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 He
  • Philippe Humbert
  • Céline Viennet
Article
  • 168 Downloads

Abstract

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.

Keywords

Skin Melanocytes Pigmentation Cell culture Extraction 

Notes

Acknowledgements

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.

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Copyright information

© 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
  • Philippe Humbert
    • 1
  • Céline Viennet
    • 1
  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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