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Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0–10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points ( n = 18, Grade A), 8 points ( n = 19, Grade B), and 7 points ( n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively ( P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation ( P < 0.0001) and reversal rate of diabetes ( P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

PURPOSE: The transplantation of pancreatic islets is a promising cell replacement therapy for type 1 diabetes. Subcutaneous islet transplantation is currently under investigation as a means to circumvent problems associated with standard intra-hepatic islet transplantation. As modifications are being developed to improve the efficacy of subcutaneous islet transplantation, it is important to have robust methods to assess engraftment. Experimentally, ATP-dependent bioluminescence imaging using luciferase reporter genes has been effective for non-invasively tracking engraftment. However, it was heretofore unknown if the bioluminescence of subcutaneously transplanted luciferase-expressing islet grafts correlates with diabetes reversal, a primary outcome of transplantation. PROCEDURES: A retrospective analysis was conducted using data obtained from subcutaneous islet transplantations in Lewis rats. The analysis included transplantations from our laboratory in which islet donors were transgenic rats ubiquitously expressing luciferase and recipients were wild type, streptozotocin-induced diabetic rats. Data from 79 bioluminescence scans were obtained from 27 islet transplantations during the post-transplant observation period (up to 6 weeks). The bioluminescence intensity of the subcutaneously transplanted grafts, captured after the intravenous administration of luciferin, was correlated with diabetes reversal. RESULTS: After subcutaneous transplantation, islet bioluminescence decreased over time, dropping >50% from 1 to 3 weeks post-transplant. Bioluminescence intensity in the early post-transplant phase (1–2 weeks) correlated with the subsequent reversal of diabetes; based on optimized bioluminescence cutoff values, the bioluminescence intensity of islets at 1 and 2 weeks predicted successful transplantations. However, intensity in the late post-transplant phase (≥4 weeks) did not reflect transplantation outcomes. CONCLUSIONS: Early-phase bioluminescence imaging of luciferase-expressing islets could serve as a useful tool to predict the success of subcutaneous islet transplantations by preceding changes in glucose homeostasis.

Background: Islet transplantation is a promising treatment for type-1 diabetes; however, donor shortage is a concern. Even when a pancreas is available, low islet yield limits the success of transplantation. Islet culture enables pooling of multiple low-yield isolations into an effective islet mass, but isolated islets rapidly deteriorate under conventional culture conditions. Oxygen depletion in the islet core, which leads to central necrosis and volume loss, is one of the major reasons for this deterioration. Methods: To promote long-term culture of human islets in PIM-R medium (used for islet research), we adjusted temperature (12, 22, and 37°C) and oxygen concentration (21% and 50%). We simulated the oxygen distribution in islets based on islet oxygen consumption rate and dissolved oxygen in the medium. We determined the optimal conditions for oxygen distribution and volume maintenance in a 2-week culture and assessed viability and insulin secretion compared to noncultured islets. In vivo islet engraftment was assessed by transplantation into diabetic NOD-SCID mouse kidneys. We validated our results using CMRL 1066 medium (used for clinical islet transplantation). Results: Simulation revealed that 12°C–50% oxygen PIM-R culture supplied oxygen effectively into the islet core. This condition maintained islet volume at >90% for 2 weeks. There were no significant differences in viability and function in vitro or diabetic reversal rate in vivo between 2-week cultured and noncultured islets. Similar results were obtained using CMRL 1066. Conclusion: By optimizing temperature and oxygen concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

OBJECTIVES The aim of this study was to determine whether the size of islets isolated from human donors-measured pretransplant-impacts transplantation outcomes in diabetic mice. METHODS Human islets (1200 islet equivalents) were transplanted into the kidney capsules of streptozotocin-induced diabetic immunodeficient mice. Data from a total of 174 mice that received islets from 45 isolations were analyzed to evaluate the correlation between pretransplant islet size and posttransplant diabetes reversal. Fluorescent images of islet clusters were used to categorize individual islets by size (small, 50-150 μm; medium, 150-250 μm; large, >250 μm), and the fractions of islets in each category were calculated. RESULTS The fraction of large islets negatively correlated with diabetes reversal rates. Mice that received islet grafts containing 0% to 5%, 5% to 10%, and more than 10% large islets had diabetes reversal rates of 75%, 61%, and 45%, respectively (P = 0.0112). Furthermore, mice that exhibited diabetes reversal received smaller fractions of large islets than mice that did not (5.5% vs 8.0%, P = 0.0003). Intriguingly, the fractions of medium and small islets did not correlate with diabetes reversal outcomes. CONCLUSIONS The fraction of large islets is a sensitive predictor of human islet transplantation outcomes in diabetic mice.

