Adjustment of the Ratio Between Collagenase Class II and I Improves Islet Isolation Outcome

Brandhorst, Daniel; Huettler, S.; Alt, A.; Raemsch-Guenther, N.; Kurfuerst, M.; Bretzel, Reinhard G.; Brandhorst, Heide

doi:10.1016/j.transproceed.2005.09.079

Cited by 18 publications

(7 citation statements)

References 6 publications

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“…while CI tends to have greater activity towards insoluble collagen and is more stable, CII acts on various peptide substrates at a greater rate (19). An early study in rat islet isolation suggested that CII plays more important role (20), while a recent study showed that equal amount of both CI and CII were required for the efficient rat islet isolation (21). Our results using human pancreata differ from those of both rodent studies: a higher islet yield was obtained with a higher CI activity.…”

Section: Adjustment Of Both Collagenase and Thermolysin Dosagesmentioning

confidence: 99%

Enhancing the Success of Human Islet Isolation Through Optimization and Characterization of Pancreas Dissociation Enzyme

Kin

Zhai

Murdoch

et al. 2007

American Journal of Transplantation

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A major obstacle to successful human islet isolation has been the variability of the enzymatic digestion phase. The aim of this study was to define optimal enzyme activity ranges normalized by the pancreas weight and to identify valid parameters for the optimal selection of successful lots of collagenase enzyme blends. Our results from 251 islet isolations showed that optimization of thermolysin dosage based on Caseinase unit/g pancreas contributed considerably to islet isolation outcome but that collagenase dosage measured by the manufacturer (Wünsch unit/g pancreas) was not a major determinant of islet isolation outcome. We also found that lot-to-lot inconsistency of enzyme performance was not explained by the activity values provided by the manufacturer, but rather by an in-house assay of class I collagenase (CI) and class II collagenase (CII); using a lot with a lower CII/CI resulted in a higher success rate. The odds of successful isolation was 8.67 times higher when a vial with CII/CI ratio <0.204 was used than when a vial with CII/CI ≥ 0.204 was used, suggesting that CII/CI ratio may be a strong predictor to distinguish potential lot success. This study provides a framework for improved enzymatic digestion in human islet isolation.

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Section: Adjustment Of Both Collagenase and Thermolysin Dosagesmentioning

confidence: 99%

Enhancing the Success of Human Islet Isolation Through Optimization and Characterization of Pancreas Dissociation Enzyme

Kin

Zhai

Murdoch

et al. 2007

American Journal of Transplantation

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“…Clostridium histolyticum collagenases G and H (ColG and ColH) can easily digest collagens, regardless of their types and sizes (27). Therefore, they are used extensively as a clinical tool in the nonsurgical treatment of Dupuytren's disease (1,13) and in the isolation of cells from tissues and organs, such as in the preparation of pancreatic islet cells for transplantation (5,6,15).…”

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confidence: 99%

Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

Ohbayashi

Yamagata

Goto

et al. 2012

Appl Environ Microbiol

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e The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca 2؉ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca 2؉ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca 2؉ was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca 2؉ chelation, in comparison with the thermal melting point (T m ). The melting point changed from 54 to 49°C by the Ca 2؉ chelation, and it was restored to 54°C by the addition of excess Ca 2؉ . These results indicated that the interdomain flexibility and the domain arrangement of fulllength collagenase H are reversibly regulated by Ca 2؉ .

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“…Concerning the rat pancreas digestion, it has been reported that the CII is the most efficient at digesting exocrine tissue, whereas CI increases the digestion rate (13,14); therefore, an appropriate mix of CI and CII has been proposed to optimize islet isolation (15)(16)(17)(18). In the early stages of digestion, neutral proteases also are needed, but in excessive amounts these can destroy islet integrity (19 -21).…”

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confidence: 99%

Characterization of Collagenase Blend Enzymes for Human Islet Transplantation

Antonioli¹,

Fermo

Cainarca

et al. 2007

Transplantation

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Background. Efficient islet isolation represents a necessary requirement for successful islet transplantation as a treatment for type 1 diabetes. The choice of collagenase for pancreas digestion is critical for the isolation outcome, and Liberase™ is the most widely used enzyme, although large intra-batched variability in activity and efficiency has been observed. Methods. The aim of this study was to characterize Liberase™ components and their relative role in pancreas digestion. Liberase batches were characterized by microelectrophoresis. Results. By means of microelectrophoresis, we identified three main proteins each with different prevalences between batches. Two proteins were found to correspond to class I (CI) and one to class II (CII) collagenase. In a series of 163 islet isolations, we observed that the CII correlated with islet yield (PϽ0.001) and digestion time (PϽ0.001); additionally, CI directly correlated with purity (Pϭ0.028). Finally, when CII and one of the CI isoforms were Ͼ50 percentile, 15 of 36 preparations were transplanted, with 27 of 127 transplanted in the other cases (Pϭ0.013). Conclusion. These results represent an important step toward the characterization of enzymes, with the final aim of identifying key components for a standardized product.

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Adjustment of the Ratio Between Collagenase Class II and I Improves Islet Isolation Outcome

Cited by 18 publications

References 6 publications

Enhancing the Success of Human Islet Isolation Through Optimization and Characterization of Pancreas Dissociation Enzyme

Enhancing the Success of Human Islet Isolation Through Optimization and Characterization of Pancreas Dissociation Enzyme

Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

Characterization of Collagenase Blend Enzymes for Human Islet Transplantation

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