skip to main content

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):
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nature Medicine
Page Range / eLocation ID:
798 to 808
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Insulin-producing β cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between β cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

    more » « less
  2. Abstract

    The aim of this article is to develop, characterize, and test a novel 3D bioscaffold matrix that can accommodate pancreatic islets and provide them with a continuous, controlled, and steady source of oxygen to prevent hypoxia‐induced damage following transplantation. Hence, a collagen‐based cryogel bioscaffold that incorporates calcium peroxide (CPO) into its matrix is made. The optimal concentration of CPO integrated into bioscaffolds is 0.25 wt% and this generates oxygen at 0.21 ± 0.02 × 10‐3mday‐1(day 1), 0.19 ± 0.01 × 10‐3mday‐1(day 6), 0.13 ± 0.03 × 10‐3md‐1(day 14), and 0.14 ± 0.02 × 10‐3md‐1(day 21). Accordingly, islets seeded into cryogel‐CPO bioscaffolds have a significantly higher viability and function compared to islets seeded into cryogel alone bioscaffolds; these findings are supported by data from quantitative computational modeling. When syngeneic islets are transplanted into the epididymal fat pad (EFP) of diabetic mice, the cryogel‐0.25 wt%CPO bioscaffold improves islet function with diabetic animals re‐establishing glycemic control. Mice transplanted with cryogel‐0.25 wt%CPO bioscaffolds show faster responses to intraperitoneal glucose injections and have a higher level of insulin content in their EFP compared to those transplanted with islets alone (P< 0.05). The novel oxygen‐generating bioscaffold (i.e., cryogel‐0.25 wt%CPO) therefore provides a biostable and biocompatible 3D microenvironment for islets which can facilitate islet survival and function at extra‐hepatic sites of transplantation.

    more » « less
  3. Abstract

    Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP‐sensitive potassium channel (KATP) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATPlocally produces the ATP that inhibits KATPactivity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside‐out patches inhibited KATP. To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATPactivity using mouse beta cells and INS‐1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATPactivity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well‐known action of protons to inhibit KATP. In cell‐attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATPchannel closure by glucose, further suggesting that PK is not an important regulator of KATP. In contrast, addition of mitochondrial inhibitors robustly increased KATPactivity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATPin pancreatic beta cells.image

    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.

    more » « less
  4. Introduction Blood sugar homeostasis relies largely on the action of pancreatic islet hormones, particularly insulin and glucagon. In a prototypical fashion, glucagon is released upon hypoglycemia to elevate glucose by acting on the liver while elevated glucose induces the secretion of insulin which leads to sugar uptake by peripheral tissues. This simplified view of glucagon and insulin does not consider the paracrine roles of the two hormones modulating the response to glucose of α- and β-cells. In particular, glucose-stimulated glucagon secretion by isolated α-cells exhibits a Hill-function pattern, while experiments with intact pancreatic islets suggest a ‘U’-shaped response. Methods To this end, a framework was developed based on first principles and coupled to experimental studies capturing the glucose-induced response of pancreatic α- and β-cells influenced by the two hormones. The model predicts both the transient and steady-state profiles of secreted insulin and glucagon, including the typical biphasic response of normal β-cells to hyperglycemia. Results and discussion The results underscore insulin activity as a differentiating factor of the glucagon secretion from whole islets vs . isolated α-cells, and highlight the importance of experimental conditions in interpreting the behavior of islet cells in vitro . The model also reproduces the hyperglucagonemia, which is experienced by diabetes patients, and it is linked to a failure of insulin to inhibit α-cell activity. The framework described here is amenable to the inclusion of additional islet cell types and extrapancreatic tissue cells simulating multi-organ systems. The study expands our understanding of the interplay of insulin and glucagon for pancreas function in normal and pathological conditions. 
    more » « less
  5. null (Ed.)
    Diabetes is one of the leading causes of death globally. Currently, the donor pancreas is the only source of human islets, placing extreme constraints on supply. Hence, it is imperative to develop renewable islets for diabetes research and treatment. To date, extensive efforts have been made to derive insulin-secreting cells from human pluripotent stem cells with substantial success. However, the in vitro generation of functional islet organoids remains a challenge due in part to our poor understanding of the signaling molecules indispensable for controlling differentiation pathways towards the self-assembly of functional islets from stem cells. Since this process relies on a variety of signaling molecules to guide the differentiation pathways, as well as the culture microenvironments that mimic in vivo physiological conditions, this review highlights extracellular matrix proteins, growth factors, signaling molecules, and microenvironments facilitating the generation of biologically functional pancreatic endocrine cells from human pluripotent stem cells. Signaling pathways involved in stepwise differentiation that guide the progression of stem cells into the endocrine lineage are also discussed. The development of protocols enabling the generation of islet organoids with hormone release capacities equivalent to native adult islets for clinical applications, disease modeling, and diabetes research are anticipated. 
    more » « less