In clinical and experimental human pancreatic islet transplantations, establishing pretransplant assessments that accurately predict transplantation outcomes is crucial. Conventional in vitro viability assessment that relies on manual counting of viable islets is a routine pretransplant assessment. However, this method does not correlate with transplantation outcomes; to improve the method, we recently introduced a semi-automated method using imaging software to objectively determine area-based viability. The goal of the present study was to correlate semi-automated viability assessment with posttransplantation outcomes of human islet transplantations in diabetic immunodeficient mice, the gold standard for in vivo functional assessment of isolated human islets. We collected data from 61 human islet isolations and 188 subsequent in vivo mouse transplantations. We assessed islet viability by fluorescein diacetate and propidium iodide staining using both the conventional and semi-automated method. Transplantations of 1,200 islet equivalents under the kidney capsule were performed in streptozotocin-induced diabetic immunodeficient mice. Among the pretransplant variables, including donor factors and post-isolation assessments, viability measured using the semi-automated method demonstrated a strong influence on in vivo islet transplantation outcomes in multivariate analysis. We calculated an optimized cutoff value (96.1%) for viability measured using the semi-automated method and showed a significant difference in diabetes reversal rate for islets with viability above this cutoff (77% reversal) vs. below this cutoff (49% reversal). We performed a detailed analysis to show that both the objective measurement and the improved area-based scoring system, which distinguished between small and large islets, were key features of the semi-automated method that allowed for precise evaluation of viability. Taken together, our results suggest that semi-automated viability assessment offers a promising alternative pretransplant assessment over conventional manual assessment to predict human islet transplantation outcomes.

[68Ga]DO3A-VS-Cys40-Exendin-4, a glucagon-like peptide 1 receptor agonist, was evaluated as a potential PET tracer for the quantitation of human islets transplanted to the liver. The short-lived PET radionuclide 68Ga, available on a regular basis from a 68Ge/68Ga generator, is an attractive choice. Human C-peptide was measured to evaluate human islet function post-transplantation and prior to microPET imaging. [68Ga]DO3A-VS-Cys40-Exendin-4 was radiosynthesized and evaluated for PET imaging of transplanted human islets in the liver of healthy NOD/SCID mice. The biodistribution of the tracer was evaluated to determine the uptake into various organs, and qPCR of liver samples was conducted to confirm engrafted islet numbers after PET imaging. Measurement of human C-peptide indicated that higher engrafted islet mass resulted in higher human C-peptide levels in post-transplantation. The microPET imaging yielded high resolution images of liver-engrafted islets and also showed significant retention in mouse livers at 8 weeks post-transplantation. Biodistribution studies in mice revealed that liver uptake of [68Ga]DO3A-VS-Cys40-Exendin-4 was approximately 6-fold higher in mice that received 1000 islet equivalent (IEQ) than in non-transplanted mice. qPCR analysis of insulin expression suggested that islet engraftment numbers were close to 1000 IEQ transplanted. In conclusion, human islets transplanted into the livers of mice exhibited significant uptake of [68Ga]DO3A-VS-Cys40-Exendin-4 compared to the livers of untreated mice; and imaging of the mice using PET showed the human islets clearly with high contrast against liver tissue, enabling accurate quantitation of islet mass. Further validation of [68Ga]DO3A-VS-Cys40-Exendin-4 as an islet imaging probe for future clinical application is ongoing.

A new microelectromechanical systems (MEMS) oxygen transporter device to improve the efficacy of islet transplantation to treat type 1 diabetes is described here. The device is fabricated for biocompatibility and designed to maintain islet oxygenation levels throughout the normally hypoxic subcutaneous transplant site. Transplantation of the islets and the device to the subcutaneous location is minimally invasive. Bench-top testing and computational modelling of the device demonstrate that the device can provide sufficient oxygen to prevent hypoxia-induced islet death transplanted subcutaneously. In vitro studies show that the partial oxygen tension is well-maintained using the oxygen transporter in an anoxic environment. In vivo studies using a rat subcutaneous transplant model demonstrate that transplanted islets have more than three-times higher survival using the oxygen transporter compared with transplants not using the transporter, two weeks post-transplant. In addition, histology sections show well-maintained islet structures one month after transplantation for islets transplanted in the presence of the device. Our MEMS oxygen transporter is a novel and promising device to support the efficacy of subcutaneous islet transplantation as a potential cure for type 1 diabetes.

