Islet Purification Research Articles

Automated Analysis of Microscopic Images of Isolated Pancreatic Islets.

Clinical islet transplantation programs rely on the capacities of individual centers to quantify isolated islets. Current computer-assisted methods require input from human operators. Here we describe two machine learning algorithms for islet quantification: the trainable islet algorithm (TIA) and the nontrainable purity algorithm (NPA). These algorithms automatically segment pancreatic islets and exocrine tissue on microscopic images in order to count individual islets and calculate islet volume and purity. References for islet counts and volumes were generated by the fully manual segmentation (FMS) method, which was validated against the internal DNA standard. References for islet purity were generated via the expert visual assessment (EVA) method, which was validated against the FMS method. The TIA is intended to automatically evaluate micrographs of isolated islets from future donors after being trained on micrographs from a limited number of past donors. Its training ability was first evaluated on 46 images from four donors. The pixel-to-pixel comparison, binary statistics, and islet DNA concentration indicated that the TIA was successfully trained, regardless of the color differences of the original images. Next, the TIA trained on the four donors was validated on an additional 36 images from nine independent donors. The TIA was fast (67 s/image), correlated very well with the FMS method (R2=1.00 and 0.92 for islet volume and islet count, respectively), and had small REs (0.06 and 0.07 for islet volume and islet count, respectively). Validation of the NPA against the EVA method using 70 images from 12 donors revealed that the NPA had a reasonable speed (69 s/image), had an acceptable RE (0.14), and correlated well with the EVA method (R2=0.88). Our results demonstrate that a fully automated analysis of clinical-grade micrographs of isolated pancreatic islets is feasible. The algorithms described herein will be freely available as a Fiji platform plugin.

Evaluation of isolated caprine pancreatic islets cytoarchitecture by laser scanning confocal microscopy and flow cytometry

Pancreatic islets are composed of different hormone-secreting cell types. A finely balanced combination of endocrine cells in the islets regulates intraportal vein secretions and plasma nutrient levels. Every islet cell type is distinguished by its specific secretory granule pattern and hormone content, endocrine and cell signaling mechanisms, and neuronal interactions. The scarcity of pancreatic islet donors for patients with diabetes has caused a considerable interest in the field of islet xenotransplantation. Previous studies have shown that cell arrangement in the pancreatic islets of ruminants differs from that of other mammals; however, caprine islet cytoarchitecture has not yet been comprehensively described. This investigation aimed to characterize caprine islets in regard to better understanding of caprine islet structure and compare with previously reported species, by conducting a detailed analysis of islet architecture and composition using confocal microscopy and immunofluorescence staining for pancreatic islet hormones. After collection and purification of caprine islets with Euro-Ficoll density gradients, islets were considered for viability and functionality procedures with DTZ (dithizone) staining and GSIST (glucose-stimulated insulin secretion test) subsequently. Batches of islet were selected for immunostaining and study through confocal microscopy and flow cytometry. Histological sections of caprine pancreatic islets showed that α-cells were segregated at the periphery of β-cells. In caprine islets, α- and δ-cells remarkably were intermingled with β-cells in the mantle. Such cytoarchitecture was observed in all examined caprine pancreatic islets and was also reported for the islets of other ruminants. In both small and large caprine islets (< 150 and > 150 μm in diameter, respectively), the majority of β-cells were positioned at the core and α-cells were arranged at the mantle, while some single α-cells were also observed in the islet center. We evaluated the content of β-, α-, and δ-cells by confocal microscopy (n = 35, mean ± SD; 38.01 ± 9.50%, 30.33 ± 10.11%, 2.25 ± 1.10%, respectively) and flow cytometry (n = 9, mean ± SD; 37.52 ± 9.74%, 31.72 ± 4.92%, 2.70 ± 2.81%, respectively). Our findings indicate that the caprine islets are heterogeneous in cell composition. The difference could be attributed to species-specific interaction between endocrine cells and blood. Comparative studies of islet architecture may lead to better understanding of islet structure and cell type population arrangement. These results suggest the use of caprine islets as an addition to the supply of islets for diabetes research.

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability.

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome.

Isolation of Mouse Pancreatic Islets of Langerhans.

The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol.

Islet Transplantation in Pediatric Patients: Current Indications and Future Perspectives.

