doi: 10.1038/s41591-022-01718-1. Epub 2022 Mar 14.
Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation
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- PMID:35288694
- PMCID: PMC9018423
- DOI: 10.1038/s41591-022-01718-1
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Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation
Li Zhan et al. Nat Med.2022 Apr.
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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.
© 2022. The Author(s).
Conflict of interest statement
L.Z., J.S.R., Z.H., N.S., M.E., C.S.D., J.C.B. and E.B.F. have filed a provisional patent application (serial no. 63/270,192) relevant to this study.
Figures
a, Cryopreservation can be the cornerstone of an islet supply chain, allowing pooling, banking, and quality control before transplant. Model systems used to explore this include mouse, porcine and human islets and SC-beta islets. To achieve high recovery, viability, function and scalability simultaneously, systematic optimization of interrelated parameters, including CPA toxicity, ice formation and cooling and warming rates during VR cryopreservation, was performed in these islet systems. The achieved cooling and warming rates can adjust the balance between CPA toxicity and ice formation. Islet morphology, viability, metabolic health and in vitro and in vivo function were evaluated after VR cryopreservation. hESC, human embryonic stem cell.b, Schematic of cryomesh VR (not to scale). After CPA loading, islets in suspension were transferred to the cryomesh, and excessive CPA solution was removed before being plunged into liquid nitrogen (LN2).c, Representative measured temperature profile of cryomesh VR. Inset is the achieved cooling and warming rates (n = 6, data presented as mean ± s.d. with individual data points).d, Correlation of CWR and CPA concentration (equation (S2) in Supplementary Materials) indicates that ~44 wt% CPA is minimally required to avoid lethal ice using cryomesh VR and shows where other studies have failed to use a CPA with an adequate CWR to avoid ice under their thermal performance conditions.
a, Schematic of the microfluidic device used to measure the biophysical properties of the islets (not to scale). The morphological changes of the islets were recorded via a microscope. PDMS, polydimethylsiloxane.b, Top: When subjected to 15 wt% DMSO at 4 °C, the islet first shrinks and then swells. Upon exposure to hypertonic CPA, water exits the cells, and the islet shrinks. CPA then diffuses across the cell membranes, followed by water re-entering the cell, leading to swelling back towards their initial state. Bottom: The islet remained shrunk in NaCl solution as the cells are impermeant to salt. Scale bars, 100 µm.c, Boyle–van’t Hoff plots of mouse and SC-beta islets display the normalized islet volume (V/V0) as a function of the osmolality ratio of isotonic and hypertonic NaCl solution (π0/π). The osmotic inactive volume (Vb) that does not participate in the osmotic response can be estimated by extrapolating the linear fit toπ0/π = 0. Further details can be found in Methods (n = 4 for SC-beta islets,n = 8 for mouse islets).d, Normalized volume of mouse and SC-beta islets versus time demonstrating the shrink–swell behavior when exposed to 15 wt% DMSO at 21 °C (n = 3 for mouse islets,n = 9 for SC-beta islets).e, Summary table of mouse and SC-beta islets water (Lp) and CPA (ω) permeability at 4 °C and 21 °C (n = 3–9; the exact sample size can be found in Supplementary Fig. 3). The red color represents mouse islet in panelsc,d,g,h, and is used in panele to maintain consistency with the rest of the panels.f, Stepwise loading (steps 1–3) and unloading (steps 4–7) of 22 wt% EG + 22 wt% DMSO for islets.g, Modeled islet normalized volume change during CPA loading/unloading using the measured biophysical properties. The volume of both mouse and SC-beta islets remained in the safe region.h, Modeled CPA concentration in the mouse and SC-beta islets. Forc–e, data are presented as mean ± s.d.
a, Confocal microscope images of live and dead controls of SC-beta islets stained with AO (cyan) and PI (red).b, Confocal microscope images (AO/PI merge) of CPA-treated (CPA loading and unloading only) and VR-treated (CPA loading, VR and CPA unloading) SC-beta islets. Various CPA formulations were examined.c, Cell viability of CPA-only-treated (cyan), VR-treated (green) and live control (red) SC-beta islets. Islets were dissociated into single cells, and viability was then measured. The yellow text inb,c is used to highlight the optimal condition. One-way analysis of variance (ANOVA) with Tukey post hoc test was used to compare groups, andP values from informative pairwise comparisons are shown (n = 4).d,e, For mouse (d) and SC-beta (e) islet cell viability after different culture time (0, 3 and 24 h) after CPA-only treatment and then after VR treatment. One-way ANOVA with Tukey post hoc test was used to compare groups, and informative pairwise comparisons are shown (n = 4). Scale bars, 100 µm. Data are shown as individual data points and mean ± s.d.