Prolonged pancreas cold ischemia is known to negatively correlate with islet isolation outcomes, hindering successful islet transplantation to treat Type-1 Diabetes. Due to poor islet isolation outcome, pancreata with over 16 h cold ischemia are currently not considered for islet transplantation. Mechanisms involved in pancreas cold ischemia/rewarming mediated islet damage during islet isolation and culture are not well understood. Using an en bloc cold preserved rat pancreas preparation, we attempted to clarify possible mechanisms of islet death associated with prolonged pancreas cold ischemia and subsequent rewarming. Cold ischemia lasting 16 h decreased post-isolation islet yield and increased islet death during the initial 6 h of culture. Electron micrographs revealed swelling and severe disruption of cellular and mitochondrial membranes, as well as an enlarged endoplasmic reticulum (ER) in β-cells isolated from cold preserved pancreata. Prolonged cold ischemia of the pancreas transiently activated mitogen-activated protein kinases (MAPKs) in isolated islets and increased lipid peroxidation products 4-hydroxynonenal (HNE) and heat shock protein (Hsp) 70 after culture, indicating the activation of oxidative stress signaling pathways. The islet isolation process, irrespective of pancreas cold ischemia, activated unfolded protein response (UPR), while the ER protective chaperon BiP was further upregulated by pancreas cold ischemia/rewarming. During the first 6 h of culture following islet isolation, p53 upregulated modulator of apoptosis (Puma) and caspase-3 activation were also upregulated. Our study indicates the involvement of both apoptosis and necrosis in islet death, and suggests oxidative stress and disruption of membranes are critical mechanisms mediated by pancreas cold ischemia/rewarming.

Islet transplantation is an effective therapy that allows the achievement of insulin independence in patients with type 1 diabetes (T1D). To ensure successful transplantation, islet viability and function are of great importance. Viability assessments most often use fluorescein diacetate (FDA)/propidium iodide (PI) staining. However, results using this method often do not correlate well with graft function. Because FDA nonspecifically penetrates all cells present in the islet preparation, including islets and contaminating acinar cells, its use often complicates viability assessments of the overall cell population. Furthermore, the manual method for determining viability percentages is highly subjective. Shortcomings of the conventional islet viability assay can be potentially improved by staining cells with Newport Green (NG). NG, is a zinc-specific fluorescent dye that specifically reacts with zinc-rich β cells. Two kinds of NG dyes, NG-DCF and NG-PDX, are currently available. We examined the zinc specificity of these NG dyes and compared NG staining with traditional FDA staining to explore the potential of NG dyes to improve islet viability assessment. Of the two NGs tested, NG-DCF showed the higher specificity toward a β-cell line as well as human islets. NG-DCF accurately identified the islet area, even in low-purity islets, while neither FDA nor NG-PDX did. Although NG-DCF staining required a longer incubation time, the addition of poloxamer F127 and incubation at 37°C allowed viability assessment to take place within 30 min. Unlike FDA/PI staining, NG-DCF/PI staining allowed for islet-specific assessment. We also introduced a semiautomated measurement to determine NG-DCF/PI staining results, which enabled us to obtain objective and reproducible results. NG-DCF/PI staining is easy and reliable, and this method permits highly objective islet-specific viability assessments.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic (“Firefly”) Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in “Firefly” islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.

A major limitation in the development of radiolabeled Exendin-4 analogues (short half-life isotopes) is an inability to efficiently and rapidly separate final products from precursors. This is important as lack of purity in the final product decreases probe efficiency. The purpose of this study was to develop a method to prepare the high-purity imaging reagent [18F] PTTCO-Cys40-Exendin-4. To accomplish this, magnetic TCO-beads were incubated with the crude product to remove unlabeled Exendin-4. In rodents pre-treatment with purified [18F] PTTCO-Cys40-Exendin-4 (~1.85 MBq) allowed precise microPET imaging of ectopic insulinomas. Moreover, analogue uptake was successfully blocked by administering non-labelled “cold” Exendin-4. Biodistribution data revealed that [18F] PTTCO-Cys40-Exendin-4 accumulated specifically in GLP-1R-enriched insulinomas in mice, confirming results obtained using miroPET. Investigation of [18F] PTTCO-Cys40-Exendin-4 as a tracer to image portal vein-transplanted pancreatic islets is proceeding in animals.

