Islet Equivalents Research Articles

Early Metabolic Markers That Anticipate Loss of Insulin Independence in Type 1 Diabetic Islet Allograft Recipients

The objective of this study was to identify predictors of insulin independence and to establish the best clinical tools to follow patients after pancreatic islet transplantation (PIT). Sequential metabolic responses to intravenous (I.V.) glucose (I.V. glucose tolerance test [IVGTT]), arginine and glucose-potentiated arginine (glucose-potentiated arginine-induced insulin secretion [GPAIS]) were obtained from 30 patients. We determined the correlation between transplanted islet mass and islet engraftment and tested the ability of each assay to predict return to exogenous insulin therapy. We found transplanted islet mass within an average of 16 709 islet equivalents per kg body weight (IEQ/kg BW; range between 6602 and 29 614 IEQ/kg BW) to be a poor predictor of insulin independence at 1 year, having a poor correlation between transplanted islet mass and islet engraftment. Acute insulin response to IVGTT (AIR(GLU) ) and GPAIS (AIR(max) ) were the most accurate methods to determine suboptimal islet mass engraftment. AIR(GLU) performed 3 months after transplant also proved to be a robust early metabolic marker to predict return to insulin therapy and its value was positively correlated with duration of insulin independence. In conclusion, AIR(GLU) is an early metabolic assay capable of anticipating loss of insulin independence at 1 year in T1D patients undergoing PIT and constitutes a valuable, simple and reliable method to follow patients after transplant.

Islet isolation and purification from inbred Wuzhishan miniature pigs

To investigate the applicability of inbred Wuzhishan (WZS) miniature pigs for porcine islet isolation and purification. Islet isolation and purification was conducted on adult (1-yr-old), male inbred WZS miniature pigs and age- and sex-matched market pigs obtained from a local slaughterhouse (control group). Pancreata were excised, and islet isolation was carried out by static digestion and discontinuous gradient centrifugation. Viability of the purified islets was tested by radioimmunochemistry assay to measure glucose-induced insulin release in culture and transplantation in an in vivo study. The anatomical structure of the WZS miniature pig pancreas was more similar to the human pancreas than that of the market pig. Islet yield of the WZS miniature pigs' pancreata was significantly higher than that of the market pigs (6078 ± 1105 vs. 2500 ± 625 islet equivalents [IEQ]/g). In vitro study demonstrated that the islets isolated from WZS miniature pigs were viable, as they efficiently responded to glucose challenge. In vivo study showed that the islets from both groups could cure the diabetic rat with the survival varied from 3 to 5 days (median, 4.3 days) and 2-4 days (median, 3.6 days) in experimental group and control group, respectively. Wuzhishan miniature pig pancreas may be a feasible source of islets for xenotransplantation.

Adenine Nucleotide Levels in a Closed Enzymatic Digestion System for Porcine Islet Isolation

Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.

Improvement of Porcine Islet Isolation by Inhibition of Trypsin Activity during Pancreas Preservation and Digestion Using α1-Antitrypsin

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Rescue of a Pancreatic Islet Graft After Steroid Therapy

Rescue of a Pancreatic Islet Graft After Steroid Therapy

A replacement for islet equivalents with improved reliability and validity

Islet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R 2 value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter ≤ 100 μm) and large (diameter ≥ 200 μm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

Nerve growth factor is associated with islet graft failure following intraportal transplantation

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role in pancreatic β-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF. However, the usefulness of NGF in intraportal islet transplantation was not previously tested. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal vein of streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (≤ 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (area under the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated with increased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotic transplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islet transplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). On the other hand, viable (TUNEL-negative and not necrotic) islets in both groups did not express VEGF and HIF-1α. In the present study, pre-transplant NGF treatment was associated with impaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantly vary according to the transplant site.

A Combined Continuous Density/Osmolality Gradient for Supplemental Purification of Human Islets.

For islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein. However, islet purification may result in decreased numbers of islets recovered from digested tissue. In this study, we evaluated the effectiveness of performing supplemental purification (SP) after regular purification (RP). We designed the densities of low- and high-density solutions based on the outcome of RP. Moreover, a combined continuous osmolality/continuous density gradient for the SP was used in this study. Low-density/osmolality (1.075-1.110 g/cm3/400-410 mOsm/kg) and high-density/osmolality (1.090-1.125 g/cm3/495-505 mOsm/kg) solutions were produced by changing the volumetric ratio of iodixanol, 10 × HBSS, and RP solutions. The percentage of islet recovery (postpurification IE/prepurification IE × 100) after RP was 77.3 ± 5.6%, and the percentage of islet recovery after addition of SP was 85.3 ± 5.4%. In vitro and in vivo assessments showed that islet viability and function were not altered by the additional purification step. These data suggest that the addition of SP could contribute approximately 8% to islet recovery with viability and potency comparable to that obtained by RP and, therefore, that usage of the combined continuous density and continuous osmolality gradient for SP could efficiently improve islet equivalents in the final preparation.

