Characterization of Collagenase Blend Enzymes for Human Islet Transplantation

Antonioli, Barbara; Fermo, Isabella; Cainarca, Silvia; Marzorati, Simona; Nano, Rita; Baldissera, Michela; Bachi, Ángela; Paroni, Rita; Ricordi, Camillo; Bertuzzi, Federico

doi:10.1097/01.tp.0000295719.88525.60

Cited by 33 publications

(29 citation statements)

References 26 publications

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“…Variations in enzyme content between and within lots of collagenases, even in purified preparations, have been reported by several groups. 43–46 We speculate that higher concentrations of collagenase in M2 compared with M1 might have led to a more pronounced effect of other existing enzymes in the collagenase preparations such as clostripain, trypsin, and neutral proteases (specified by the manufacturer) on islet isolation, contributing to better yields in some isolations or resulting in lower yields in others, due to overdigestion, as previously reported. 47,48 For human islet isolations, low amounts of endogenous pancreatic enzymes can remain active throughout the isolation process 49 ; if this occurs in canine islet isolations, it might have also influenced in part our results.…”

Section: Discussionmentioning

confidence: 59%

Simplified Method to Isolate Highly Pure Canine Pancreatic Islets

Woolcott

Bergman²,

Richey³

et al. 2012

Pancreas

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Objectives The canine model has been used extensively to improve the human pancreatic islet isolation technique. At the functional level, dog islets show high similarity to human islets and thus can be a helpful tool for islet research. We describe and compare 2 manual isolation methods, M1 (initial) and M2 (modified), and analyze the variables associated with the outcomes, including islet yield, purity, and glucose-stimulated insulin secretion (GSIS). Methods Male mongrel dogs were used in the study. M2 (n = 7) included higher collagenase concentration, shorter digestion time, faster shaking speed, colder purification temperature, and higher differential density gradient than M1 (n = 7). Results Islet yield was similar between methods (3111.0 ± 309.1 and 3155.8 ± 644.5 islets/g, M1 and M2, respectively; P = 0.951). Pancreas weight and purity together were directly associated with the yield (adjusted R2 = 0.61; P = 0.002). Purity was considerably improved with M2 (96.7% ± 1.2% vs 75.0% ± 6.3%; P = 0.006). M2 improved GSIS (P = 0.021). Independently, digestion time was inversely associated with GSIS. Conclusions We describe an isolation method (M2) to obtain a highly pure yield of dog islets with adequate β-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans.

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Section: Discussionmentioning

confidence: 59%

Simplified Method to Isolate Highly Pure Canine Pancreatic Islets

Woolcott

Bergman²,

Richey³

et al. 2012

Pancreas

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“…Two major factors that affect human islet yields are donor organ characteristics and tissue dissociation enzymes used for islet isolation (8–10). Determining suitable enzyme blends—specifically the combination, concentration, and optimal ratio of collagenases and proteases—is critical for obtaining proper islet release from the surrounding acinar tissue (11–13). …”

Section: Introductionmentioning

confidence: 99%

A New Enzyme Mixture to Increase the Yield and Transplant Rate of Autologous and Allogeneic Human Islet Products

et al. 2012

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Background The optimal enzyme blend which maximizes human islets yield for transplantation remains to be determined. In this study, we evaluated 8 different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact, or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch) as well as neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). Methods We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP instead of using thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from 8 different Ecs were statistically compared using multivariate analysis. Results The NEM consistently achieved higher islet yields from pancreatitis (p<0.003) and deceased donor pancreases (p<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510±2150 IEQ/g pancreas and 694,681±147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded >200,000 IEQ from 19 of 21 pancreases (averaging 422,893±181,329 total IEQ and 5979±1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. Conclusions A new enzyme mixture composed of Clostridium histolyticum neutral protease with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

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“…1 Three important factors contributed to this success. First, an improvement in the islet isolation procedure, by use of highly purified collagenase in conjunction with thermolysin or neutral protease followed by purification with continuous density gradients, which yielded an improvement in islet quantity and quality [2][3][4] and in turn, enabled a positive clinical outcome in some patients with the use of 1-3 cadaveric pancreata. Second, was the application of low-dose immunosuppressive regiments to prevent allograft rejection.…”

Section: Introductionmentioning

confidence: 99%