Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Biological hypoxia in pre-transplant human pancreatic islets induces transplant failure in diabetic mice.

Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation.

A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

Risk factors for postoperative complications in patients with Parkinson disease: A single center retrospective cohort study.

Surgical treatment for patients with Parkinson disease (PD) under general anesthesia has become frequent. PD is a significant predictor of postoperative complications. However, the factors that predict complications in patients with PD remain unknown. We retrospectively recruited patients with PD who underwent surgery between April 2015 and March 2019. The prevalence of postoperative complications was analyzed. We compared the patient characteristics, medical data, and surgical data between patients with and without postoperative complications. We also estimated the odds ratios (OR) for postoperative complications in patients with PD who underwent surgery. Sixty-five patients were enrolled. Eighteen patients presented with 22 complications, including urinary tract infections (UTI) (n = 3; 5%), pneumonia (n = 1; 2%), surgical site infections (SSI) (n = 3; 5%), postoperative delirium (POD) (n = 7; 10%), and others (n = 8; 12%). Four patients presented with 2 complications each. The operation time, the red blood cell transfusion and the rate of rotigotine usage were higher in patients with complications than those without (314 ± 197 min vs 173 ± 145 min, P = .006; 0 [0-560] mL vs 0 [0-0] mL, P = .02; 39% vs 6%, P = .003, respectively) (mean ± standard deviation or median [interquartile range]). Preoperative rotigotine usage (OR: 9.33; 95% confidential interval [CI]: 2.07-42.07; P = .004) was an independent risk factors for postoperative complications. The findings indicate that clinicians should closely monitor postoperative complications when patients with PD who have received transdermal dopamine agonists undergone longer time surgery.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures. Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested. Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic ß-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary ß-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice.

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.

Chronic marijuana usage by human pancreas donors is associated with impaired islet function.

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

Early-Phase Luciferase Signals of Islet Grafts Predicts Successful Subcutaneous Site Transplantation in Rats.

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.

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet.

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

Semi-Automated Assessment of Human Islet Viability Predicts Transplantation Outcomes in a Diabetic Mouse Model.

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.

High Fractions of Large Islets in Human Islet Preparations Detrimentally Affect Posttransplant Outcomes in Streptozotocin-Induced Diabetic Immunodeficient Mice.

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.

A subcutaneous pancreatic islet transplantation platform using a clinically applicable, biodegradable Vicryl mesh scaffold - an experimental study.

Pancreatic islet transplantation into the liver is an effective treatment for type 1 diabetes but has some critical limitations. The subcutaneous site is a potential alternative transplant site, requiring minimally invasive procedures and allowing frequent graft monitoring; however, hypoxia is a major drawback. Our previous study without scaffolding demonstrated post-transplant graft aggregation in the subcutaneous site, which theoretically exacerbates lethal intra-graft hypoxia. In this study, we introduce a clinically applicable subcutaneous islet transplantation platform using a biodegradable Vicryl mesh scaffold to prevent aggregation in a diabetic rat model. Islets were sandwiched between layers of clinically proven Vicryl mesh within thrombin-fibrin gel. In vitro, the mesh prevented islet aggregation and intra-islet hypoxia, which significantly improved islet viability. In vivo rat syngeneic islet transplantations into a prevascularized subcutaneous pocket demonstrated that the mesh significantly enhanced engraftment, as measured by assays for graft survival and function. Histological examination at 6 weeks showed well-vascularized grafts sandwiched in a flat shape between the mesh layers. The biodegradable mesh was fully absorbed by three months, which alleviated chronic foreign body reaction and fibrosis, and supported long-term graft maintenance. This simple graft shape modification approach is an effective and clinically applicable strategy for improved subcutaneous islet transplantation.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function.

The pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NODscid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Depth inversion with a 3D structure influences brightness perception.

Whether or not depth perception influences brightness and/or lightness perception has been repeatedly discussed, and some studies have emphasized its importance. In addition, a small number of studies have empirically tested and shown the effect of depth inversion, such as seen in the Mach card illusion, on perceived lightness, and they interpreted such results in terms of lightness constancy. However, how perceived brightness changes contingent on depth inversion remains unexplained. Therefore, this study used the matching method to examine changes in brightness perception when depth inversion is observed. We created and used a three-dimensional (3D) concave object, composed of three sides made of card stock, which could be perceived as having two different shapes in 3D; it could be perceived as a horizontal concave object, corresponding to its actual physical structure, and as a convex standing object, similar in shape to a building. Participants observed this object as both a concave object and as a convex object, and judged the brightness of its surfaces during each observation. Our results show that the perception of the brightness of the object's surfaces clearly changed depending on the perception of depth. When the object was seen as convex, one part of the surface was perceived as darker than when the object was seen as concave, but the other part of the surface remained unchanged. Here we discuss the relationship between depth perception and brightness perception in terms of perceptual organization.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets.

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 (O2) 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°C, 22°C, and 37°C) and O2 concentration (21% and 50%). We simulated the O2 distribution in islets based on islet O2 consumption rate and dissolved O2 in the medium. We determined the optimal conditions for O2 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 nonobese diabetic-severe combined immunodeficiency mouse kidneys. We validated our results using CMRL 1066 medium (used for clinical islet transplantation). RESULTS: Simulation revealed that 12°C of 50% O2 PIM-R culture supplied O2 effectively into the islet core. This condition maintained islet volume at greater than 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. CONCLUSIONS: By optimizing temperature and O2 concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

Surfactants Improve Live Cell Imaging of Human Pancreatic Islets.

OBJECTIVES: Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich ß-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection. The aim of this study was to develop a practical method of live islet imaging by using surfactants to enhance the penetration efficiency. METHODS: Surfactants (F127, Tween 20, and Triton X-100) were co-incubated with Newport Green for fluorescent imaging of live isolated human islet and nonislet tissues. Toxicity, enhancement of Newport Green fluorescence, and effects on specificity to islets were examined. RESULTS: Newport Green fluorescent intensity was increased after co-incubation with all surfactants tested (0.2-3.2 mM); however, surfactants were toxic to islets at high concentrations. Within the nontoxic range, high specificity to islets was observed when co-incubated with Tween 20 at 0.2-0.4 mM, compared with F127 and Triton X-100. This optimized range successfully distinguished islets from nonislet tissues using statistically calculated cutoff value of Newport Green fluorescent intensity. CONCLUSIONS: Surfactants, particularly Tween 20 in the optimized range, effectively and selectively enhanced Newport Green fluorescence in live islets without increasing islet toxicity.

Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata.

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.