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