P-195

A comparison of islets isolated from neonatal pigs using Sigma and Vitacyte tissue dissociation enzymes

K. Bayrack¹, B.B.F. Alavi¹, D. Mihalicz¹, A. Breite², R. McCarthy², R. Rajotte¹, G. Rayat^1
1 University of Alberta, Surgery, Edmonton, Canada; ² Vitacyte LLC, Indianapolis, USA

Objective: Tissue dissociation enzymes such ascollagenase are an important component in the isolation of islets because they affect the yield and quality of islets that will be used for transplantation. In this study, we compared the yield and in vivo function of neonatal porcine islets isolated using tissue dissociation enzyme from Sigma and Vitacyte.

Methods: Islets were isolated from 3-day-old neonatal pigs following our standard procedure using enzymes from Sigma (collagenase Type XI at 1 mg/ml, n=6) or Vitacyte (0.26 mg/ml collagenase and 0.02 mg/ml BP protease, n=6). The isolated islets were cultured in HAM’s F10 medium and purified using our standard protocol. The amount of islets isolated per pancreas was determined and some islets were placed inside an alginate microcapsule. Two thousand islet equivalents (IEQ) of non-encapsulated or encapsulated neonatal porcine islets were transplanted under the kidney capsule or into the peritoneum of streptozotocin-induced diabetic immune-deficient NOD.SCID or B6 rag-/- mice, respectively. The blood glucose levels of these mice were measured two times a week for >150 days post-transplantation.

Results: The yield of islets isolated using tissue dissociation enzymes from Sigma and Vitacyte were comparable (26,794±4,394 vs. 26,182±2,293 IEQ, respectively). Two of four mice that received non-encapsulated islets isolated using Sigma enzymes achieved normoglycemia at 78 and 81 days post-transplantation and the two mice are still diabetic (116 days post-transplantation). While 8/8 mice that received non-encapsulated islets isolated using Vitacyte enzymes achieved normoglycemia within 30 to 96 days post-transplantation. Mice (n=3) that received encapsulated islets have not achieved normoglycemia as it has only been 14 days after transplantation but the blood glucose levels were at 13.4, 14.8 and 27.9 mmol/l (Sigma) and 11.8, 14.7 and 20.6 mmol/l (Vitacyte).

Conclusion: These results indicate that Vitacyte tissue dissociation enzymes could be used effectively in the isolation of neonatal porcine islets.

P-195

A comparison of islets isolated from neonatal pigs using Sigma and Vitacyte tissue dissociation enzymes

K. Bayrack¹, B.B.F. Alavi¹, D. Mihalicz¹, A. Breite², R. McCarthy², R. Rajotte¹, G. Rayat^1
1 University of Alberta, Surgery, Edmonton, Canada; ² Vitacyte LLC, Indianapolis, USA

Objective: Tissue dissociation enzymes such ascollagenase are an important component in the isolation of islets because they affect the yield and quality of islets that will be used for transplantation. In this study, we compared the yield and in vivo function of neonatal porcine islets isolated using tissue dissociation enzyme from Sigma and Vitacyte.

Methods: Islets were isolated from 3-day-old neonatal pigs following our standard procedure using enzymes from Sigma (collagenase Type XI at 1 mg/ml, n=6) or Vitacyte (0.26 mg/ml collagenase and 0.02 mg/ml BP protease, n=6). The isolated islets were cultured in HAM’s F10 medium and purified using our standard protocol. The amount of islets isolated per pancreas was determined and some islets were placed inside an alginate microcapsule. Two thousand islet equivalents (IEQ) of non-encapsulated or encapsulated neonatal porcine islets were transplanted under the kidney capsule or into the peritoneum of streptozotocin-induced diabetic immune-deficient NOD.SCID or B6 rag-/- mice, respectively. The blood glucose levels of these mice were measured two times a week for >150 days post-transplantation.

Results: The yield of islets isolated using tissue dissociation enzymes from Sigma and Vitacyte were comparable (26,794±4,394 vs. 26,182±2,293 IEQ, respectively). Two of four mice that received non-encapsulated islets isolated using Sigma enzymes achieved normoglycemia at 78 and 81 days post-transplantation and the two mice are still diabetic (116 days post-transplantation). While 8/8 mice that received non-encapsulated islets isolated using Vitacyte enzymes achieved normoglycemia within 30 to 96 days post-transplantation. Mice (n=3) that received encapsulated islets have not achieved normoglycemia as it has only been 14 days after transplantation but the blood glucose levels were at 13.4, 14.8 and 27.9 mmol/l (Sigma) and 11.8, 14.7 and 20.6 mmol/l (Vitacyte).

Conclusion: These results indicate that Vitacyte tissue dissociation enzymes could be used effectively in the isolation of neonatal porcine islets.