The first islet transplantation in diabetes mellitus was performed more than 20 years ago. Since then, clinical results have progressively improved. Nowadays, islet transplantation can be considered a real therapeutic option after pancreatectomy for painful chronic pancreatitis (autotransplantation) and in selected adult patients affected by type 1 diabetes mellitus (allotransplantation). Better results are mainly due to the advances in the standardization of islet isolation and purification procedures as well as in the pharmacological treatment of recipients. Anti-inflammatory treatments facilitate islet engraftment and prevent metabolic exhaustion and functional β-cell apoptosis; new strategies better control islet graft rejection. As a consequence, islet transplantation activities are no longer confined to few centers only, rather thousands of transplants are now performed all over the world. Many attempts are actually undertaken to find solutions to current problems of islets transplantation, from toxicity of immunosuppressive therapy to the limited engraftment, function and duration. There is general hope that these procedures will offer a safe and feasible therapeutic option for an increasing number of patients suffering from diabetes mellitus, including pediatric patients.

IMPACT OF PHARMACOLOGICAL AGENTS ON INSULIN SECRETION CELLS TRANSPLANTED IN DIABETIC RATS

Cyclosporin A is an immunosuppressive agent that improves survival of transplant. This exciting immunosuppressive agent was first used clinically in renal transplantation (Ferguson and Fidlus 1982).The aim of this study was the isolation , purification and transplantation of hamster pancreatic islet as xenograft transplantation for the treatment of diabetic rats. It is also aimed to study the effect of cyclosporin A as an immunosuppressive agent on some biochemical parameters e.g blood glucose and blood insulin levels to induce maximum suppression of mice immune system and consequently realizing maximum survival of transplanted islets. A total of 30 streptozotocin induced rat were randomized to receive islets xenografts from golden syrian hamsters by three different approaches ,the first group of ten rats received islets only in the renal subcapsular space, the 2nd of ten rats received islets in the renal subcapsular space and was given cyclosporin A in a dose of 30 mg/kg/day for three days only.The 3rd group of ten rats were received islets in the renal subcapsular space and were given cyclosporin A orally every day at a dose of 30 mg/kg/day for three days which was gradually decreased to 10 mg/kg/day. Non of rats of group 1 became normoglycemic. All rats of group 2 became normoglycemic for 228± ( range 12-36 days) . Only rats of group 3 enjoyed normoglycemic as long as cyclosporin-A was administrated. Consequantly, prolonged survival of islets xenografts may be achieved with administration of cyclosporin-A (Springer,et al., 2015).

51. Humidified oxygen gas perfusion (persufflation) in organ preservation and cryobanking

It is widely recognized that exposure to ischemia and its metabolic and sequelae during procurement and preservation results in tissue injury and/or death and that normothermic reperfusion during transplantation further amplifies the damage. This is also now a recognized issue in organ cryopreservation with data suggesting that inadequately preserved (damaged) organs may not be successfully cryopreserved and rewarmed for transplantation. Published literature reports demonstrate that enhanced oxygenation during organ preservation for kidney, heart, pancreas, and liver, results in improved organ viability, leading to better short and long-term outcomes including limiting chronic rejection in a porcine kidney and heart transplant model. Persufflation (PSF) the flow of hypothermic humidified oxygen gas through the vasculature of organs has been shown to be effective in oxygenating and preserving as well as reconditioning a variety of organs including kidneys, livers, hearts, and pancreata from expanded criteria donors (including Donors after Cardiac Death) in a variety of small and large animal models with excellent outcomes. Persufflation is currently in a clinical trial for reconditioning (after hypothermic static storage) and transplanting livers that would have otherwise been discarded in Europe. PSF also extended significantly the allowable window for organ preservation (for example 14 h instead of 4–6 for the heart and 24 h instead of 8–12 h for the pancreas) without compromising outcomes. However, perhaps due to the unavailability of a “turnkey” portable persufflator and the requirement for use of pressurized oxygen gas cylinders, PSF has not been widely used clinically and has been until recently limited to reconditioning organs after prolonged cold ischemia times (shipped on hypothermic static storage). The development and current availability of a novel portable battery operated electrochemical oxygen generator (“persufflator”) that can be used during flight and deliver humidified oxygen gas to the vasculature of organs may change that. This PSF has been extensively tested with porcine and human pancreata and to a limited extent with porcine kidneys, livers, hearts and composite tissues. It is currently in pilot clinical testing in Canada for pancreas preservation prior to rewarming for digestion and islet purification for clinical islet allo transplantation. In this paper we present we discuss the utility of PSF in organ preservation and in particular organ (re) conditioning prior to cryopreservation as well as during thawing and rewarming prior to transplantation. Financial Support: NIH/NIDDK SBIR R44DK070400.

