EVALUATION OF A PURIFIED ENZYME BLEND FOR THE RECOVERY AND FUNCTION OF CANINE PANCREATIC-ISLETS

Recently developed technologies enabling the production of a reproduci ble, purified enzyme blend for optimal human pancreatic islet isolatio n has renewed interest in clinical islet transplantation. The canine m odel has been an ideal preclinical model for the development of islet transplantation protocols, As seen in other species, the application o f crude collagenase for isolating canine islets resulted in highly var iable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crud e collagenases with that of Liberase(TM)-CI purified enzyme blend for canine islet isolation, We also compared two manufacturing runs of Lib erase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and functio n of islets isolated using Liberase-CI enzyme. No difference in dog ag e, mean body weight, or pancreas weight were observed between the expe rimental groups, We observed a significantly higher postpurification r ecovery of islet equivalent number (IE) from pancreases processed usin g two lots of Liberase-CI enzyme (189 +/- 20 x 103 IE, n = 4) and lot 2 (234 +/- 39 x 10(3) IE, n = 7) than that obtained from pancreases pr ocessed with Sigma Type V (116.8 +/- 0.27 x 10(3) IE, n = 5), Serva co llagenase (49 +/- 11.6 x 10(3) IE, n = 5,p < 0.05) or Boehringer-Mannh eim (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 recove ry from pancreases processed using the two production lots of Liberase -CI enzyme. Islet survival after 48 h in culture at 37 degrees C was s ignificantly higher from islets isolated using Liberase-CI enzyme (88 +/- 3.7% survival) when compared to Sigma Type V (81.8 +/- 3.3%), Serv a (71.7 +/- 2.8%), and BM Type P (77 +/- 7.2%) (p < 0.05), Islet funct ional testing in vitro demonstrated islets isolated using crude collag enase had an increased insulin basal release and a reduced insulin sti mulated 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 purifi ed 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 formula ted for canine islet isolation, can consistently yield large numbers o f islets that survive longer in culture and demonstrate an improved in sulin response in vitro. (C) 1998 Elsevier Science Inc.