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO_2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO_2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes.

Background. A significant loss of islet function observed in type 1 diabetic recipients after intraportal islet transplantation raises a question about the suitability of the liver as a transplant site. We hypothesize that natural killer (NK) cells in the liver play a role in the islet graft destruction. Methods. Phenotypical and functional differences between liver and splenic NK cells isolated from mice were examined by flow cytometry and in vitro cytotoxicity assays. In vivo, the role of liver NK cells was determined by examining the function of intraportally administered islet iso- and allografts by treating recipients with anti-asialo GM1 to deplete NK cells. Results. NK cell-depleted diabetic C57BL/6 mice receiving 400 syngeneic islets into the liver rapidly ameliorated hyperglycemia, whereas control recipients did not. The same number of BALB/c islets grafted in the liver of diabetic nonimmunosuppressed C57BL/6 mice failed to function, whereas NK cell-depleted recipients reversed hyperglycemia for up to 10 days. NK cells from the liver of naive C57BL/6 mice showed significantly higher cytotoxicity than splenic NK cells as tested with a β-cell line and allogeneic islets. The cell proportion and the expression level of activation markers on liver NK cells significantly increased after intraportal islet transplantation as compared with the control. Liver NK cells also increased anti-islet cytotoxicity, but not splenic NK cells, after islet transplantation. Conclusions. Our results clearly show the destructive activity of liver NK cells toward islets, suggesting that NK cells play a role in early islet graft loss after intraportal islet transplantation.

TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Successful human islet isolation is essential for clinical and research applications,1-4 and is greatly influenced by selecting appropriate enzyme(s) for pancreas digestion.5-10 Hence, developing and using consistently high-quality enzymes is a key element for islet isolations. Advancements in obtaining non-good manufacturing practice liberase HI indeed made major progress to achieve successful islet isolation11,12 and allowed for an increase in the number of patients receiving islet transplants.13 However, in 2007, the use of liberase HI for human islet isolations was ceased due to safety concerns of possible prion contamination from bovine tissue-derived raw materials.14 Liberase HI was a mixture of collagenase and neutral protease (NP) and not of highly purified enzymes and hence, lot-to-lot variability was a major concern due to failure to consistently achieve successful islet isolation and long term storage was sporadic.9,12,15 Therefore, there is a great demand for manufacturing GMP-grade, highly pure, and low endotoxin digestion enzyme to replace liberase HI among islet isolation specialists and commercial manufacturers to consistently obtain high quality islets.6,16-18 Consequently, collagenase NB1 supplemented with NP was adopted by many centers globally as an alternative enzyme for pancreas digestion for islet isolation.6,19,20 Indeed, several patients have been transplanted with islets isolated using this enzyme, suggesting that this may be a promising product for islet isolation replacing liberase HI. However, the limitation of collagenase NB1 for its efficacy to isolate islets from younger donors influenced the ultimate islet isolation outcome from different donor populations. Furthermore, it has been reported that the collagenase NB1 enzyme contains degraded collagenase; therefore, higher doses are required to achieve successful isolations.8,21 Collagenase, and NP, and clostripain are produced from Clostridium histolyticum,15 whereas thermolysin is purified from Bacillus thermoproteolyticus rokko.12 Collagenase (class I and II isoforms) supplemented with either Thermolysin or NP is currently used for pancreas digestion. Hence, the combination of CIzyme collagenase HA (containing nondegradable class I [60%] and II [40%]) and NP was found to be effective in isolating islets for transplantation, but are not GMP products.21 Indeed, liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) has been shown to be a promising enzyme for human islet isolation for clinical islet transplantation compared to collagenase NB1/NP.6,22 All types of aforementioned enzymes have been used in islet isolations for research and transplantation purposes. However, the advantage of using a particular enzyme from a specific supplier over another is debatable and often subjectively determined by an isolation team's experiences.21,23 It was reported that the overall isolation outcome of collagenase NB1/NP was comparable with that of liberase HI.5,19 In this retrospective analysis, islet isolations from 221 donor pancreata using 3 commercially available enzymes, liberase HI, collagenase NB1/NP, and liberase MTF C/T enzymes, were compared.

