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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

Research output: Contribution to journalJournal articleResearchpeer-review

DOI

  1. Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

    Research output: Contribution to journalJournal articleResearchpeer-review

  • Reza Yarani
  • Kamran Mansouri
  • Hamid Reza Mohammadi-Motlagh
  • Mitra Bakhtiari
  • Ali Mostafaie
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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.

Original languageEnglish
JournalCell Proliferation
Volume46
Issue number3
Pages (from-to)348-55
Number of pages8
ISSN0960-7722
DOIs
Publication statusPublished - Jun 2013
Externally publishedYes

    Research areas

  • Actinidia, Cell Culture Techniques, Cell Proliferation, Cell Separation, Cells, Cultured, Colforsin, Culture Media, Cysteine Endopeptidases, Epidermis, Humans, Leukemia Inhibitory Factor, Melanocytes, Journal Article, Research Support, Non-U.S. Gov't

ID: 51973574