Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend (2011)

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), an enzyme blend containing Clostridium histolyticum 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±9.8 g pancreas weight. The enzyme solution was prepared by combining one vial of Collagenase MA (750 Wunsch units) and one vial of Thermolysin (1.69 million neutral protease units) and bringing to a volume of 125 mL with phase 1 solution containing heparin (10,000 units/L). Enzyme distention was performed by manually injecting 2 to 2.5 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 COBE 2991 cell processor (COBE Laboratories, Lakewood, CO) using continuous iodixanol (OptiPrep; Axis-Shield, Oslo, Norway) gradients (1.030–1.110 g/cm3). The purified islets were cultured in CMRL-1066 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. Table 1 shows the isolation characteristics 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 4430±1622 IEQ/g pancreas postculture on the day of transplant. The percent recovery after COBE purification was 87.1%±36.7% with an average purity of 83%±7%. The minimum of 5000 IEQ/kg recipient body weight for transplantation was achieved in 10 of 16 NHP isolations (failed isolations were due to smaller donor pancreas) with an average of 8110±2398 IEQ/kg per transplant. One recipient received a second infusion of 4604 IEQ/kg receiving a total of 102,847 IEQ (10,024 IEQ/kg).TABLE 1: Nonhuman primate islet isolation outcomes by enzyme preparationPreviously, LHI was used to isolate islets from 34 rhesus macaques and 10 baboons. A statistical comparison of the results from NHP isolations using LHI or PEB enzymes showed no significant differences. Table 1 illustrates the digest IEQ/g, culture recovery percentage by DNA, postpurification IEQ/g, and postculture IEQ/g results with relative P values. Islet yields achieved with the PEB (3550±1313 IEQ/g pancreas) were comparable to isolations with LHI on rhesus macaques (3306±1818 IEQ/g, P=0.82) and on baboons (2813±1763 IEQ/g, P=0.25). Quality control data showed that the average oxygen consumption rates by DNA (OCR/DNA) was 188.2±37.4 nmol/min/mg (n=4); stimulation index calculated from glucose-stimulated insulin release was 1.96 (n=3). The conclusions are limited, however, by the lack of available islets to establish a quality control assessment across all the groups being compared. Table 2 shows the transplant recipient characteristics and outcomes. Consistent with the criteria outlined by the International Xenotransplantation Association consensus statement for conduct of preclinical trials, the diabetic state was confirmed by a fasting C-peptide below 0.5 ng/mL or one third the value of the preinduction period, and the absence of a stimulated C-peptide response during an intravenous glucose tolerance test or arginine stimulation test (<0.3 ng/mL) in combination with persistent hyperglycemia (blood glucose ≥200 mg/dL) before the initiation of exogenous insulin. Diabetes induction was only considered successful if the diabetic state was documented, and there were no complications that prevented enrollment onto a subsequent transplant study.TABLE 2: Recipient characteristics and posttransplant engraftment by enzyme preparationEvaluation of engraftment is important to assess the success of islet transplantation. We found that fasting blood C-peptide and glucose levels correlated well after islet transplantation, and C-peptide to glucose ratio (C-peptide×100/blood glucose) is predictive of long-term function. PEB was slightly superior to LHI using the C-peptide to glucose ratio as a measure of engraftment and by percent insulin independent at 30 days. The conclusion generated as a result of the analysis, summarized in Table 2, should be interpreted with caution, because different immunosuppressive protocols were used across the groups, providing an important confounding factor for the analysis of the data. Several methods of pancreatic islet transplantation have been tested in various animal models. However, the limited availability of NHP tissue prohibits expansive experiments in this field. The PEB not only consistently produced a high islet yield but also resulted in successful allograft transplants. These findings indicate that the optimal ratio of collagenase to thermolysin was identified and that the PEB is an effective enzyme blend for NHP islet isolation. Janine Abouaish 1 Melanie Graham 1 Pratima Bansal-Pakala 1 Gopalakrishnan Loganathan 1 Sajjad M. Soltani 1 Mukesh Tiwari 1 Takeshi Yuasa 1 Klearchos K. Papas 1 David E. R. Sutherland 1 Robert C. McCarthy 2 Bernhard J. Hering 1 A. N. Balamurugan 1 1Department of Surgery Schulze Diabetes Institute University of Minnesota Minneapolis, MN 2VitaCyte LLC Indianapolis, IN