Human Islet Distribution Program for Basic Research at a Single Center

The Clinical Islet Laboratory at the University of Alberta/Alberta Health Services distributes human islets for basic research when islet preparations fail to meet defined release criteria for transplantation. This report highlights our islet distribution activity for diabetes research over a 3-year period. Shipments of the acinar-enriched fraction for research were not included in this report. In 2010, we distributed 6.3 million islet equivalents (IEQs) of islets through 127 shipments to 8 researchers, locally, nationally, and internationally. The number of preparations for research use was stable over the 3-year period (26, 23, and 29 preparations in 2008, 2009, and 2010, respectively). Islet yield distributed for research per isolation was 201, 212, and 218 × 10 3 IEQs, respectively. The number of basic researchers was stable as well, although there were only 2 researchers before 2007. Recently, each researcher has received fewer islets per shipment (49,820 IEQs in 2010 vs 75,635 IEQs in 2008) but more frequently (21.5 in 2010 vs 11.2 times per year in 2008). This paradigm shift would be desirable for researchers, because in our experience, most require <30,000 IEQs per shipment, and more frequent islet shipments results in a larger sample size for experimentation. After an initial expansion in the number of researchers requesting islets, our islet distribution activity has remained stable over the years in terms of total productivity of islets utilized for research. The current supply-versus-demand ratio in our program appears to be appropriate.

Islet vs. pancreas transplantation in Brazil: Defining criteria for pancreas allocation decision

Background: Many studies have evaluated whether there are characteristics related to pancreas donors and the islet isolation process that can influence in pancreatic islet yield. However, this analysis has not yet been performed in Brazil, one of the world leaders in whole pancreas organ transplantation (WOPT), where pancreas allocation for pancreatic islet transplantation (PIT) has no officially defined criteria. Definition of parameters that would predict the outcome of islet isolation from local pancreas donors would be useful for defining allocation priority in Brazil. Objective: To analyze the relationship between multiple donor-related and islet isolation variables with the total number of isolated pancreatic islet equivalents (IEQ) in a Brazilian sample of pancreas donors. Methods: Several variables were analyzed in 74 pancreata relative to the outcome of total IEQs obtained at the end of the process. Results: In univariate analysis, body mass index (BMI) (p = 0.003), the presence of fatty infiltrates in the pancreas as observed during harvesting (p = 0.042) and pancreas digestion time (p = 0.046) were identified as variables related to a greater IEQ yield. In a multivariate analysis a statistically significant contribution to the variability of islet yield was found only for the BMI (p = 0.017). A ROC curve defined a BMI = 30 as a cut-off point, with pancreata from donors with BMI > 30 yielding more islets than donors with BMI < 30 (p < 0.001). Conclusion: These data reinforces the importance of the donor BMI as a defining parameter for successful islet isolation and establishes this variable as a potential pancreas allocation criterion in Brazil, where there is an unequal competition for good quality organs between WOPT and PIT.

Assessment of Tissue-Engineered Islet Graft Viability by Fluorine Magnetic Resonance Spectroscopy

Introduction Despite significant progress in the last decade, islet transplantation remains an experimental therapy for a limited number of patients with type 1 diabetes. Tissue-engineered approaches may provide promising alternatives to the current clinical protocol and would benefit greatly from concurrent development of graft quality assessment techniques. This study was designed to evaluate whether viability of tissue-engineered islet grafts can be assessed using fluorine magnetic resonance spectroscopy ( 19F-MRS), by the noninvasive measurement of oxygen partial pressure (pO 2) and the subsequent calculation of islet oxygen consumption rate (OCR). Methods Scaffolds composed of porcine plasma were seeded with human islets and perfluorodecalin. Each graft was covered with the same volume of culture media in a Petri dish. Four scaffolds were seeded with various numbers (0–8000) of islet equivalents (IE) aliquoted from the same preparation. After randomizing run order, grafts were examined by 19F-MRS at 37°C using a 5T spectrometer and a single-loop surface coil placed underneath. A standard inversion recovery sequence was used to obtain characteristic 19F spin-lattice relaxation times (T1), which were converted to steady-state average pO 2 estimates using a previously determined linear calibration ( R 2 = 1.000). Each condition was assessed using replicate 19F-MRS measurements ( n = 6–8). Results Grafts exhibited IE dose-dependent increases in T1 and decreases in pO 2 estimates. From the difference between scaffold pO 2 estimates and ambient pO 2, the islet preparation OCR was calculated to be 95 ± 12 (mean ± standard error of the mean) nmol/(min · mg DNA) using theoretical modeling. This value compared well with OCR values measured using established methods for human islet preparations. Conclusions 19F-MRS can be used for noninvasive pre- and possibly posttransplant assessment of tissue-engineered islet graft viability by estimating the amount of viable, oxygen-consuming tissue in a scaffold.

