Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.
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PMID:Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol. 896 93

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.
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PMID:Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation. 1020 19