title: Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.
abstract: Four techniques for dissociation of skin biopsies were compared to identify the method of choice for optimal expansion of isolated keratinocytes. Equivalent biopsies were obtained from 4 healthy human subjects and each divided into four parts. One part was minced and placed in a trypsinizing flask containing 0.05% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA). Released cells were harvested hourly. With the other parts, the epidermis was separated from the dermis after treatment with 0.5 mg/nml thermolysin, 2.5 mg/ml Dispase, or 0.17% trypsin and the epidermal portions were minced and incubated for 1 h in trypsin:EDTA. The cells were cocultivated with irradiated 3T3 fibroblasts to study the keratinocytes proliferative capacity. Freshly isolated cells were immunostained with anti-vimentin antibodies or grown in fibroblast-supportive conditions to detect the presence of human dermal fibroblasts. The mean number of cells dissociated per cm2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0x 10(6) cells/cm2) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8x 10(6) cells/cm2), Dispase (2.3x 10(6) cells/cm2) or trypsin (1.1 x 10(6) cells/cm2). Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy were human fibroblasts. This comprised more than twice the number of fibroblasts obtained by using epidermal/dermal split techniques. The proliferative capacity in primary and secondary culture was higher in cells isolated by trypsin:EDTA incubation in the trypsinizing flask or after epidermal-dermal separation using thermolysin, suggesting that Dispase or trypsin may have a more detrimental effect on the isolated keratinocytes. Our results show that dissociating the cells by trypsin:EDTA incubation in a trypsinizing flask or after epidermal-dermal separation using thermolysin, are preferable methods for isolating keratinocytes from human skin.
authors: Department of Plastic Surgery, Uppsala University Hospital, Sweden.
authors: S
authors: S
authors: Hybbinette
authors: M
authors: M
authors: Boström
authors: K
authors: K
authors: Lindberg
mesh_terms: D016475:3T3 Cells; D000328:Adult; D000818:Animals; D001706:Biopsy; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D020405:Dermis / Q000033:anatomy & histology / Q000187:drug effects; D010450:Endopeptidases / Q000494:pharmacology*; D004817:Epidermis / Q000033:anatomy & histology / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D012867:Skin / Q000166:cytology / Q000187:drug effects*; D013820:Thermolysin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D010450:Endopeptidases; D014357:Trypsin; D013820:Thermolysin; C023139:dispase