Application of Digital Image Analysis to Determine Pancreatic Islet Mass and Purity in Clinical Islet Isolation and Transplantation.

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely assessed by estimation only. In this study, we validated our digital image analysis (DIA) system by using the software of Image Pro Plus and a custom-designed Excel template to assess islet mass and purity to better comply with current good manufacturing practice (cGMP) standards. Human islet samples (60 collected from a single isolation and 24 collected from 12 isolations) were captured as calibrated digital images for the permanent record. Seven trained technicians participated in determination of IEQ and purity by the manual counting method (manual image counting, Manual I) and DIA. IEQ count showed statistically significant correlations between the Manual I and DIA in all sample comparisons (r > 0.819 and p < 0.0001). A statistically significant difference in IEQ between Manual I and DIA was not found in all sample groups (p > 0.05). In terms of purity determination, statistically significant differences between assessment and DIA measurement were found in high-purity 100-µl samples (p < 0.005) and low-purity 100-µl samples (p < 0.001) of the single isolation. In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between Manual I and DIA. In conclusion, the DIA used in this study is a reliable technique to determine IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training, and information exchange among islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islet counting and purity estimation.

Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation.

Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure. Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age. There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases. The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Automated Digital Image Analysis of Islet Cell Mass Using Nikon's Inverted Eclipse Ti Microscope and Software to Improve Engraftment may Help to Advance the Therapeutic Efficacy and Accessibility of Islet Transplantation across Centers

Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method ( p &lt; 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number ( r2 = 0.91) and total islet number ( r2 = 0.88) and thus increased to r2 = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method ( p &lt; 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method ( p &lt; 0.001). The ADIA method also detected small islets between 10 and 50 μm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

Islet Product Characteristics and Factors Related to Successful Human Islet Transplantation From the Collaborative Islet Transplant Registry (CITR) 1999–2010

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999–2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p < 0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007–2010 when compared to 1999–2002 (445.4 ± 156.8 vs. 421.3 ± 155.4 ×103 IEQ; p < 0.05). Islet purity and total number of β cells significantly improved over the study period (p < 0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999–2010, and these parallel improvements in clinical outcomes over the same period.

Preface

It is my great pleasure and honor to participate in the launching of the Volume 3, issue 01 of our journal “Current Tissue Engineering”. I would like to thank and pay tribute to the hard work and dedication of the editorial board members and staff. Without them this would not be possible. Tissue engineering is a rapidly growing interdisciplinary field involving the biological, chemical, physical, and engineering sciences. It uses a combination of cells and biomaterials to generate new functional tissues for implantation to repair or replace damaged tissues due to trauma, disease, or aging. In this issue, we have 3 review articles on the updated information of use of adipose tissue for tissue engineering: “ Recent advances and challenges in adipose tissue engineering: Adipose derived stem cell-based approaches: (Author: A. Cimpean): Autologous Fat Transfer and Cell-Based Strategies for Soft Tissue Reconstruction (Authors: Senarath-Yapa et al.) and “The future of neuroregenerative therapy for Parkinson’s disease” (Authors: Sugaya & Hendrix). We also have 1 review article on Biomaterial: “Potential of tissue engineered blood vessel as model to study effect of flow and wall thickness on cellular communication” (Authors: Ragaseema et al.). We have the first time 3 research articles: “Tubulin, F-box/leucine-rich repeat protein 14, and type 1 procollagen C-peptide in bovine periosteal cells β Association” (Author: M. Akiyama); Islet purification for clinical islet transplantation. (Author: H. Noguchi): “NMR-Relaxation and PFG NMR Studies of Water Dynamics in Oriented Collagen Fibres with different degree of cross-linking”. (Authors: Rodin and Nikerov). In future issues, it is my hope that we will expand our scope to include both review and full-length articles on biomechanics, mechano-biology and cellular therapies in both animal models and clinical trials.

Impact of Hypothermic Preservation on Tissue Yield and Viability in Pig Pancreata