A Precise Analysis of C5a Inhibitory Peptide on Inflammatory Mediators Induced After Islet Transplantation

Objective We recently reported that C5a inhibitory peptide (C5aIP) prevents the instant blood-mediated inflammatory reaction by attenuating cross talk between complement and coagulation cascades. C5aIP has also been shown to possess a broad range of anti-inflammatory effects. Due to methodological limitations, it is difficult to perform detailed analyses on wide range of inflammatory mediators in rat model. Therefore, we examined whether C5aIP suppressed various inflammatory cytokines induced after islet transplantation using a mouse model. Methods Six islet equivalents per gram of syngeneic mouse islet grafts were transplanted intraportally into two groups of streptozotocin-induced diabetic mice: control group ( n = 8) and C5aIP group ( n = 6). The C5aIP group was treated with a bolus of 4 mg/kg just after islet infusion and a continuous infusion of 0.4 mg/kg/h, whereas the control group was injected with equivalent amounts of saline. Serum samples were collected at 0, 6, and 24 hours after transplantation. We analyzed 23 types of cytokines: interleukins-1a, -1b, -3, -4, -5, -6, -9, -10, -12, -13, and -17; eotaxin; G-CSF; GM-CSF; interferon (INF) gamma; KC; MCP-1; MIP-1 and -1b, RANTES and tumor necrosis factor-alpha. Leukocytes in the recipient liver were isolated at 6 hours after transplantation to examine IFN gamma production by fluorescence activated cell sorting (FACS). Results No significant difference was detected in terms of the major inflammatory cytokines between the two groups. INF gamma production on CD11b +Gr-1 + cells in the liver was not significantly inhibited by C5aIP (control 30.0% vs C5aIP 24.1%). Conclusions These data suggested that beneficial effects of C5aIP on islet engraftment are mainly due to blockade of cross talk between complement and coagulation cascades, rather than the suppression of inflammatory mediators.

Cellular Immunoisolation for Islet Transplantation by a Novel Dual Porosity Electrospun Membrane

Immunoisolation strategies have the potential to impact the treatment of several diseases, such as hemophilia, Parkinson's and endocrine disorders, such as parathryroid disorders and diabetes. The hallmark of these disease states is the amelioration of the disease process by replacement of the deficient protein. Naturally, several cellular therapeutic strategies like genetically modified host cells, stem cells, donor cells, or even complex tissues like pancreatic islets have been investigated. Current evidence suggests that successful strategies must incorporate considerations for local hypoxia, vascularity, and immunoisolation. Additional regulatory concerns also include safe localization of implanted therapeutic cells to allow for monitoring, dose adjustment, or removal when indicated. Local hypoxia and cellular toxicity can be detrimental to the survival of freshly implanted pancreatic islets, leading to a need for a larger initial number of islets or repeated implantation procedures. The lack of adequate donors and the large number of islet equivalents needed to achieve euglycemic states amplify the nature of this problem. We have developed a novel immunoisolation device based on electrospun nylon, primarily for islet transplantation, such that the inner component functions as a cellular barrier while allowing diffusion, whereas the outer component can be optimized for tissue integration and accelerated vascularization. Devices explanted after subcutaneous implantation in wild-type B6 mice after a period of 30 days show vascular elements in the outer layer of the electrospun device. The inner layer when intact functioned as an effective barrier to cellular infiltration. The preimplantation of such a device, with a relatively thin inner barrier membrane, will allow for adequate vascularization and reduce postimplantation hypoxia. This study demonstrates the feasibility of an electrospun isolation device that can be easily assembled, modified by varying the electrospinning parameters, and functionalized with surface-active molecules to accelerate vascularization.

Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend

Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend

Glial Cell Line–Derived Neurotrophic Factor Enhances Human Islet Posttransplantation Survival

Development of pretransplantation islet culture strategies that preserve or enhance β-cell viability would eliminate the requirement for the large numbers of islets needed to restore insulin independence in type 1 diabetes patients. We investigated whether glial cell line-derived neurotrophic factor (GDNF) could improve human islet survival and posttransplantation function in diabetic mice. Human islets were cultured in medium supplemented with or without GDNF (100 ng/mL) and in vitro islet survival and function assessed by analyzing β-cell apoptosis and glucose stimulated insulin release. In vivo effects of GDNF were assessed in streptozotocin-induced diabetic nude mice transplanted under the kidney capsule with 2000 islet equivalents of human islets precultured in medium supplemented with or without GDNF. In vitro, human islets cultured for 2 to 10 days in medium supplemented with GDNF showed lower β-cell death, increased Akt phosphorylation, and higher glucose-induced insulin secretion than islets cultured in vehicle. Human islets precultured in medium supplemented with GDNF restored more diabetic mice to normoglycemia and for a longer period after transplantation than islets cultured in vehicle. Our study shows that GDNF has beneficial effects on human islet survival and could be used to improve islet posttransplantation survival.

Validation of Islet Transport From a Geographically Distant Isolation Center Enabling Equitable Access and National Health Service Funding of a Clinical Islet Transplant Program for England.

Islet transplantation has become established as a successful treatment for type 1 diabetes complicated by recurrent severe hypoglycemia. In the UK access has been limited to a few centrally located units. Our goal was to validate a quality-assured system for safe/effective transport of human islets in the UK and to successfully undertake the first transplants with transported islets. Pancreases were retrieved from deceased donors in the north of England and transported to King's College London using two-layer method (TLM) or University of Wisconsin solution alone. Islets were isolated and transported back to Newcastle in standard blood transfusion or gas-permeable bags with detailed evaluation pre- and posttransport. In the preclinical phase, islets were isolated from 10 pancreases with mean yield of 258,000 islet equivalents. No significant differences were seen between TLM and University of Wisconsin solution organ preservation. A significant loss of integrity was demonstrated in islets shipped in gas-permeable bags, whereas sterility, number, purity, and viability were maintained in blood transfusion bags. Maintenance of secretory granules and glucose-stimulated insulin secretion was confirmed following transport. A Standard Operating Procedure enabling final pretransplant quality control from a simple side-arm sample was validated. Moreover, levels of insulin and cytokines in transport medium were low, enabling transplant without centrifugation/resuspension at the recipient site. Six clinical transplants of transported islets were undertaken in five recipients with 100% primary graft function and resolution of severe hypoglycemia. Safe and clinically effective islet transport has been established facilitating sustainable NHS funding of a clinical islet transplant program for the UK.

Paramagnetic microparticles do not elicit islet cytotoxicity with co‐culture or host immune reactivity after implantation

Paramagnetic microparticles (MPs) may be useful in pancreatic islet purification, in particular purification of porcine islets as a potential xenotransplantation product. We assessed whether MPs affect islet function or induce an adverse effect following implantation. Porcine islets were co-cultured with 0, 500, and 1500 MPs per islet equivalent (IE) for 1 day and with 0 and 1500 MPs/IE for 7 days. Fractional viability was assessed using oxygen consumption rate normalized to DNA content (OCR/DNA) and after 7-day co-culture by perifusion glucose-stimulated insulin secretion (GSIS) and by transplantation under the renal capsule of diabetic nude mice. To assess an inflammatory response or immune reaction, MPs (∼10(7)) were implanted under the renal capsule of C57BL/6 mice. No statistically significant differences were measured in OCR/DNA (mean ± SE) following 1-day co-culture with 0, 500, or 1500 MPs/IE (243.3 ± 4.5, 211.3 ± 8.1, or 230.6 ± 11.3 nmol/min·mgDNA, respectively) or following 7-day co-culture with 0 or 1500 MPs/IE (248.5 ± 1.4 or 252.9 ± 4.7 nmol/min·mgDNA, respectively). GSIS was not affected by the presence of MPs; first- and second-phase insulin area-under-the-curve (mean ± SE) reflected no statistically significant differences after 7-day co-culture between 0 and 1500 MPs/IE (8.36 ± 0.29 and 8.45 ± 0.70 pg/ml·min·ngDNA for first-phase; 69.73 ± 2.18 and 65.70 ± 4.34 pg/ml·min·ngDNA for second-phase, respectively). Islets co-cultured with MPs normalized hyperglycemia in diabetic nude mice, suggesting no adverse effects on in vivo islet function. Implantation of MPs did not elicit tissue injury, inflammatory change or immune reactivity. MPs do not adversely affect islet viability or function during co-culture, and MPs are not immune reactive following implantation.

