Islet surface modification with urokinase through DNA hybridization

Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)(20) (oligo(dT)(20)-PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)(20). The oligo(dT)(20)-PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)(20) on the cell and complementary oligo(dA)(20) on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.     

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.     

Chemical coupling of a monoclonal antisurfactant protein-B antibody to human urokinase for targeting surfactant-incorporating alveolar fibrin

Intraalveolar fibrin formation is a common histopathological finding in acute inflammatory and chronic interstitial lung diseases. Incorporation of hydrophobic surfactant components into polymerizing fibrin results in a severe loss of surface activity, altered mechanical and structural clot properties, and a reduced susceptibility toward fibrinolytic degradation. Such events have been implicated in atelectasis formation, impairment of gas exchange, and provocation of fibroproliferative changes. In an effort to address the unique features of alveolar fibrin, we designed a hybrid molecule consisting of a monoclonal antibody against surfactant protein SP-B (8B5E) and the catalytic domain of urokinase (B-chain), which was termed MABUC. The urokinase B-chain was prepared by limited reduction of human two-chain-urokinase and subsequent affinity purification and coupled to the antibody using a heterobifunctional cross-linker. Purification of the chimeric protein included gel filtration chromatography and affinity chromatography. An ELISA-like microtiter plate assay, based on the immunological detection of the SP-B moiety and the fibrinolytic activity of the u-PA domain, was developed for the detection of the hybrid molecule. Chromogenic substrate assays, (125)I-based fibrin plate assays, and active site titration were performed to analyze the specific fibrinolytic activity of the conjugate. MABUC was found to fully retain the ability of SP-B binding and the fibrinolytic activity of u-PA. In addition, MABUC was noted to be 1.5-2-fold more effective in the dissolution of surfactant embedding clots and to be approximately 3-fold more resistant against PAI-1, the predominant fibrinolysis inhibitor in the alveolar compartment, as compared to the native u-PA. The superiority of MABUC was particularly prominent (>5-fold efficacy) when investigating clot material incorporating both PAI-1 and surfactant, as a mimicry of alveolar fibrin. We conclude that urokinase and 8B5E can be cross-linked chemically, thus yielding a fibrinolytic enzyme with enhanced substrate specifity for surfactant-containing clots and higher PAI-1 resistance as compared to native u-PA.     

Cryopreservation of islets of Langerhans

Development of techniques for cryopreservation of pancreatic islets of Langerhans could potentially allow for increased freedom from the time restrictions presently affecting viability in islet cell transplantation. While several investigators have attempted islet cell freezing and have obtained favorable in vitro results after thawing, there have been few reported in vivo successes with islets transplanted after freezing. We have developed a simple system for freezing islet cell pancreatic fragments to ?196 °C and have either stored them in liquid nitrogen for 24 hr or immediately thawed the islets prior to transplantation. In addition, antilymphoblast globulin has been used as graft pretreatment modality in order to modify islet cell immunogenicity. We found that ALG was effective in prolongation of graft survival after freezing as well as on fresh nonfrozen transplants. The use of freezing and ALG appears, therefore, to have a favorable effect on the immunogenicity of the pancreatic islet cell allograft.     

Maintenance of Islet Morphology Is Beneficial for Transplantation Outcome in Diabetic Mice

We have previously shown that co-transplantation of islets and Mesenchymal Stem Cells (MSCs) improves islet graft function and revascularisation, which was associated with the maintenance of normal islet morphology. The aim of the current study was to determine whether maintaining islet morphology in the absence of additional islet-helper cells would improve transplantation outcome in diabetic mice. Islets were isolated from C57BL/6 mice. Recipient streptozotocin-diabetic C57BL/6 mice were transplanted with a minimal mass of 150 islets as a single pellet or islets that were either manually dispersed or dispersed within a matrigel plug beneath the kidney capsule. Blood glucose concentrations were monitored for one month. Islet graft morphology and vascularisation were analysed by histology. Islets dispersed either alone or within matrigel plugs maintained near normal morphology, in contrast to pelleted islets, where individual islets fused to form large endocrine aggregates. The vascularisation of manually dispersed islets and islets dispersed within matrigel plugs was increased relative to respective control pelleted islet grafts. After one month 1/6 mice transplanted with pelleted islets cured compared to 5/6 mice transplanted with manually dispersed islets. The curative capacity of islets dispersed in matrigel was also better than that of pelleted islets (5/8 islet-matrigel implanted mice vs. 1/7 mice transplanted with pelleted islets cured by one month). Therefore, this study demonstrates that the maintenance of islet morphology is associated with improved graft function and revascularisation in diabetic mice.     

