (human), bovine pituitary extract, and epinephrine. As noted, epinephrine has surprisingly been discovered to enhance the growth of primary keratinocytes in tissue culture.

Problems solved by technology

However, while numerous groups have reported immortalized keratinocyte cell lines, and their usage in in vitro assays, previous immortalized keratinocyte cell lines and melanocyte cell lines have typically exhibited one or more properties which render their usage disadvantageous.

Images