Scientific Director of Islet Transplant Program
Department of Surgery
University of Louisville
Improving porcine islet yield with new recombinant class I (rC1) and class II (rC2) collagenases in combination with BP Protease, an animal-free enzyme mixture.
Balamurugan Appakalai1,2, Michael L Green3, Andrew G Breite3, Gopalakrishnan Loganathan1,2, Joshua J Wilhelm1, Jeffrey D Ansite1, Thomas Gilmore1, Benjamin T Tweed2, Bernhard J Hering1, Francis E Dwulet3, Robert C McCarthy3.
1Department of Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 2Department of Surgery, Clinical Islet Cell Laboratory, Cardiovascular Innovation Institute, University of Louisville, Louisville, MN, United States; 3VitaCyte LLC, Indianapolis, MN, United States
Background: Isolating quality islets from the porcine pancreas is difficult due to the intrinsic fragility of porcine islets. Besides donor characteristics and procurement techniques, the composition and ratio of digestive enzyme blends are important determinants for maximizing islet yield and viability. Recently, we successfully manufactured recombinant class I (rC1) and class II (rC2) collagenases and tested the islet isolation efficacy utilizing 16 human pancreases. Varying amounts of rC1 and rC2 , in combination with BP-protease (a dispase equivalent neutral protease), were tested using a statistically designed experiment (DOE) in a split-pancreas model. Results were compared with isolations performed using natural collagenase (C. histolyticum collagenase) and neutral protease. In this study, we successfully utilized recombinant rC1 and rC2 collagenases to improve porcine islet yield.
Materials and Methods: Donor selection and pancreas procurement was done according to institutional protocols. An enzyme blend consisting of rC1rC2 and BP-Protease (VitaCyte, Indianapolis, IN) was evaluated in four minipigs using our standard porcine islet processing and quality control protocols. Islet isolation results using the rC1rC2 and BP-rotease combination were compared with results of porcine isolations performed with the historical enzyme blend (n=4). Islet quality was assessed with viability (FDA/PI), insulin secretion (GSIR), and oxygen consumption rat (OCR) assay measurements. All statistical comparisons were made using student’s t-test.
Results: The results are summarized in the table below. The islet morphology, acinar cell releaing pattern, digestion time and other digestion profiles were normal when compared to traditional enzyme formulations. At digest level, the total islet yield was higher for the rC1rC2 combination when compared to the historical enzyme blend (P<0.0191). However, the final islet yield per gram pancreas after purification was not statistically significant compared to historical isolations (2018 ± 865 vs 1331 ± 333, respectively P<0.0188), possibly due to the small sample size. The OCR/DNA results indicate a very high fractional viability of islets (>175) processed with the new animal-free enzyme mixture.
Conclusion: These preliminary findings suggest that the new rC1rC2 collagenase in combination with BP-Protease facilitate porcine islet processing with results comparable to those previously achievable with our historical enzyme combination. Continued evaluation of donor factors and enzyme ratio optimization are underway to further improve the quality of porcine islet therapy products. By utilizing different ratios of rC1:rC2, it is possible to obtain high islet yields from young and adult porcine donors.
Muhamad Abdullah. Kate Mueller. David Heller.
 Anazawa T, Balamurugan AN, Matsumoto S, Lafreniere SA, O'Brien TD, Sutherland DE, Hering BJ. Rapid quantitative assessment of the pig pancreas biopsy predicts islet yield. Transplant Proc. 2010 Jul-Aug; 42(6): 2036-9.
 Anazawa T, Balamurugan AN, Papas KK, Avgoustiniatos ES, Ferrer J, Matsumoto S, Sutherland DE, Hering BJ. Improved method of porcine pancreas procurement with arterial flush and ductal injection enhances islet isolation outcome. Transplant Proc. 2010 Jul-Aug; 42(6): 2032-5.
 Papas KK, Suszynski TM, Colton CK. Islet assessment for transplantation. Curr Opin Organ Transplant. 2009 Dec;14(6):674-82.
13:00 - 16:15
|Islet Isolation and Transplantation: where are we now and where are we going?||Which donor and isolation factors predict successful islet isolations and successful islet transplant outcomes?||Plenary Room 1|
11:00 - 12:30
|Islet Xenotransplantation||Improving porcine islet yield with new recombinant class I (rC1) and class II (rC2) collagenases in combination with BP Protease, an animal-free enzyme mixture.||Room 109|
17:30 - 18:30
|Joint Poster Session||Modified islet isolation techniques can isolate high islet yield even from alcoholic pancreatitis pancreases intended for clinical islet auto-transplantion||Main Foyer 1 & 2|