The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation

Brandhorst, Heide; Friberg, Andrew; Andersson, Helena H.; Felldin, Marie; Foss, Aksel; Salmela, K.; Lundgren, Torbjörn; Tibell, Annika; Tufveson, Gunnar; Korsgren, Olle; Brandhorst, Daniel

doi:10.1097/tp.0b013e31819499f0

Cited by 50 publications

(52 citation statements)

References 26 publications

(26 reference statements)

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“…In recent years, it has also been applied to the isolation of many other highyield homogenous and viable cells (27). Because of the persistent renewal of human fetal intestinal cells, continuous epithelial cell cultures can be generated relatively easily from the human fetal intestine in a reproducible manner (17,19).…”

Section: Discussionmentioning

confidence: 99%

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Liu

Zhang

Zhou

et al. 2010

Braz J Med Biol Res

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Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

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Section: Discussionmentioning

confidence: 99%

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Liu

Zhang

Zhou

et al. 2010

Braz J Med Biol Res

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“…Isolation, purification, and culture of human islets were performed as previously described utilizing collagenase NB1 and neutral protease (Serva, Heidelberg, Germany) for pancreas digestion performed at 37 C in a continuous digestion-filtration device [16,19]. Enzyme activity and perfusion volume were adjusted according to the trimmed pancreas weight and administered by means of continuous ductal perfusion as previously reported [20]. Islet purification was performed in an automated closed purification system utilizing a continuous hyperosmolaric Ficoll gradient for islet separation from non-endocrine tissue [19,21].…”

Section: Pancreas Processingmentioning

confidence: 99%

Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation

Caballero-Corbalan

Brandhorst

Malm

et al. 2012

Journal of Surgical Research

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“…CP cleaves the carboxyl terminal side peptide bonds of Arg residues, like trypsin, from the pancreatic exocrine. CP had been largely ignored, as the enzyme did not seem to be involved in the decomposition of ECM; however, the enzyme has recently attracted attention due to the fact that a higher trypsin-like activity in the C. histolyticum proteolytic enzyme mixture improves the success rate of islet isolation (Brandhorst et al 2009). NP, in the meanwhile, has been thought to play an important role in islet isolation, because islet preparation by collagenases without NP requires longer digestion times (Wolters et al 1992;Bucher et al 2004).…”

Section: Introductionmentioning

confidence: 98%

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Maeda

Nakagawa

Murayama

et al. 2015

Appl Microbiol Biotechnol

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Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

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The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation

Abstract: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

Cited by 50 publications

References 26 publications

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

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