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Presenter: Efstathios, Avgoustiniatos, Lauderdale, United States
Authors: Efstathios S. Avgoustiniatos1, Phillip R. Rozak1, Kate R. Mueller1, Connor A. Lyons1, William E. Scott III1, Eric J. Falde1, Jennifer P. Kitzmann1, John R. Wilson2, Bernhard J. Hering1, Klearchos K. Papas1
Silicone rubber membrane devices allow islet culture at 20 times the standard surface density with no adverse effects on viability, recovery, or potency
Efstathios S. Avgoustiniatos1, Phillip R. Rozak1, Kate R. Mueller1, Connor A. Lyons1, William E. Scott III1, Eric J. Falde1, Jennifer P. Kitzmann1, John R. Wilson2, Bernhard J. Hering1, Klearchos K. Papas1
1Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis; 2Wilson Wolf Manufacturing, New Brighton, MN, United States
Introduction: Islet culture in T-flasks requires seeding the islets at surface densities below 200 IE/cm2 to provide sufficient oxygenation through the media. More than 20 T-175 flasks are needed for large porcine or human islet preparations, especially when impure. Culturing islets on top of gas-permeable (GP) silicone rubber membranes increases proximity of islets to ambient oxygen and allows entire preparations to be cultured in 1-2 flasks. In this work we summarize the characterization of 2-day porcine islet culture in GP silicone rubber devices (Wilson Wolf, New Brighton, MN).
Methods: Porcine islets from 30 donors were cultured for 2 days along with standard culture (T-flask, 200 IE/cm2, 1000 IE/mL) paired controls. Conditions were seeded at 100-4000 IE/cm2 measured by DNA (1 IE = 10.4 ng DNA). The primary outcomes were islet viability, measured by oxygen consumption rate to DNA ratio (OCR/DNA), DNA recovery, viable tissue (OCR) recovery, and islet potency, assessed with the diabetic nude mouse bioassay (2000 IE under the kidney capsule).
Results: For GP devices, OCR/DNA, DNA recovery, and OCR recovery were similar to paired controls, even at 4000 IE/cm2. In contrast, all these outcomes deteriorated with increasing surface density for non-GP devices. For surface densities over 1000 IE/cm2 and relative to paired controls, OCR/DNA was 58 ± 6% (mean ± SEM, n = 13, P = 1.1x10-5) for non-GP devices and 95 ± 4% (n = 25, P = 0.27) for GP devices; DNA recovery was 67 ± 6% (n = 8, P = 2.2x10-4) for non-GP devices and 114 ± 10% (n = 12, P = 0.21) for GP devices; and OCR recovery was 34 ± 4% (n = 8, P = 8.2x10-7) for non-GP devices and 113 ± 14% (n = 12, P = 0.36) for GP devices. Islets from 5 preparations cultured at 1000 or 4000 IE/cm2 in non-GP and GP devices were transplanted into nude diabetic mice. Diabetes reversal rates were similar for islets cultured at high densities in GP devices (14/16) as for paired control islets (14/15). In sharp contrast, only 1 of 13 mice (P=1.0x10-5) exhibited delayed diabetes reversal after being transplanted with islets cultured at high densities in non-GP devices.
Conclusion: Culturing islets in GP devices allows the increase of islet seeding density from 200 to 4000 IE/cm2 while maintaining islet viability, recovery, and potency relative to paired standard controls. Islets cultured at similar high surface densities in non-GP devices exhibit decreased viability and recovery and fail to reverse diabetes in nude mice.
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