Evaluation of a purified enzyme blend for the recovery and function of canine pancreatic islets.

Abstract

Recently developed technologies enabling the production of a reproducible, purified enzyme blend for optimal human pancreatic islet isolation has renewed interest in clinical islet transplantation. The canine model has been an ideal preclinical model for the development of islet transplantation protocols. As seen in other species, the application of crude collagenase for isolating canine islets resulted in highly variable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crude collagenases with that of Liberase-CI purified enzyme blend for canine islet isolation. We also compared two manufacturing runs of Liberase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and function of islets isolated using Liberase-CI enzyme. No difference in dog age, mean body weight, or pancreas weight were observed between the experimental groups. We observed a significantly higher postpurification recovery of islet equivalent number (IE) from pancreases processed using two lots of Liberase-CI enzyme (189 +/- 20 x 10(3) IE, n = 4) and lot 2 (234 +/- 39 x 10(3) IE, n = 7) than that obtained from pancreases processed with Sigma Type V (116.8 +/- 27 x 10(3) IE, n = 5), Serva collagenase (49 +/- 11.6 x 10(3) IE, n = 5, p < 0.05) or Boehringer-Mannheim (BM) Type P collagenase (85.4 +/- 25 x 10(3) IE, n = 5, p < 0.05, ANOVA). No significant differences were observed in islet yield recovery from pancreases processed using the two production lots of Liberase-CI enzyme. Islet survival after 48 h in culture at 37 degrees C was significantly higher from islets isolated using Liberase-CI enzyme (88 +/- 3.7% survival) when compared to Sigma Type V (81.8 +/- 3.3%), Serva (71.7 +/- 2.8%), and BM Type P (77 +/- 7.2%) (p < 0.05). Islet functional testing in vitro demonstrated islets isolated using crude collagenase had an increased insulin basal release and a reduced insulin stimulated response when compared with islets isolated using the two lots of Liberase-CI enzyme. The calculated stimulation index was 7.8 +/- 1.7, 3.1 +/- 0.6, and 4.8 +/- 1.1 for Sigma Type V, Serva, and BM Type P isolated islets, respectively, compared to 15.7 +/- 1.6 and 16.2 +/- 1.9 for islets isolated with Liberase-CI enzyme production lots 1 and 2, respectively (p < 0.05). This evaluation demonstrates that a purified enzyme blend can significantly improve islet recovery and function. It also demonstrates the manufacturing reproducibility of Liberase-CI enzyme lots resulting in the isolation of canine islets with the same degree of efficacy. A blend of purified enzymes, specifically formulated for canine islet isolation, can consistently yield large numbers of islets that survive longer in culture and demonstrate an improved insulin response in vitro.