406 High human islet yield with recombinant class I (rC1) and class II (rC2) collagenases in combination with BP-Protease: Efficacy of a low dose of an animal-free enzyme mixture on human pancreas digestion
Tuesday November 17, 2015 from 07:00 to 08:00
Room 110

Michael G. Hughes, United States

Assistant Professor of Surgery

Department of Surgery; Division of Transplant Surgery

University of Louisville/Jewish Hospital


High human islet yield with recombinant class I (rC1) and class II (rC2) collagenases in combination with BP-Protease: Efficacy of a low dose of an animal-free enzyme mixture on human pancreas digestion

Benjamin Tweed1, Gopalakrishnan Loganathan1, Michael G Hughes1, Stuart K Williams1, Michael L Green2, Andrew G Breite2, Francis E Dwulet2, Robert C McCarthy2, Balamurugan Appakalai1.

1Department of Surgery, Clinical Islet Cell Laboratory, Cardiovascular Innovation Institute, University of Louisville, Louisville, KY, United States; 2VitaCyte LLC, Indianapolis, IN, United States

Background: The dose and composition of the enzymes used in the islet isolation process is a critical factor that impacts the number and quality of islets released from tissue. Recently, we successfully manufactured recombinant class I (rC1) and class II (rC2) collagenases and tested their efficacy for isolating islets utilizing 16 human pancreases.  Low and high target doses of rC1 and rC2, in combination with BP-protease, were tested using a statistically designed experiment (DOE) in a split-pancreas model. Four different enzyme formulations with divergent C1:C2 collagenase mass ratios (about 27:73, 38:62, 43:57, and 55:45) were assessed for their effectiveness to recover functional human islets while keeping the neutral protease activity constant. Results from the DOE indicated that isolation outcomes and islet functional data were similar among all formulations tested, suggesting enzyme target activities could be dramatically reduced without a consequential effect on isolation outcome.  In the present study, we have selected and tested the low activity target formulation (rC1rC2; 100,000 CDA Unit/g, 12 Wunsch Unit/g) in whole human research pancreases.
Methods: Human islets were isolated according to our institution's standard clinical islet isolation protocol using four deceased donors. Low dose (rC1rC2; 100,000 CDA Unit/g, 12 Wunsch Unit/g) rC1rC2 enzyme  was prepared in Hank’s balanced salt solution (350 ml) and injected into the pancreatic duct. Pancreas digestion profiles were carefully monitored and compared to isolations performed with traditional enzyme formulations (>200,000 CDA U/g, 20 Wunsch U/g). Islet cell viability was assessed by FDA/PI after overnight culture.
Results: Our results confirmed the effectiveness of BP-Protease in combination with recombinantly-sourced collagenase for isolating islets from whole human pancreases. The digestion profiles, including the digestion times (19.7±0.6), were normal, although we used enzyme target activities that were only 50-60% those of a traditional formulation. Islet morphology, acinar cell release profiles, and percentage of undigested pancreatic tissue were similar to standard islet isolation.  Islet yield (IEQ/gram pancreas) at pre- and post-purification was 5,318 ± 718 and 4,553 ± 825, respectively, for the low dose rC1rC2 enzyme blend.  Islet viability was >90% in all isolations.
Conclusion: A lower dose recombinant collagenase in combination with BP-protease was successfully used to recover >5000 IEQ/g tissue from four consecutive islet isolations using research grade human pancreases.  Results from this small set of isolations indicate the effectiveness of this animal-free enzyme mixture to recover islets at doses of collagenase that are ≈50% of those used commonly for human islet isolation. By utilizing different ratios of rC1:rC2, it is possible to obtain high islet yields from young donors and sub-optimal pancreases.


© 2018 Melbourne2015