Sajjad M. Soltani

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.

Prediction of pancreatic tissue densities by an analytical test gradient system before purification maximizes human islet recovery for islet autotransplantation/allotransplantation.

Background. Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. Methods. Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. Results. Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. Conclusions. Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.

Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of α-1 antitrypsin treatment.

Background. Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of &agr;-1 antitrypsin (A1AT). Methods. Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and &bgr;-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets. Results. Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced &bgr;-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity. Conclusion. Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.

Factors affecting transplant outcomes in diabetic nude mice receiving human, porcine, and nonhuman primate islets: Analysis of 335 transplantations

Background In the absence of a reliable islet potency assay, nude mice (NM) transplantation is the criterion standard to assess islet quality for clinical transplantation. There are factors other than islet quality that affect the transplant outcome. Methods Here, we analyzed the transplant outcomes in 335 NM receiving islets from human (n=103), porcine (n=205), and nonhuman primate (NHP; n=27) donors. The islets (750, 1000, and 2000 islet equivalents [IEQ]) were transplanted under the kidney capsule of streptozotocin-induced diabetic NM. Results The proportion of mice that achieved normoglycemia was significantly higher in the group implanted with 2000 IEQ of human, porcine, or NHP islets (75% normoglycemic) versus groups that were implanted with 750 IEQ (7% normoglycemic) and 1000 IEQ (30% normoglycemic). In this study, we observed that the purity of porcine islet preparations (P⩽0.001), islet pellet size in porcine preparations (P⩽ 0.01), and mice recipient body weight for human islet preparations (P=0.013) were independently associated with successful transplant outcome. NHP islets of 1000 IEQ were sufficient to achieve normoglycemic condition (83%). An islet mass of 2000 IEQ, high islet purity, increased recipient body weight, and high islet pellet volume increased the likelihood of successful reversal of diabetes in transplanted mice. Also, higher insulin secretory status of islets at basal stimulus was associated with a reduced mouse cure rate. The cumulative incidence of graft failure was significantly greater in human islets (56.12%) compared with porcine islets (35.57%; P⩽0.001). Conclusion Factors affecting NM bioassay were identified (islet mass, islet purity, pellet size, in vitro insulin secretory capability, and mouse recipient body weight) and should be considered when evaluating islet function.

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