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

The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy.
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PMID:Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro. 2112 25

The reproducible isolation and accurate characterization of thymic epithelial cell (TEC) subsets is of critical importance to the ongoing study of thymopoiesis and its functional decline with age. The study of adult TEC, however, is significantly hampered due to the severely low stromal to hematopoietic cell ratio. Non-biased digestion and enrichment protocols are thus essential to ensure optimal cell yield and accurate representation of stromal subsets, as close as possible to their in vivo representation. Current digestion protocols predominantly involve diverse, relatively impure enzymatic variants of crude collagenase and collagenase/dispase (col/disp) preparations, which have variable efficacy and are often suboptimal in their ability to mediate complete digestion of thymus tissue. To address these issues we compared traditional col/disp preparations with the latest panel of Liberase products that contain a blend of highly purified collagenase and neutral protease enzymes. Liberase enzymes revealed a more rapid, complete dissociation of thymus tissue; minimizing loss of viability and increasing recovery of thymic stromal cell (TSC) elements. In particular, the recovery and viability of TEC, notably the rare cortical subsets, were significantly enhanced with Liberase products containing medium to high levels of thermolysin. The improved stromal dissociation led to numerically increased TEC yield and total TEC RNA isolated from pooled digests of adult thymus. Furthermore, the increased recovery of TEC enhanced resolution and quantification of TEC subsets in both adult and aged mice, facilitating flow cytometric analysis on a per thymus basis. We further refined the adult TEC phenotype by correlating surface expression of known TEC markers, with expression of intracellular epithelial lineage markers, Keratin 5 and Keratin 8. The data reveal more extensive expression of K8 than previously recognized and indicates considerable heterogeneity still exists within currently defined adult TEC subsets.
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PMID:Purified enzymes improve isolation and characterization of the adult thymic epithelium. 2291 2