*Purpose: A limitation to potential cure for diabetes through islet transplantation is insufficient islet equivalence (IEQ) from a single donor to achieve insulin independence in the recipient. We propose cryopreservation by vitrification that stabilizes islets at an ultralow temperature (<-150°C) and achieves pooling of high quality islets from multiple donors over time to attain IEQ for a successful transplant with insulin independence in the recipient.

*Methods: Mouse islets were isolated from C57BL/6 retired breeders, cultured in a bioreactor with optimized culture media at 37°C and 95% CO₂ for 16 hours. Cryoprotective Agent (CPA) was optimized & volumetric properties measured using microfluidics. Islets were loaded with CPA, vitrified-rewarmed (VR) & unloaded using a cryomesh. Post VR protocol optimization was performed at 0, 3 & 24h. Brightfield & Transmission Electron Microscopy (TEM), viability, apoptosis, ATP content, mitochondrial reactivation, mitochondrial stress/ Oxygen Consumption Rate (OCR), Glucose Stimulated Insulin Secretion (GSIS) were performed. HUES-8 Human Stem Cell Derived Beta Cells (SC-Beta), porcine, non-human primate (cynomolgus) & human isolated islets were also evaluated. Syngeneic C57BL/6 transplants were performed under kidney capsule and Blood Glucose (BG) monitored daily for 150 days. SC-beta, porcine and NHPs grafts were transplanted into Nod-Scid-Gamma mice. Recipients were subjected to an intra-peritoneal glucose stimulation test. Nephrectomies were performed on defined post-operative days & imaged using antibody labeling for insulin & glucagon. Volumetric assessments were analyzed using MATLAB 2018ba/2019a. R version 4.0.3 was used for statistical analyses (Shapiro-Wilk, Levene, Tukey, Games-Howell, Kruskal, Kaplan Meier tests). A p value of <0.05 was considered significant.

*Results: Viability of 90.5, 92.1, 87.2, 88.4, 87.4% was achieved with VR (~59% with conventional cryopreservation) for mouse, SC-Beta, porcine, NHP and human islets respectively at 3h of conditioning. Microscopy of VR islets show well preserved cellular architecture. Apoptosis, ATP content, mitochondrial reactivation, OCR, GSIS showed significant difference compared to conventional cryopreservation in all five species. 9/10 VR transplants (250 islets) normalized BG (1/10 had a partial cure) in 48h with good glycemic control for 150 days and thereafter with acceptable responses to glucose stress tests, compared to all conventional cryopreserved transplants (450 islets) that failed to normalize BG. Extended cryopreservation was assessed by vitrifying mouse islets for nine months and transplanted to have similar graft function and survival. Serum insulin from porcine & SC-Beta VR xenografts were comparable to controls.

*Conclusions: The optimized CPA loading/unloading, VR protocol has potential in pooling islets from multiple donors for a single islet transplant to cure diabetes.

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