Elsevier

Analytical Biochemistry

Volume 318, Issue 2, 15 July 2003, Pages 260-269
Analytical Biochemistry

Reconstituted 3-dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation

https://doi.org/10.1016/S0003-2697(03)00172-6Get rights and content

Abstract

Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte–keratinocyte co-cultures, melanocyte–keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (α-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.

Section snippets

Chemicals and solvents used

MSH, DOPA, hydroquinone, arbutin, kojic acid, niacinamide, phorbol 12-myristate 13-acetate (PMA), and β-mercaptoethanol were from Sigma Chemical (St. Louis, MO). l-[U-14C]tyrosine was from Amersham Biosciences (Piscataway, NJ). All compounds were dissolved in water and were protected from light at −20 °C until used, except PMA, which was dissolved in DMSO.

Culture and treatment of pigmented reconstituted human epidermis

MelanoDerm (MatTek Corp., Ashland, MA) is a viable reconstituted 3-dimensional human epidermis containing melanocytes and keratinocytes that

General considerations

We have previously established streamlined protocols (the STOPR and the co-culture models) that use immortalized mouse melanocytes with or without keratinocytes in monolayer culture to measure effects on pigmentation [18], [19]. These 2-dimensional models do not completely reflect the physiological effects of bioactive compounds, because melanocyte or melanocyte–keratinocyte interactions are limited spatially in 2-dimensional monolayers, rather than the 3-dimensions that occur in the skin. In

Conclusions

Numerous pigmented melanoma or melanocyte culture models have been used (some with co-cultures of keratinocytes) for evaluating the efficacy of melanogenic compounds [18], [19], [35], [36], but each has only limited value when compared with physiologically relevant, pigmented 3-dimensional skin equivalents. The 3-dimensional skin models have the advantage that they show an accurate histological structure and allow one to assess not only pigment synthesis, but pigment translocation and melanin

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  • Cited by (0)

    1

    Present address: Department of Dermatology, Gyeongsang National University Hospital, 90 Chilam-Dong, Chinju, Gyeongnam 660-702, South Korea.

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