title: New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
authors: Reza
authors: R
authors: Yarani
authors: Kamran
authors: K
authors: Mansouri
authors: Hamid Reza
authors: HR
authors: Mohammadi-Motlagh
authors: Mitra
authors: M
authors: Bakhtiari
authors: Ali
authors: A
authors: Mostafaie
mesh_terms: D029042:Actinidia / Q000201:enzymology; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D000078404:Epidermal Cells; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology*; D008544:Melanocytes / Q000166:cytology* / Q000187:drug effects
chemical_list: D003470:Culture Media; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D005576:Colforsin; D003546:Cysteine Endopeptidases; C018745:actinidain
references: J Invest Dermatol. 1989 Apr;92(4):565-72
references: 2467948
references: J Am Acad Dermatol. 1989 Aug;21(2 Pt 1):257-64
references: 2768575
references: Dev Biol. 1987 Apr;120(2):329-47
references: 3549390
references: Pigment Cell Res. 2000 Apr;13(2):60-9
references: 10841026
references: Exp Pathol. 1990;40(2):105-9
references: 2079104
references: Cell. 1975 Nov;6(3):331-43
references: 1052771
references: Biosci Biotechnol Biochem. 2004 Apr;68(4):861-7
references: 15118315
references: Arch Virol. 1996;141(6):1011-20
references: 8712919
references: Skin Pharmacol Physiol. 2009;22(2):114-21
references: 19188759
references: In Vitro. 1984 May;20(5):447-50
references: 6724622
references: J Surg Res. 2008 Sep;149(1):27-30
references: 18028952
references: Exp Cell Res. 1979 Aug;122(1):137-47
references: 113230
references: J Cell Physiol. 2002 Sep;192(3):315-26
references: 12124777
references: Endocr Rev. 2000 Jun;21(3):313-45
references: 10857556
references: Pigment Cell Res. 2001 Aug;14(4):236-42
references: 11549105
references: J Cell Biol. 1988 Oct;107(4):1611-9
references: 2459134
references: Proc Natl Acad Sci U S A. 1982 Mar;79(6):2018-22
references: 6952249
references: J Cell Biol. 1988 Nov;107(5):1919-26
references: 2460471
references: Dermatol Clin. 2007 Jul;25(3):271-81, vii
references: 17662893
references: Cancer Res. 1980 Jul;40(7):2465-74
references: 6992987
references: Arch Dermatol Res. 2007 Jul;299(4):191-200
references: 17334773
references: Dev Biol. 1973 Sep;34(1):39-46
references: 4595498
references: Biochim Biophys Acta. 1959 May;33(1):242-4
references: 13651208
references: Methods Mol Med. 2005;107:13-28
references: 15492362
references: J Supramol Struct. 1979;12(2):195-208
references: 397370
references: Pigment Cell Res. 2005 Feb;18(1):2-12
references: 15649147
references: Dev Biol. 2003 Jul 1;259(1):162-75
references: 12812796
references: Arch Dermatol Res. 1992;284(7):424-6
references: 1337693
references: Appl Biochem Biotechnol. 2008 Feb;144(2):123-31
references: 18456944
references: Pigment Cell Res. 2007 Oct;20(5):380-4
references: 17850511
references: J Invest Dermatol. 1957 Jan;28(1):15-32
references: 13416673
references: J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2005;23(2):105-61
references: 16291526
references: Curr Biol. 2009 Dec 29;19(24):R1116-7
references: 20064420
references: Arch Dermatol Res. 1993;285(7):385-92
references: 8304778
references: PLoS One. 2011 Feb 23;6(2):e17197
references: 21383848
references: Yale J Biol Med. 1973 Dec;46(5):384-96
references: 4779127