Human mesenchymal stem cells protect human islets from pro-inflammatory cytokines

Transplantation of human islets is an attractive alternative to daily insulin injections for patients with type 1 diabetes. However, the majority of islet recipients lose graft function within five years. Inflammation is a primary contributor to graft loss, and inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. As mesenchymal stem cells (MSCs) possess numerous immunoregulatory properties, we hypothesized that MSCs could protect human islets from pro-inflammatory cytokines. Five hundred human islets were co-cultured with 0.5 or 1.0 × 10(6) human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-γ, tumor necrosis factor-α and interleukin 1β. Controls include islets cultured alone (± cytokines) and with human dermal fibroblasts (± cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet β cell apoptosis, and potential cytoprotective factors secreted in the culture media were determined. Cytokine exposure disrupted human islet GSIS based on stimulation index and percentage insulin secretion. Conversely, culture with 1.0 × 10(6) bMSCs preserved GSIS from cytokine treated islets. Protective effects were not observed with fibroblasts, indicating that preservation of human islet GSIS after exposure to pro-inflammatory cytokines is MSC dependent. Islet β cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented β cell apoptosis after cytokine treatment. Hepatocyte growth factor (HGF) as well as matrix metalloproteinases 2 and 9 were also identified as putative secreted cytoprotective factors; however, other secreted factors likely play a role in protection. This study, therefore, demonstrates that MSCs may be beneficial for islet engraftment by promoting cell survival and reduced inflammation.     

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

Background aimsCo-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.MethodsMouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed.ResultsIslets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group.ConclusionsMSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.     

Overexpression of suppressor of cytokine signaling 1 in islet grafts results in anti-apoptotic effects and prolongs graft survival

AimsA significant portion of islet grafts are destroyed by apoptosis and fail to become functional after transplantation. Strategies that enhance islet resistance to apoptosis may prevent graft loss. The aim of this study was to investigate whether overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts could achieve an anti-apoptotic effect and prolong graft survival.Main methodsWe used a chimeric adenovirus vector (Ad5F35) to enhance SOCS1 expression in isolated rat islets, and assessed its protective action against TNF-α-induced apoptosis. After transplanting SOCS1-overexpressing islets into allogeneic recipients with streptozotocin-induced diabetes, graft survival and in situ apoptosis were analyzed using immunohistochemistry.Key findingsThe isolated rat islets infected with Ad5F35–SOCS1 showed significantly higher SOCS1 expression than Ad5F35–EGFP and mock infected islets. The Ad5F35 transfection and SOCS1 overexpression on islets did not affect their insulin secretory function. After treatment with rat TNF-α and cycloheximide in vitro, Ad5F35–-SOCS1 infected islets exhibited a lower apoptotic ratio than controls (Ad5F35–EGFP and mock infected islets). The diabetic recipients transplanted with Ad5F35–SOCS1 infected islets displayed longer time of normoglycemia than recipients transplanted with mock infected islets. Furthermore, histological analysis indicated that the infected grafts with local overexpression of SOCS1 showed decreased apoptosis in the early post-transplant period.SignificanceThese results demonstrate that overexpression of SOCS1 in islet grafts prior to transplantation can significantly protect them from apoptotic loss and prolong their survival. This approach might find a clinical counterpart.     

