Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24–48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.

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Introduction: Beta cells have been successfully differentiated from human pluripotent stem cells (hPSCs) by mimicking the pancreatic developmental process, provide a virtually unlimited source of cells for curative diabetes treatment. Current protocols to generate hPSC-derived insulin-producing cells (IPCs) consist of exogenous non-physiological high glucose levels (≥20 mM) for the final steps. These glucose levels have been considering a key contributor to cell fate definition and maturation. However, long-term exposure to high glucose levels has been linked to beta-cell dedifferentiation and less robust glucose-stimulated insulin secretion (GSIS) in human islets in vitro. This study aims to investigate hiPSCs to IPCs differentiation under physiological glucose (5.5mM) compared with a high glucose level (20mM) after the pancreas progenitor stage. Methods: The hiPSC lines (Babk2, WTC11, INSULIN-H2B-Cherry (InsCherry)) were differentiated following the seven-stages protocol (Rezania et al., 2014) illustrated in Figure 1A. Stage 6 cells were differentiated under 20mM or 5.5mM glucose, followed by culturing in stage 7 media contains 5.5mM glucose, and then cultured in the same media in suspension for 7-10 days (Stage 7+ cells) until collecting for GSIS assay, oxygen consumption rate (OCR) measurement, and calcium flux analyzing. Stage 6 cells were analyzed by immunofluorescences staining. Stage 6 and Stage 7+ cells were collected for gene expressions and mitochondria contents analysis. Results: Stage 6 Cells (Babk2 and WTC11) differentiated under 20mM glucose showed significantly reduced NKX6.1 gene expression and co-localization of PDX1+/NKX6.1+ (Figures 1B and C, P<0.001, n=5). For osmotic pressure control, the cells under 5.5mM media were supplemented with mannitol which supports a direct effect of the glucose (Figure 1C). The GSIS showed no functionality in either 20 mM or 5.5mM glucose differentiated cells (Figure 1E). But differentiated InsCherry-iPS in 20mM glucose obtained functionality demonstrated by the GSIS (Figures 1D and F). The disallowed gene LDHA was more successfully suppressed in 20mM condition (Figure 1G), and K ATP channel subunits gene ABCC8 was increased in high glucose-induced differentiation from stage 6 to 7+ (Figure 1H). Calcium flux analysis showed an improved K ATP channel activity in high glucose-induced cells (Figure 1I). The mitochondrial biogenesis was suppressed temporally in stage 6 cells exposed to 20 mM (Figure 1J). The OCR analysis suggests that high glucose negatively impacts mitochondrial spare respiratory capacity (Figure 1K). Conclusion:The impact of high glucose levels on IPC differentiation is complex and cell line dependent. We found that high glucose level benefits K ATP channel activity and helps obtain functionality of the differentiated cells. In contrast, it suppresses mitochondrial respiration ability. Our study supports the use of high glucose for IPC generation, but this seems to be on behalf of mitochondrial respiration ability. New and better solutions to bypass...