Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling

Background All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. Methods Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose stimulated insulin secretion as a measure of β cell function and RNA sequencing to elucidate transcriptomic changes. Results Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. Conclusions These data demonstrate that pancreas preservation by persufflation rather than SCS prior to islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved β cell function. Corresponding Author: Klearchos K. Papas, PhD, Institute for Cellular Transplantation, Department of Surgery, University of Arizona, Medical Research Building, 1656 E. Mabel Street, Room 122, Tucson, AZ 85724, Tel: (520) 626-5853, Fax: (520) 626-3770. E-Mail: kkpapas@surgery.arizona.edu Author Contributions K.K.P., A.C.K., K.E.S., S.W.L, and R.M.L. designed the study. Pancreas persufflation and islet isolations were conducted by C.G.M., J.J., K.E.S., N.D.P, D.S.M., I.G.G., T.L., C.P.H, S.P., W.E.S., D.M., J.A.S., D.O., T.K., G.L.S., A.M.P., P.G.S., T.K., W.J.S., B.P.W., and R.C.H. Functional islet assessments at the University of Arizona were conducted by K.E.S., C.W., W.G.P., C.G.M., D.S.M., and N.D.P. Technical aspects of sequencing and subsequent data analysis were conducted by A.C.K., A.M.C, L.E.C., F.M.M., R.M.L., and S.W.L. Traditional RNA quantification was conducted by A.C.K., K.E.S., L.E.C., M.J.A. All authors contributed to data interpretation, reviewed the manuscript and approved the final version. Funding This work was supported by the National Institutes of Health grants NIH/SBIR 5R44DK070400-04 (K.K.P., Co-Principal Investigator with Dr. Linda Templeman, Giner Inc.) and R01DK084842 (S.W.L., Principal Investigator) and NIH/NIDDK DP3 grant 1DP3DK106933-01 (K.K.P., Principal Investigator). Duality of Interest Klearchos K. Papas, PhD served as Consultant/ Chair SAB Giner Inc, is a co-inventor on pending patents related to persufflation with University of Minnesota and Giner Inc., and was the PI or co-PI on the NIH/NIDDK grants that partially funded the work. He is no longer affiliated with Giner Inc. "The remaining authors declare no conflicts of interest". Guarantee K.K.P. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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