The role of endothelial cells on islet function and revascularization after islet transplantation

Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation.     

pH is decreased in transplanted rat pancreatic islets

Recent studies of transplanted pancreatic islets have indicated incomplete revascularization. We investigated the pH, in relation to oxygen tension (Po(2)), in endogenous islets and islets syngeneically transplanted to the renal subcapsular site of nondiabetic and streptozotocin-diabetic recipients. Tissue pH and Po(2) were measured using microelectrodes. In the endogenous islets, tissue pH was similar to that in arterial blood. In the transplanted islets, tissue pH was 0.11-0.15 pH units lower. No differences in islet graft pH were seen between nondiabetic and diabetic animals, and none if the islet grafts were investigated 1 day or 1 mo posttransplantation. The Po(2) in the endogenous islets was approximately 35 mmHg. Transplanted islets had a markedly lower tissue Po(2) both 1 day and 1 mo after transplantation. A negative correlation between the tissue Po(2) and the hydrogen ion concentration was seen in the 1-mo-old islet transplants in diabetic animals. In conclusion, decreased Po(2) in transplanted islets is associated with a decreased tissue pH, suggesting a shift toward more anaerobic glucose metabolism after transplantation.     

Quantitative micro positron emission tomography (PET) imaging for the in vivo determination of pancreatic islet graft survival

Islet transplantation is an attractive approach for treating type-1 diabetes, but there is a massive loss of transplanted islets. It is currently only possible to estimate islet mass indirectly, through measurement of circulating C-peptide and insulin levels. This type of estimation, however, is not sufficiently sensitive or reproducible for follow-up of individuals who have undergone islet transplantation. Here we show that islet graft survival could be assessed for 1 month in diabetic NOD mice using 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG)-positron emission tomography (PET) technology, the PET signal reflecting insulin secretory capacity of transplanted islets. Expression of the gene encoding viral interleukin-10 (vIL-10), was measurable in real time with PET scanning. Additionally, we addressed the clinical potential of this approach by visualizing transplanted islets in the liver, the preferred clinical transplantation site. We conclude that quantitative in vivo PET imaging is a valid method for facilitating the development of protocols for prolonging islet survival, with the potential for tracking human transplants.     

Size distribution of mouse Langerhans islets

Pancreatic beta-cells are clustered in islets of Langerhans, which are typically a few hundred micrometers in a variety of mammals. In this study, we propose a theoretical model for the growth of pancreatic islets and derive the islet size distribution, based on two recent observations: First, the neogenesis of new islets becomes negligible after some developmental stage. Second, islets grow via a random process, where any cell in an islet proliferates with the same rate regardless of the present size of the islet. Our model predicts either log-normal or Weibull distributions of the islet sizes, depending on whether cells in an islet proliferate coherently or independently. To confirm this, we also measure the islet size by selectively staining islets, which are exposed from exocrine tissues in mice after enzymatic treatment. Indeed revealed are skewed distributions with the peak size of approximately 100 cells, which fit well to the theoretically derived ones. Interestingly, most islets turned out to be bigger than the expected minimal size (approximately 10 or so cells) necessary for stable synchronization of beta-cells through electrical gap-junction coupling. The collaborative behavior among cells is known to facilitate synchronized insulin secretion and tends to saturate beyond the critical (saturation) size of approximately 100 cells. We further probe how the islets change as normal mice grow from young (6 weeks) to adult (5 months) stages. It is found that islets may not grow too large to maintain appropriate ratios between cells of different types. Our results implicate that growing of mouse islets may be regulated by several physical constraints such as the minimal size required for stable cell-to-cell coupling and the upper limit to keep the ratios between cell types. Within the lower and upper limits the observed size distributions of islets can be faithfully regenerated by assuming random and uncoordinated proliferation of each beta-cell at appropriate rates.     

Combined Strategy of Endothelial Cells Coating,Sertoli Cells Coculture and Infusion Improves Vascularization and Rejection Protection of Islet Graft

Improving islet graft revascularization and inhibiting rejection become crucial tasks for prolonging islet graft survival. Endothelial cells (ECs) are the basis of islet vascularization and Sertoli cells (SCs) have the talent to provide nutritional support and exert immunosuppressive effects. We construct a combined strategy of ECs coating in the presence of nutritious and immune factors supplied by SCs in a co-culture system to investigate the effect of vascularization and rejection inhibition for islet graft. In vivo, the combined strategy improved the survival and vascularization as well as inhibited lymphocytes and inflammatory cytokines. In vitro, we found the combinatorial strategy improved the function of islets and the effect of ECs-coating on islets. Combined strategy treated islets revealed higher levels of anti-apoptotic signal molecules (Bcl-2 and HSP-32), survival and function related molecules (PDX-1, Ki-67, ERK1/2 and Akt ) and demonstrated increased vascular endothelial growth factor receptor 2 (KDR) and angiogenesis signal molecules (FAk and PLC-γ). SCs effectively inhibited the activation of lymphocyte stimulated by islets and ECs. Predominantly immunosuppressive cytokines could be detected in culture supernatants of the SCs coculture group. These results suggest that ECs-coating and Sertoli cells co-culture or infusion synergistically enhance islet survival and function after transplantation.     

