title: Testing of viable human skin cell dilution cultures as an approach to validating microsampling.
abstract: Skin biopsies are a valuable technique in the diagnosis of cutaneous inflammatory and neoplastic conditions. We were interested to test the minimal size or equivalent volume by dilution of proteolytically disassociated skin tissue required to allow the isolation and propagation of cutaneous cells, for freezing, storage and biochemical analysis. It was possible to propagate with 100% efficiency fibroblast and melanocytic cells from a 0.1 to 0.5 mm3equivalent neonatal foreskin sample using a combination of DispaseII and CollagenaseIV. The smallest tissue dilution that allowed melanocytic cell culture was 0.01 mm3, which equated to approximately 16 cells based on the average skin density of melanocytes. However, passaging of cells to create frozen stocks was achieved routinely only from 1 mm3skin, equating to 1560 cells. Tissue-specific antigen expression of these cultures was tested by western blot of total protein extracts. There was no pigmentation antigen expression in fibroblast cultures; however, smooth muscle actin protein expression was high in fibroblast but absent from melanocytic cell strains. Melanocytic cells expressed pigmentation antigens and E-cadherin, but these were not detected in fibroblasts. Moreover, maturation of these melanocytic cells resulted in a decrease of Dopachrome Tautomerase antigen expression and induction of Tyrosinase protein consistent with the differentiation potential seen in cell cultures derived routinely from large sections of skin tissue.
authors.affiliation: Dermatology Research Centre, School of Medicine, Level 5, Translational Research Institute (TRI), The University of Queensland, 37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia. s.ainger@uq.edu.au.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ainger
authors.affiliation: Dermatology Research Centre, School of Medicine, Level 5, Translational Research Institute (TRI), The University of Queensland, 37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia.
authors.forename: X L Hilary
authors.initials: XL
authors.lastname: Yong
authors.affiliation: Dermatology Research Centre, School of Medicine, Level 5, Translational Research Institute (TRI), The University of Queensland, 37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia.|Department of Dermatology, Princess Alexandra Hospital, Brisbane, Australia.
authors.forename: H Peter
authors.initials: HP
authors.lastname: Soyer
authors.affiliation: Dermatology Research Centre, School of Medicine, Level 5, Translational Research Institute (TRI), The University of Queensland, 37 Kent Street, Woolloongabba, Brisbane, QLD, 4102, Australia.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Sturm
mesh_terms: D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D008544:Melanocytes / Q000502:physiology*; D008904:Miniaturization; D014442:Monophenol Monooxygenase / Q000378:metabolism; D012867:Skin / Q000166:cytology* / Q000378:metabolism; D012880:Skin Pigmentation
chemical_list: D015820:Cadherins; D014442:Monophenol Monooxygenase; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase
keywords: Cell isolation; Fibroblast; Keratinocyte; Melanoblast; Melanocyte; Skin biopsies