a, Morphology of mouse islets from live control, VR (cryopreserved by VR) and conventional (cryopreserved by conventional slow freezing) was evaluated by brightfield microscopy, hematoxylin and eosin (H&E) histology, and TEM. For the conventional group, a mixture of intact and disrupted islets was observed. Examples of disrupted islet gross morphology due to ice formation are shown in the brightfield and H&E histology.b, Viability (percentage of live control) of mouse, SC-beta, porcine and human islets from treatment groups including live control, VR, VR 9 months (islets stored in LN2 for 9 months before rewarming), conventional (cryopreserved by conventional slow freezing) and dead control (treated by 75% ethanol). ND, not done. One-way ANOVA with Games–Howell post hoc test was used to compare groups, andP values from informative pairwise comparisons are shown (n = 3–34 per group; exact number in Supplementary Table 3).c, Confocal microscope images (AO/PI) of mouse, SC-beta, porcine and human islets from treatment groups, including live control, VR and conventional.d, TUNEL-stained images of mouse, SC-beta and human islets from treatment groups, including live control, VR and conventional. Bottom panel is Annexin V staining of mouse islets from the same treatment groups. Scale bars represent 2 µm for TEM, 50 µm for brightfield images, 70 µm for histology and TUNEL images and 100 µm for all fluorescence images. Data are shown as individual data points and mean ± s.d.
a, Mitochondrial membrane potential (via TMRE staining) of mouse, SC-beta, porcine and human islets from treatment groups, including live control, VR and conventional.b, Left: Quantification of TMRE staining intensity. Comparisons shown between live control and treatment groups were performed by Kruskal–Wallis and pairwise Wilcoxon tests (n = 3–16 per group). Right: Measurement of ATP levels of four types of islets from live and dead control groups and cryopreservation groups (VR and conventional). One-way ANOVA with Games–Howell post hoc test was used to compare groups, andP values from informative pairwise comparisons are shown (n = 3–12/group).c, Example OCR curve showing the change in OCR during Mito Stress testing in SC-beta islets and comparing live control, VR, conventional cryopreservation and dead control islets (n = 5–8 per group at each time point).d, Compilation of the metabolic OCR parameters for each islet type and each treatment group. One-way ANOVA with Tukey post hoc test was used to compare groups, and significant (P < 0.05) pairwise differences are shown (n = 3–33 per group).e, In vitro GSIS assay for mouse, SC-beta and human islets from treatment groups, including live control, VR and conventional. One-way ANOVA with Tukey post hoc test was used to compare groups, and informative pairwise comparisons are shown (n = 3–12/group). Inb,d ande, relevant statistical comparisonP values are included within the plots. Scale bars, 100 µm. Data are shown as individual data points and mean ± s.d. Forb–e, the exact number of replicates can be found in Supplementary Table 3. RLU, relative light units.
a, Blood glucose levels of streptozotocin-induced diabetic mice after syngeneic transplant of marginal mass mouse islets (250 islets per recipient) from treatment groups, including live control, VR, VR with 9-month cryopreserved storage (islets stored in LN2 for 9 months) and conventional cryopreservation (450 islets per recipient). All pairwise comparisons withP value <0.05 are shown (*P < 0.05), as determined by one-way ANOVA with Games–Howell post hoc test [(n = 10 (control and conventional cryopreservation), 9 (VR), 1 (VR partial function) and 3 (9-month storage and VR)).b, Insulin (red) and glucagon (green) staining in syngeneic (mouse) and xenogeneic (porcine and SC-beta) mouse transplant models. Treatment groups of islets include live control, VR and conventional. 4′,6-Diamidino-2-phenylindole staining (blue) is shown in the merged images.c, IPGTT of wild-type (WT) mice, diabetic mice and diabetic mice transplanted with live control, VR and conventional cryopreserved islets (left). Area under the curve (AUC) of IPGTT (right panel). Groups were compared by one-way ANOVA and Tukey post hoc test, and only informative pairwise comparisons are shown (n = 9–10 per group).d, Xenotransplant of SC-beta islets in NSG mice with nonfasting plasma human insulin levels at 4, 8 and 12 weeks after transplant.e, At 14 weeks, plasma insulin level of NSG mice after fasting and 30 min following stimulated insulin production by intraperitoneal glucose injection. Ford ande, group comparison is by one-way ANOVA and Tukey post hoc test with informative comparisons shown (n = 3–5/treatment group). Scale bars, 200 µm. Data are shown as individual data points and mean ± s.d. Forc–e, the exact number of replicates can be found in Supplementary Table 3.
Comment in
- Awaking sleeping islets for a cure of diabetes.Scheibner K, Lickert H.Scheibner K, et al.Med. 2022 May 13;3(5):279-280. doi: 10.1016/j.medj.2022.04.004. Epub 2022 May 13.Med. 2022.PMID:35584645No abstract available.
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