Keratinocyte-melanocyte co-cultures and pigmented reconstructed human epidermis: Models to study modulation of melanogenesis

Normal human melanocytes were amplified and cultured in a new defined cultu re medium without phorbol esters or cholera toxin. The medium decreased con siderably the doubling time and increased the possible passage number. Mela nocytes were co-seeded with normal human keratinocytes into 24 well culture dishes to screen potentially active modulators of melanogenesis. For the a ssay, the co-cultures were exposed to the compounds under investigation in the presence of C-14-thiouracil and H-3-leucine. Control cultures contain L -tyrosine or kojic acid, modulators which served as internal calibration st andards. Changes in the rate of melanin synthesis were measured on the basi s of C-14-thiouracil incorporation into newly synthesized melanin. A reduct ion or increase in 3H-leucine incorporation was taken as an indication of c ytotoxicity or induction of proliferation, respectively. The NHK-NHM co-cul ture screening assay provides a useful tool to compare the activity of know n modulators of melanogenesis and to perform structure-activity studies wit h newly identified modulators to improve their activity. The efficacy of pa rticularly interesting new compounds was further evaluated on reconstructed pigmented epidermis after repeated topical application. The same model was used to assess the anti-pigmenting effect of sunscreens on W-induced pigme ntation. Integration of melanocytes from different ethnic origin resulted i n pigmented epidermis reflecting different skin phenotypes, Caucasian, Asia n and African.