Advances in long-term islet culture

Long-term culture of human islets provides opportunity for improving results of islet transplantation. The techniques of long-term culture are reproducible and can result in improved function of the islet after transplantation into NOD-SCID mice. We have been able to cure streptozotocin-induced diabetes by islets cultured for more than 6 mo. Culture conditions play an important role in the success of the procedure. Culture success is dependent on the media type, additives, type of colloid or protein used, purity of the islets, and concentration and volume of the tissue. Cellular and structural changes occur over time in culture. These changes may explain the improved efficacy of the islet graft after short and intermediate culture periods. Further research into long-term culture of islets is necessary to fully explore the potential of the technique.     

Early autoimmune destruction of islet grafts is associated with a restricted repertoire of IGRP-specific CD8+ T cells in diabetic nonobese diabetic mice

beta cell replacement via islet or pancreas transplantation is currently the only approach to cure type 1 diabetic patients. Recurrent beta cell autoimmunity is a critical factor contributing to graft rejection along with alloreactivity. However, the specificity and dynamics of recurrent beta cell autoimmunity remain largely undefined. Accordingly, we compared the repertoire of CD8+ T cells infiltrating grafted and endogenous islets in diabetic nonobese diabetic mice. In endogenous islets, CD8+ T cells specific for an islet-specific glucose-6-phosphatase catalytic subunit-related protein derived peptide (IGRP206-214) were the most prevalent T cells. Similar CD8+ T cells dominated the early graft infiltrate but were expanded 6-fold relative to endogenous islets. Single-cell analysis of the TCR alpha and beta chains showed restricted variable gene usage by IGRP206-214-specific CD8+ T cells that was shared between the graft and endogenous islets of individual mice. However, as islet graft infiltration progressed, the number of IGRP206-214-specific CD8+ T cells decreased despite stable numbers of CD8+ T cells. These results demonstrate that recurrent beta cell autoimmunity is characterized by recruitment to the grafts and expansion of already prevalent autoimmune T cell clonotypes residing in the endogenous islets. Furthermore, depletion of IGRP206-214-specific CD8+ T cells by peptide administration delayed islet graft survival, suggesting IGRP206-214-specific CD8+ T cells play a role early in islet graft rejection but are displaced with time by other specificities, perhaps by epitope spread.     

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Competition of activated protein C and urokinase for a heparin-dependent inhibitor

Human urine contains a hitherto unrecognized heparin-dependent inhibitor of activated protein C (APC) (Mr approximately 50,000) that coelutes from heparin-Sepharose together with the only observed peak of urokinase inhibitory activity at a position (0.35 M NaCl) similar to that of plasma protein C (PC) inhibitor. Based on functional assays and immunoblot studies, urokinase and APC compete for this crude inhibitor in the absence or presence of heparin. These results suggest that the same heparin-dependent urinary inhibitor that is immunologically different from several known protease inhibitors is responsible for the observed inhibition of APC and urokinase. In the absence of heparin this inhibitor inhibits APC and urokinase with similar rates, and heparin enhances its inhibitory activity toward both enzymes with more pronounced stimulation of its PC inhibitory activity than its urokinase inhibitory activity. Half-maximal stimulation of inhibition of APC occurs at about 2 mU/ml and maximal stimulation (approximately 10-fold increase of the pseudo-first-order rate constant) at greater than or equal to 50 mU/ml of heparin. This is the first demonstration of competition between APC and urokinase for a heparin-dependent inhibitor. These results may therefore represent a new link between the two major antithrombotic pathways, the PC pathway and the fibrinolytic system.     

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3–5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4–5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.