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.

Islet transplantation offers a promising treatment for type 1 diabetes (T1D). However, a major hurdle in this treatment is the rapid loss of functional islets during culture and after transplantation. The liver site, currently utilized for transplantation, is suboptimal for achieving long-term insulin independence due to a rapid islet loss followed by a chronic decline in islet function after transplantation. Herein, we report a synthetic saccharide-peptide (SP) hydrogel that allows suspending islets in liquid and injecting for in situ polymerization without forming islet clumps, indicating its potential in extrahepatic islet transplantation. In vitro, rat islets in SP hydrogel maintained a 3D structure and high glucose-stimulated insulin release similar to that observed in freshly isolated islets for 4 weeks, while control islets cultured in suspension lost their 3D structure and insulin release responses by 2 weeks. Biocompatibility of SP hydrogel was shown by the absence of cytokine mRNA activation in peripheral blood mononuclear cells (PBMCs) exposed to hydrogel in vitro and by the absence of cellular infiltrates in and around the hydrogel implanted subcutaneously. Syngeneic Lewis rat islets transplanted in SP hydrogel in various extrahepatic sites stained strongly for insulin, and more effectively reversed diabetes than unencapsulated islets when transplanted in an omental pocket. In conclusion, the SP hydrogel is non-cytotoxic and supports normal islet structure and function both in vitro and in vivo. Specifically, the ability of the hydrogel to separate individual islets after transplantation is important for maintaining their function in vivo. This important property, combined with the versatility and biocompatibility, makes our SP hydrogel a promising synthetic scaffold that can facilitate transplantation of organized heterogeneous cells to preserve their micro-structure and function.

Background. Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent β-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing β-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%± 1.2% insulin-positive, 33.9%± 1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic β cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic β cells correlated well (P

BACKGROUND Bone marrow-derived mesenchymal stem cells (MSCs) are known to produce vascular endothelial growth factor. We hypothesize that co-transplantation of MSCs and islets promotes revascularization and improves islet graft function. METHODS Lewis rat islets were infused into the liver of streptozotocin-diabetic syngeneic recipients or transplanted under the renal capsule of nonobese diabetic severe combined immunodeficiency (NOD SCID) mice with MSCs isolated from Lewis bone marrow and expanded in culture. RESULTS Co-transplantation of 500 islets and 107 MSCs (islet-MSCs) reversed diabetes in all eight recipients, whereas islet-alone transplantation achieved euglycemia in 3 of 10 recipients. With 300 islets, five of nine islet-MSCs and 1 of 10 islets-alone recipients reversed diabetes. Results of intravenous glucose tolerance tests performed on day 56 were significantly better in islet-MSCs than islet-alone recipients. One week after transplantation, well-preserved islet structure and higher number of capillaries were found in the liver of islet-MSCs recipients, whereas islet-alone grafts were fragmented with very few capillaries. Islets showed a similar morphology when transplanted with MSCs in nonobese diabetic severe combined immunodeficiency mice with a significantly higher capillary per [beta]-cell ratio than that in islet-alone grafts (0.135+/-0.046 vs. 0.052+/-0.028 capillary segments per [beta]-cell, P

Background. The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a microassay that evaluates the potency of human islets by measuring changes in glucose-induced human insulin gene (INS) expression using a single islet in octuplicate samples. Methods. Poly (A) + messenger RNA (mRNA) was purified from a set of single handpicked human islets. Glucose-induced mature (postspliced) and premature (prespliced) insulin mRNA were quantified by reverse-transcriptase polymerase chain reaction using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction. Results. The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 vs. 4 hr (P

BACKGROUND The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. METHODS Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. RESULTS p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134+/-297 vs. 1477+/-145 IEQ/g or 65,012+/-9385 vs. 45,700+/-5103 IEQ/pancreas; P

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (DeltaOCR(glc)) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of DeltaOCR(glc) obtained at both institutions were compared. Islet specificity was determined by comparing DeltaOCR(glc) in islet and nonislet tissue. The DeltaOCR(glc) ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (DeltaOCR(glc) was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The DeltaOCR(glc) was statistically independent of percent beta cells, demonstrating that DeltaOCR(glc) is governed more by islet quality than by islet composition. The DeltaOCR(glc), but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing DeltaOCR(glc) as a measurement of islet quality for human islet transplantation.

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.