Abstract
Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (3∶1) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m2 over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.
Similar content being viewed by others
References
Bertaux B, Molière P, Moreno G, Courtalon A, Massé JM, Dubertret L (1988) Growth of melanocytes in a skin equivalent model in vitro. Br J Dermatol 119: 503–512
DeLeo YA, Scheide S, Meshulam J, Hanson D, Cardullo A (1988) Ultraviolet radiation alters choline phospholipid metabolism in human keratinocytes. J Invest Dermatol 91: 303–308
De Luca M, D'Anna F, Bondanza S, Fraryi AT, Cancedda R (1988) Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J Cell Biol 107: 1919–1926
Donatien P, Surlève-Bazeille JE, Thody AJ, TaÏeb A 1993 Growth and differentiation of normal human melanocytes in a TPA free, cholera toxin free, low serum medium and keratinocyte influence. Arch Dermatol Res 285: 385–392
Fitzpatrick TB, Breathnach AB (1963) Das epidermal melanin-einheit-system. Dermatol Wchnschr 147: 481–489
Friedmann PS, Gilchrest BA (1987) Ultraviolet radiation directly induces pigment production by cultured human melanocytes. J Cell Physiol 133: 88–94
Gilchrest BA, Albert LS, Karassik RL, Yaar M (1985) Substrate influences human epidermal melanocyte attachment and spreading in vitro. In Vitro Cell Dev Biol 21: 114–120
Gordon PR, Gilchrest BA (1989) Human melanogenesis is stimulated by diacylglycerol. J Invest Dermatol 93: 700–702
Haake AR, Scott GA (1991) Physiologic distribution and differentiation of melanocytes in human foetal and neonatal skin equivalents. J Invest Dermatol 96: 71–77
Halaban R, Langdon R, Birchall N et al. (1988) Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol 107: 1611–1619
Hearing VJ, Ekel TM (1975) A comparison of tyrosine hydroxylation and melanin formation. Biochem J 157: 549–557
Hearing VJ, Jiménez M (1989) Analysis of mammalian pigmentation at the molecular level. Pigment Cell Res 2: 75–85
Hunt G, Cresswell J, Donatien P, Thody A (1991) Effects of αMSH and Ca2+ on the attatchment of human melanocytes to laminin and fibronectin. Br J Dermatol 125: 487
Krasagakis K, Garbe C, Krüger S, Orfanos CE (1991) Effect of interferons on cultured human melanocytes in vitro: interferon Β but not α or γ inhibit proliferation and all interferons significantly modulate the cell phenotype. J Invest Dermatol 97: 364–372
Kupper TS, Chua AO, Flood P, McGuire J, Gubler U (1987) Interleukin-1 gene expression in cultured human keratinocytes is augmented by ultraviolet iradiation. J Clin Invest 80: 430–436
Laidlaw GF, Blackberg SN (1932) Melanoma studies II. A simple technique for the Dopa reaction. Am J Pathol 8: 491–498
Liu SC, Karassik M (1978) Isolation and growth of adult human epidermal keratinocytes in cell culture. J Invest Dermatol 71: 157–162
Macaig T, Cerundolo J, Ilsley S, Kelley PR, Forand R (1979) An endothelial cell growth factor from bovine hypothalamus: identification and partial purification. Proc Natl Acad Sci USA 76: 5674–5678
Naeyaert JM, Yaar M, Peacocke M, Gilchrest B (1990) Human melanocytes synthesise and express genes for basement membrane components. J Invest Dermatol 95: 481A
Oxholm A, Oxholm P, Staberg B, Bendtzen K (1988) Immunohistological detection of interleukin-like molecules and tumor necrosis factor in human epidermis before and after UVB-irradiation. Br J Dermatol 118: 369–376
Ponec M (1991) Reconstruction of human epidermis on de-epidermised dermis: expression of differentiation-specific protein markers and lipid composition. Toxicol In Vitro 5: 597–606
Ponec M, Weerheim A, Kempenaar J, Mommaas A-M, Nugteren DH (1988) Lipid composition of cultured human keratinocytes in relation to their differentiation. J Lipid Res 29: 949–961
Prunieras M, Regnier M, Schlotterer M (1979) Nouveau procédé de culture des cellules epidermiques humaines sur derme homologue ou heterologue: préparation de greffons recombinés. Ann Chir Plast 24: 357–362
Ranson M, Posen S, Mason RA (1988) Extracellular matrix modulates the function of human melanocytes but not melanoma cells. J Cell Physiol 136: 281–288
Regnier M, Prunieras M, Woodley D (1981) Growth and differentiation of adult human epidermal cells on dermal substrates. Front Matrix Biol 9: 4–35
Regnier M, Asselineau D, Lenoir MC (1990) Human epidermis reconstructed on dermal substrates in vitro: an alternative to animals in skin pharmacology. Skin Pharmacol 3: 70–85
Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331–334
Scott GA, Haake AR (1991) Keratinocytes regulate melanocyte numbers in human foetal or neonatal skin equivalents. J Invest Dermatol 97: 776–781
Shu SY, Ju G, Fan LZ (1988) The glucose oxidase-DAB-nikkel method in peroxidase histochemistry of the nervous system. Neurosic Lett 85: 169–171
Swope VB, Abdel-Malek Z, Kassem LM, Nordlund JJ (1991) Interleukins 1α and 6 and tumor necrosis factor α are paracrine inhibitors of human melanocyte proliferation and melanogenesis. J Invest Dermatol 96: 180–185
Topol BM, Haimes HB, Dubertret L, Bell E (1986) Transfer of melanosomes in a skin equivalent model in vitro. J Invest Dermatol 87: 642–647
Valyi-Nagy IT, Murphy GF, Mancianti M, Whitaker D, Herlyn M (1990) Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model. Lab Invest 62: 314–324
Yaar M, Woodley DT, Gilchrest B (1988) Human naevocellular naevus cells are surrounded by basement membrane components: immunohistologic studies of human naevus cells and melanocytes in vivo and in vitro. Lab Invest 58: 157–162
Yohn JJ, Critelli M, Lyons MB, Norris DA (1990) Modulation of melanocyte intercellular adhesion molecule-1 by immune cytokines. J Invest Dermatol 90: 233–237
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Todd, C., Hewitt, S.D., Kempenaar, J. et al. Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation. Arch Dermatol Res 285, 455–459 (1993). https://doi.org/10.1007/BF00376817
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00376817