, Volume 70, Issue 4, pp 1167–1176 | Cite as

Development and validation of a simple method for the extraction of human skin melanocytes

  • Yinjuan Wang
  • Marion Tissot
  • Gwenaël Rolin
  • Patrice Muret
  • Sophie Robin
  • Jean-Yves Berthon
  • Li HeEmail author
  • Philippe Humbert
  • Céline ViennetEmail author


Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE™ product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE™ incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm2) whatever the method used. In conclusion, this new procedure based on TrypLE™ incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.


Skin Melanocytes Pigmentation Cell culture Extraction 



This work was financially supported by Program for Innovative Research Team in University of Ministry of Education of China (Grant No. IRT13067), the Fund of Yunnan Province Chinese Academy of Sciences Cooperation (Grant No. 2014IB008) and Greentech SA (France).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Duval C, Schmidt R, Regnier M, Facy V, Asselineau D, Bernerd F (2003) The use of reconstructed human skin to evaluate UV-induced modifications and sunscreen efficacy. Exp Dermatol 12:64–70CrossRefPubMedGoogle Scholar
  2. Eisinger M, Marko O (1982) Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA 79:2018–2022CrossRefPubMedGoogle Scholar
  3. Gilchrest BA, Vrabel MA, Flynn E, Szabo G (1984) Selective cultivation of human melanocytes from newborn and adult epidermis. J Invest Dermatol 83:370–376CrossRefPubMedGoogle Scholar
  4. Godwin LS, Castle JT, Kohli JS, Goff PS, Cairney CJ, Keith WN, Sviderskaya EV, Bennett DC (2014) Isolation, culture, and transfection of melanocytes. Curr Protoc Cell Biol 63:1.8.1–1.8.20CrossRefGoogle Scholar
  5. Guerra L, Primavera G, Raskovic D, Pellegrini G, Golisano O, Bondanza S, Paterna P, Sonego G, Gobello T, Atzori F, Piazza P, Luci A, De Luca M (2003) Erbium: YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 139:1303–1310CrossRefPubMedGoogle Scholar
  6. Halaban R, Ghosh S, Baird A (1987) bFGF is the putative natural growth factor for human melanocytes. In Vitro Cell Dev Biol 23:47–52CrossRefPubMedGoogle Scholar
  7. Hsu MY, Li L, Herlyn M (2005) Cultivation of normal human epidermal melanocytes in the absence of phorbol esters. Methods Mol Med 107:13–28PubMedGoogle Scholar
  8. Hu F, Staricco RJ, Pinkus H, Fosnaugh R (1957) Human melanocytes in tissue culture. J Invest Dermatol 28:15–32CrossRefPubMedGoogle Scholar
  9. Imokawa G, Yada Y, Morisaki N, Kimura M (1998) Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes. Biochem J 330:1235–1239CrossRefPubMedPubMedCentralGoogle Scholar
  10. Kormos B, Belso N, Bebes A, Szabad G, Bacsa S, Széll M, Kemény L, Bata-Csörgo Z (2011) In Vitro dedifferentiation of melanocytes from adult epidermis. PLoS One 6:e17197CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kumar R, Parsad D, Kanwar A, Kaul D (2012) Development of melanocyte-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes. Indian J Dermatol Venereol Leprol 78:599–604CrossRefPubMedGoogle Scholar
  12. Manira M, Khairul Anuar K, Seet WT, Ahmad Irfan AW, Ng MH, Chua KH, Mohd Heikal MY, Aminuddin BS, Ruszymah BH (2014) Comparison of the effects between animal-derived Trypsin and recombinant Trypsin on human skin cells proliferation, gene and protein expression. Cell Tissue Bank 15:41–49CrossRefPubMedGoogle Scholar
  13. Nielsen HI, Don P (1984) Culture of normal adult human melanocytes. Br J Dermatol 110:569–580CrossRefPubMedGoogle Scholar
  14. Normand J, Karasek MA (1995) A method for the isolation and serial propagation of keratinocytes, endothelial cells and fibroblasts from a single punch biopsy of human skin. In Vitro Cell Dev Biol Animal 31:447–455CrossRefGoogle Scholar
  15. Peacocke M, Yaar M, Mansur CP, Chao MV, Gilchrest BA (1988) Induction of nerve growth factor receptors on cultured human melanocytes. Proc Natl Acad Sci USA 85:5282–5286CrossRefPubMedGoogle Scholar
  16. Shi Z, Ji K, Yang S, Zhang J, Yao J, Dong C, Fan R (2016) Biological characteristics of mouse skin melanocytes. Tissue Cell 48:114–120CrossRefPubMedGoogle Scholar
  17. Tang J, Li Q, Cheng B, Jing L (2014a) Primary culture of human face skin melanocytes for the study of hyperpigmentation. Cytotechnology 66:891–898CrossRefPubMedGoogle Scholar
  18. Tang X, Richardson WJ, Fitch RD, Brown CR, Isaacs RE, Chen J (2014b) A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells. Cytotechnology 66:979–986CrossRefPubMedGoogle Scholar
  19. Tsuji T, Karasek M (1983) A procedure for the isolation of primary cultures of melanocytes from newborn and adult human skin. J Invest Dermatol 81:179–180CrossRefPubMedGoogle Scholar
  20. Verma G, Varkhande SR, Kar HK, Rani R (2015) Evaluation of repigmentation with cultured melanocyte transplantation (CMT) compared with non-cultured epidermal cell transplantation in vitiligo at 12th week reveals better repigmentation with CMT. J Invest Dermatol 135:2533–2535CrossRefPubMedGoogle Scholar
  21. Virador VM, Kobayashi N, Matsunaga J, Hearing VJ (1999) A standardized protocol for assessing regulators of pigmentation. Anal Biochem 270:207–219CrossRefPubMedGoogle Scholar
  22. Yada Y, Higuchi K, Imokawa G (1991) Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266:18352–18357PubMedGoogle Scholar
  23. Yarani R, Mansouri K, Mohammadi-Motlagh HR, Bakhtiari M, Mostafaie A (2013) New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin. Cell Prolif 46:348–355CrossRefPubMedGoogle Scholar
  24. Yoon TJ, Lei TC, Yamaguchi Y, Batzer J, Wolber R, Hearing VJ (2003) Reconstituted 3-dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation. Anal Biochem 318:260–269CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Yinjuan Wang
    • 1
  • Marion Tissot
    • 1
  • Gwenaël Rolin
    • 1
    • 5
  • Patrice Muret
    • 1
  • Sophie Robin
    • 2
  • Jean-Yves Berthon
    • 3
  • Li He
    • 4
    Email author
  • Philippe Humbert
    • 1
  • Céline Viennet
    • 1
    Email author
  1. 1.University of Bourgogne Franche-ComtéINSERM, EFS BFC, UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et GéniqueBesançonFrance
  2. 2.Bioexigence S.A.R.LBesançonFrance
  3. 3.Greentech SABiopôle Clermont LimagneSaint BeauzireFrance
  4. 4.Department of DermatologyFirst Affiliated Hospital of Kunming Medical UniversityKunmingChina
  5. 5.Clinical Investigation Center, Inserm CICB 1431University HospitalBesançonFrance

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