Robert C. McCarthy

A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products

Background. The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight 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), and 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 in place of 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 eight 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 islet equivalent number/gram (IEQ/g) pancreas and 694,681±147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 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 NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay.

Background. Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. Methods. Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. Results. These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100±150,900 islet equivalent number (IEQ) (4147±1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500±152,400 IEQ (2134±1524 IEQ/g pancreas). Conclusions. These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administrations oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

Background Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. Methods We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Results Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. Conclusions A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

Successful isolation and transplantation of nonhuman primate islets using a novel purified enzyme blend

Pancreatic islet transplantation has emerged as a viable treatment option for type 1 diabetes mellitus in humans, spurred on by recent improvements to immunosuppression protocols which have achieved similar rates of insulin independence after islet transplants as whole organ pancreas transplants (1, 2). Nonhuman primate (NHP) preclinical models are needed to test new therapeutic agents/treatments or to analyze the factors that affect islet engraftment. NHPs are the closest model to humans both phylogenetically and immunologically (3–9), but in view of ethical considerations and the limited availability of NHPs, it is critical that a sufficient number of high-quality islets are obtained from every processed donor pancreas (10, 11). Earlier studies showed that consistent islet isolation results were obtained with Liberase-HI (LHI; Roche Applied Sciences, Indianapolis, IN), anenzymeblendcontainingClostridiumhistolyticum class I and class II collagenase, and thermolysin, a bacterial neutral protease isolated from Bacillus thermoproteolyticus (8, 12). After 2007, this product was no longer manufactured because of the potential risk of transmissible spongiform encephalopathy (13), leaving many isolation groups to find another source of purified enzymes capable of producing consistently high islet yields from NHPs. In this study, we carefully customized the optimal composition and ratio of collagenase and thermolysin enzymes to maximize islet yield from NHPs using our experience from human and porcine islet isolations. After careful selection, we tested the efficacy of a new purified enzyme blend (PEB; VitaCyte’s CIzyme Collagenase MA and CIzyme Thermolysin) and compared our results to earlier isolation outcomes obtained with LHI. NHP pancreatic islet isolations (n 16) were performed at our center, and the donor pancreases were obtained from male, rhesus macaques (Macaca mulatta) averaging 13.7 1.5 kg body weight and 26.9 9.8gpancreasweight.Theenzymesolution was prepared by combining one vial of Collagenase MA (750 Wunsch units) and one vial of Thermolysin (1.69 million neutralproteaseunits)andbringingtoavolume of 125 mL with phase 1 solution containing heparin(10,000units/L).Enzymedistention wasperformedbymanuallyinjecting2to2.5 mL of enzyme solution per gram of pancreas. The tissue was digested using a 250-mL Ricordi chamber and a modified semiautomated digestion method previously described (14). Isolated islets were purified by isopycnic gradient separation on a COBE2991cellprocessor(COBELaboratories, Lakewood, CO) using continuous iodixanol (OptiPrep; Axis-Shield, Oslo, Norway) gradients (1.030–1.110 g/cm). The purified islets were cultured in CMRL1066 supplemented media for approximately 92 hr before transplantation. On the day of transplant, islet samples were taken for counting by DNA measurement. Finally, results from these isolations were compared with those isolated with LHI, and a statistical analysis was performed by Student’s t test.Table1showstheisolationcharacteristics and outcomes with islet yield presented as islet equivalents (IEQ). The PEB consistently provided high islet yields in NHP islet isolations, averaging 4405 1922 IEQ/g pancreas postdigest and 443

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM‐1200 WU rC2 and 10 million collagen‐degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100‐g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100‐g pancreas. Low‐dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low‐dose NCEM (12WU/g). Additionally, low‐dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 μU/mL; P < .05) compared with standard‐dose NCEM. Nude mouse bioassay demonstrated better islet function for low‐dose RCEM (area under the curve [AUC] 24 968) compared with low‐dose (AUC–38 225) or standard‐dose NCEM (AUC–38 685), P < .05. This is the first report indicating that islet function can be improved by using low‐dose rC1rC2 (RCEM).

Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets

ABSTRACT One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase‐protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a “trisected” pancreas model to overcome the donor‐to‐donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase‐protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

A NEW ENZYME MIXTURE TO CONSISTENTLY ACHIEVE HIGH HUMAN ISLET YIELD AND IMPROVED ALLOGENIC AND AUTOGRAFT ISLET TRANSPLANTATION OUTCOME: 3390

A.N. Balamurugan1, G. Loganathan1, M. Bellin1, J.J. Wilhelm1, J. Harmon1, T. Anazawa2, D. Radosevich3, T. Yuasa1, M. Tiwari1, K.K. Papas1, R.C. Mccarthy4, D.E. Sutherland1, B.J. Hering2 1Surgery, Schulze Diabetes Institute, Minneapolis/MN/UNITED STATES OF AMERICA, 2Surgery, Schulze Diabetes Institute, Minneapolis/UNITED STATES OF AMERICA, 3Surgery, University of Minnesota, Minneapolis/MN/UNITED STATES OF AMERICA, 4R&d, VitaCyte LLC, Indianapolis/IN/UNITED STATES OF AMERICA