A Novel Method for Quantitative Monitoring of Transplanted Islets of Langerhans by Positive Contrast Magnetic Resonance Imaging

The Automatic Quantitative Ultrashort Echo Time imaging (AQUTE) protocol for serial MRI allows quantitative in vivo monitoring of iron labeled pancreatic islets of Langerhans transplanted into the liver, quantifying graft implantation and persistence in a rodent model. Rats (n = 14), transplanted with iron oxide loaded cells (0-4000 islet equivalents, IEQ), were imaged using a 3D radial ultrashort echo time difference technique (dUTE) on a Siemens MAGNETOM 3T clinical scanner up to 5 months postsurgery. In vivo 3D dUTE images gave positive contrast from labeled cells, suppressing liver signal and small vessels, allowing automatic quantification. Position of labeled islet clusters was consistent over time and quantification of hyperintense pixels correlated with the number of injected IEQs (R² = 0.898, p < 0.0001), and showed persistence over time (5 months posttransplantation). Automatic quantification was superior to standard imaging and manual counting methods, due to the uniform suppressed background and high contrast, resulting in significant timesavings, reproducibility and ease of quantification. Three-dimensional coverage of the whole liver in the absence of cardiac/respiratory artifact provided further improvement over conventional imaging. This imaging protocol reliably quantifies transplanted islet mass and has high translational potential to clinical studies of transplanted pancreatic islets.

Agent-based model of instant blood-mediated inflammatory reaction effects on immediate graft loss after intraportal islet cell transplantation

The purpose of this study was to investigate the mechanisms involved in the development of instant blood-mediated inflammatory reaction (IBMIR) triggered by pancreatic islet transplantation in an in vitro system.Pancreatic islets were prepared and blood was taken from male and female Wistar rats. An in vitro heparinized tubing loop model on a rocking device that was placed in a 37°C incubator was used to generate blood flow inside the loops. Rat blood was added to the device, and after a 60-minute incubation period, 800 islet equivalents were added to the system. Rat blood samples were taken for immediate hematologic analysis before circulation, before the addition of islets, and 5, 15, 30, and 60 minutes after the addition of islets. After 60 minutes, the blood in the tubing was filtered, and the filtrate was stained using hematoxylin and eosin for microscopic analysis.Following the addition of islets, the counts of platelets, white blood cells, and mononuclear cells were significantly decreased at 60 min (P < .05), and the plasma levels of thrombin-antithrombin complexes and complement C3a were significantly increased (P < .05). At 60 minutes after introduction of the islets, the thrombi and tissue in the remaining blood in the device consisted of a few islets with serious structural damage and incomplete capsules, and there were many microthrombi with a large number of infiltrating neutrophils around the islets.Our in vitro model of IBMIR was characterized by platelet consumption, activation of the coagulation and complement cascade systems, and leukocytic infiltration.

Quantification of the Islet Product: Presentation of a Standardized Current Good Manufacturing Practices Compliant System With Minimal Variability

Accurate islet quantification has proven difficult to standardize in a good manufacturing practices (GMP) approved manner. The influence of assessment variables from both manual and computer-assisted digital image analysis (DIA) methods were compared using calibrated, standardized microspheres or islets alone. Additionally, a mixture of microspheres and exocrine tissue was used to evaluate the variability of both the current, internationally recognized, manual method and a novel GMP-friendly purity- and volume-based method (PV) evaluated by DIA in a semiclosed, culture bag system. Computer-assisted DIA recorded known microsphere size distribution and quantities accurately. By using DIA to evaluate islets, the interindividual manually evaluated percent coefficients of variation (CV%; n=14) were reduced by almost half for both islet equivalents (IEs; 31% vs. 17%, P=0.002) and purity (20% vs. 13%, P=0.033). The microsphere pool mixed with exocrine tissue did not differ from expected IE with either method. However, manual IE resulted in a total CV% of 44.3% and a range spanning 258 k IE, whereas PV resulted in CV% of 10.7% and range of 60 k IE. Purity CV% for each method were similar approximating 10.5% and differed from expected by +7% for the manual method and +3% for PV. The variability of standard counting methods for islet samples and clinical quantities of microspheres mixed with exocrine tissue were reduced with DIA. They were reduced even further by use of a semiclosed bag system compared with standard manual counting, thereby facilitating the standardization of islet evaluation according to GMP standards.