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Title: Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation
Abstract 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.  more » « less
Award ID(s):
1941543
NSF-PAR ID:
10325706
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nature Medicine
Volume:
28
Issue:
4
ISSN:
1078-8956
Page Range / eLocation ID:
798 to 808
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    Key points

    Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATPactivity in excised patches. PEP solutions only inhibit KATPactivity if the pH is unbalanced.

    Modulating pyruvate kinase has minimal effects on KATPactivity.

    Mitochondrial inhibition, in contrast, robustly potentiates KATPactivity in cell‐attached patches.

    Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP.

    Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATPactivity of pancreatic beta cells.

     
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