Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation

Dendo, Mami; Maéda, Hiroshi; Yamagata, Yuriko; Murayama, Kazutaka; Watanabe, K.; Imura, Takehiro; Inagaki, Akiko; Igarashi, Yasuhiro; Katoh, Yohei; Ebina, Masayuki; Fujimori, Keisei; Igarashi, Kazuhiko; Ohuchi, Noriaki; Satoh, Susumu; Goto, Masafumi

doi:10.1097/tp.0000000000000662

Cited by 20 publications

(28 citation statements)

References 32 publications

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“…Pancreatic islet transplantation is currently regarded as one of the treatment options for severe diabetes. 26 Considering that the clinical applications of HTx remain limited, 2,7 a number of experiences originating from islet transplantation, including cell isolation methods, 21,27,28 culture systems, 21,29 cell preservation, 18,21,22 cell graft evaluation, 30-32 and engraftment 33-35 could be applicable for improving the outcomes of HTx. Per the above-noted concepts, in the present study, the TD was applied to hepatocytes and was found to be effective for maintaining the viability and function of hepatocytes during a short-term preservation period.…”

Section: Discussionmentioning

confidence: 99%

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

Fukuoka

Inagaki

Nakamura

et al. 2017

Transplantation Direct

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BackgroundNo optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.MethodsHepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed.ResultsThe survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups.ConclusionsTD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

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Section: Discussionmentioning

confidence: 99%

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

Fukuoka

Inagaki

Nakamura

et al. 2017

Transplantation Direct

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“…The recombinant clostridial collagenases, ColG and ColH, were obtained from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan). The recombinant CP was purified as described previously (Dendo et al 2015). Collagenase V (Sigma-Aldrich Co.…”

Section: Methodsmentioning

confidence: 99%

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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“…A recent study in rats confirmed these previous observations, and found nearly triplicated islet yields when collagenase and neutral protease were additionally supplemented with clostripain. Interestingly, combining clostripain with TL did not enhance islet yield, and resulted in increased islet fragmentation in contrast to the combination with neutral protease [80]. An incompatibility between TL and clostripain extracted from different species is assumed to cause the problem.…”

Section: Enzyme Characterizationmentioning

confidence: 99%

Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation?

Brandhorst¹,

Brandhorst²,

Prv.³

2017

Rev Diabet Stud

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In comparison to procedures used for the separation of individual cell types from other organs, the process of human pancreatic islet isolation aims to digest the pancreatic exocrine matrix completely without dispersing the individual cells within the endocrine cell cluster. This objective is unique within the field of tissue separation, and outlines the challenge of islet isolation to balance two opposing priorities. Although significant progress has been made in the characterization and production of enzyme blends for islet isolation, there are still numerous areas which require improvement. The ultimate goal of enzyme production, namely the routine production of a consistent and standardized enzyme blend, has still not been realized. This seems to be mainly the result of a lack of detailed knowledge regarding the structure of the pancreatic extracellular matrix and the synergistic interplay between collagenase and different supplementary proteases during the degradation of the extracellular matrix. Furthermore, the activation of intrinsic proteolytic enzymes produced by the pancreatic acinar cells, also impacts on the chance of a successful outcome of human islet isolation. This overview discusses the challenges of pancreatic enzymatic digestion during human islet isolation, and outlines the developments in this field over the past 5 decades.

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Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation

Abstract: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Cited by 20 publications

References 32 publications

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

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

Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation?

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