Search results for "Collagenase"
showing 5 items of 45 documents
Isolation of Adult Rat Cardiomyocytes Using Recombinant Collagenases
2018
Direct isolation of primary cells from tissues and organs allows for the maintenance of important cell characteristics and properties for in vitro studies and a plethora of biomedical applications. Dissociation of cells from the organ of interest is possible due to the enzymatic activity of collagenases. The choice and the dose of these enzymes is the critical step to obtain the maximum number of cells with intact structure and function. In this contest, Abiel collagenases class I (Col G) and class II (Col H) were synthesised using recombinant DNA technologies and their ability to degrade collagen in cell isolation from different tissues was tested. Examples of cells isolated with these enz…
Optimization of a Biotechnological Process for Production and Purification of Two Recombinant Proteins: Col G and Col H
2017
Different strategies can be used for increasing production of heterologous recombinant proteins in Escherichia coli. Protein size is often critical for obtaining the best quantity/quality ratio of recombinant protein expression. This study focuses on two recombinant proteins; Class I and class II Collagenases, namely Col G and Col H. Their size is about 150 kDa each. We have developed a method to obtain high levels of cell growth and intracellular expression of each Collagenases in recombinant E. coli BL21(DE3). Batch and Fed-batch fermentation procedures have been performed. Results show that Fed-batch technique was most effective in obtaining the highest cell density for each recombinant …
A new method to value efficiency of enzyme blends for pancreatic tissue digestion.
2010
Islet transplantation, since the 90’s, has been resulting to be one of the best successful example of human cell therapy. Nevertheless, islet isolation procedure is not completely standardized; in fact, more than fifty percent of islets procedures don’t arrive to their transplantation. This is due both to the variability of donor’s pancreas and to an unpredictable enzymatic blend efficiency. Enzymes used in pancreas digestion are extracted from Clostridium histolyticum bacteria and digest several substrates. In particular they have strong collagenolytic activity compared to vertebrate collagenases. However, several impediments persist in human islet isolation success probably due to the var…
Tissue Dissociation and Primary Cells Isolation Using Recombinant Collagenases Class I and II
2014
Collagenases class I (Col G) and class II (Col H) currently available for tissue dissociation are produced from Clostridium histolyticum (human pathogen) strains. In the processes of extraction of the cells from the tissue, combined activity of both classes of enzymes is required. CI and CII are complementary in degrading collagen. ABIEL recently produced the collagenase class I and II using the recombinant DNA technologies (PCT WO 2011/073925 A9). The enzymes were produced in E. coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity an…
Isolation and colture of beta-like cells from porcine Wirsung duct
2009
We sought to develop a protocol to isolate and culture porcine Wirsung duct cells in order to determine their potency to differentiate into insulin-expressing beta-like cells. The porcine Wirsung duct isolated by a surgical microdissection was digested with collagenase P and trypsin to dissociate ductal cells. These elements were cultured in serum-free supplemented media: for 2 weeks. Thereafter the cells were exposed to varying concentrations of glucose (0, 5.6, 17.8, and 25 mmol/L) to induce a beta-like phenotype, as identified by immunohistochemical staining. Cell growth proceeded slowly for the first 2 weeks of culture. After glucose induction for 2 weeks, they formed pancreatic islet-l…