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New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin

Authors

  • Reza Yarani,

    1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
    2. Department of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran
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  • Kamran Mansouri,

    1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
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  • Hamid Reza Mohammadi-Motlagh,

    1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
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  • Mitra Bakhtiari,

    1. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
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  • Ali Mostafaie

    Corresponding author
    1. Department of Immunology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
    • Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
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Correspondence: A. Mostafaie, Medical Biology Research Center, Kermanshah University of Medical Sciences, sorkheh lizheh, Kermanshah, Iran, PO. Box: 6714869914. Tel:+98 831 4276473; Fax:+98-831-4276471; E-mail: amostafaie@kums.ac.ir

Abstract

Objectives

Conventional isolation of epidermis from the dermis and disruption of epidermal sheets to liberate the cells, are performed using proteolytic enzymes such as thermolysin or collagenase. Selective population expansion of melanocytes is achieved by suppressing proliferation of keratinocytes and fibroblasts in epidermal cell suspensions, using phorbol esters and cholera toxin. Here, we introduce a new procedure for isolation of epidermal cells, using proteolytic activity of kiwi fruit actinidin, and also an improved growth medium for melanocytes in the presence of leukaemia inhibitory factor (LIF) and forskolin.

Materials and methods

Dermo-epidermal separation and epidermal sheet cell dispersion were performed using actinidin compared to conventional proteases including collagenase, thermolysin or trypsin. Thereafter, melanocyte culture was performed in two common media and one modified medium to discover optimization for these cells.

Results

We found that dermo-epidermal separation and epidermal sheet cell dispersion using kiwi fruit actinidin were considerably better than previously used methods, both from the aspect of less fibroblast and keratinocyte contamination, and of more viable native cells. Also, melanocytes proliferated better in phorbol ester- and cholera toxin-free proliferation medium supplemented with LIF and forskolin.

Conclusion

Less contamination and higher numbers of viable cells were actinidin preferential for separation of epidermis and isolation of epidermal cells. Supplementation of LIF and forskolin to new medium increased proliferation potential of melanocytes in comparison to exogenous mitogens.

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