Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation
| Autor: | S. Hybbinette, M. Boström, K. Lindberg |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Adult
Keratinocytes Biopsy Cell Culture Techniques Thermolysin Human skin Cell Count Dermatology Cell Separation Biology Biochemistry Mice Dermis Dispase Endopeptidases medicine Animals Humans Trypsin Molecular Biology Skin integumentary system Epidermis (botany) medicine.diagnostic_test Epithelial Cells 3T3 Cells Fibroblasts Molecular biology Coculture Techniques medicine.anatomical_structure Female Epidermis Keratinocyte Cell Division medicine.drug |
| Zdroj: | Experimental dermatology. 8(1) |
| ISSN: | 0906-6705 |
| Popis: | 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/ml 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 cm 2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0×10 6 cells/cm 2 ) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8×10 6 cells/cm 2 ), Dispase (2.3×10 6 cells/cm 2 ) or trypsin (1.1×10 6 cells/cm 2 ). 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. |
| Databáze: | OpenAIRE |
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