IntroductionChronic shortage of quality human cadaveric pancreata limits islet transplantation. Porcine islet xenotransplantation is being explored to increase the donor pool. For clinical-ready islets, centralized animal husbandry, Current Good Manufacturing Practice–regulated processing facilities, and organ transportation support are required. Amount of cold ischemia time (CIT) before isolation significantly affects transplantation. The goal of this study was to determine the maximum safe CIT of whole pancreata before islet isolation. Materials and MethodsPancreata were rapidly removed from Yorkshire pigs (age, 14–22 days) and stored in modified University of Wisconsin solution or in EuroCollins solution at 4°C. Pancreata were processed with <1 hour CIT (control) or stored for 4 or 12 hours before isolation. Islet yield and percent purity and viability were determined after 7 days of in vitro tissue culture and maturation. Samples from nonprocessed pancreata were collected and snap-frozen in liquid nitrogen at 0, 3, 6, 9, 12, 15, and 24 hours of preservation, then analyzed for adenosine diphosphate/adenosine triphosphate ratio as a measure of tissue energetics. ResultsUp to 12 hours in cold storage had no significant impact on overall islet yield after 7 days of in vitro culture compared with controls; islet yield at the end of the maturation process was 28,700 ± 500 islet equivalents per pancreas (mean ± SEM control yield, 30,300 ± 900 islet equivalents per pancreas); islet purity was 75 ± 5% compared with 74 ± 5% in controls. Islet viability was significantly reduced at 12 hours compared with controls (80 ± 6% vs 96 ± 5%; P < .05). The tissue adenosine diphosphate/adenosine triphosphate ratio was maintained within the first 6 hours (1.6 ± 0.1 to 1.8 ± 0.2; P = NS) but was markedly increased during the 24-hour study (3.3 ± 0.1 at 24 hours), indicating a progressive loss of adenosine triphosphate tissue stores. ConclusionsYoung pig pancreata can be hypothermically stored for up to 12 hours without affecting islet yield and purity; however, islet viability is reduced. These data highlight the need for uniform shipping parameters to standardize islet quality, ideally with CIT <6 hours.

Islet Preparation Purity Is Overestimated, and Less Pure Fractions Have Lower Post-Culture Viability Before Clinical Allotransplantation

BackgroundReplacement of β-cells with the use of isolated islet allotransplantation (IT) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36–72-hour culture period before IT. We examined 13 clinical islet preparations with ≥2 purity fractions to determine the effect of culture on viability. MethodsAfter standard islet isolation and purification, pure islet fractions were placed at 37°C with 5% CO2 for 12–24 hours and subsequently moved to 22°C, whereas less pure fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100–200 islet equivalents (IEQ)/cm2 adjusted for purity. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability (oxygen consumption rate normalized to DNA content [OCR/DNA] and membrane integrity). ResultsResults indicated that purity was overestimated, especially in less pure fractions. This was evidenced by significantly larger observed pellet sizes than expected and tissue amount as quantified with the use of a dsDNA assay when available. Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared with pure. The difference in viability between the 2 purity fractions may be due to a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin in the culture media. ConclusionsCurrent clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability before transplantation. Even though relatively small, the difference in viability is important because the amount of dead or dying tissue introduced into recipients may be dramatically increased, especially with less pure preparations.

Improved islet purity by the hypertonic-hypotonic method.

Islet purification is usually performed using the density gradient separation method, but the purity of islets is low because exocrine cells and the embedded islets are hard to remove by using only the density gradient method. The aim of this study was to establish a new islet purification process comprising a hypertonic-hypotonic treatment step followed by a density gradient centrifugation step to improve the purity of islets. The Plackett-Burman method was used to determine which factors had a significant influence on the purity of islets obtained after the hypertonic-hypotonic treatment step. The hypertonic solution concentration and the incubation time were both found to have a significant effect on islet purity. The purity of islets obtained using the modified purification process was significantly higher than that of islets obtained by density gradient alone (97% vs. 87.23%). Importantly, good cell viability and normal insulin secretion ability of islets were maintained following the modified purification. The new purification process allows isolation of islets with improved purity and does not compromise the viability or function of the islets.

Adult Porcine Islet Isolation Using a Ductal Preservation Method and Purification With a Density Gradient Composed of Histidine-Tryptophan-Ketoglutarate Solution and Iodixanol

BackgroundGiven the fragility of adult porcine islets, reduction of shearing stress in islet purification using histidine-tryptophan-ketoglutarate (HTK) solution and iodixanol could be an effective strategy. We examined the effect of ductal preservation with HTK solution and an islet purification protocol that utilizes HTK solution and iodixanol in adult porcine islet isolation. MethodsIslets were isolated with a modified Ricordi method using adult Prestige World Genetics (PWG) and Yucatan pigs. The discontinuous density gradient was composed of either HTK solution/iodixanol (n = 23, iodixanol group) or Hank's balanced salt solution (HBSS)/Ficoll (n = 17, Ficoll group). In the iodixanol group, ductal injection of HTK solution was performed before purification. ResultsIn PWG pigs, significantly higher islet yield after purification (3480 ± 214.2 islet equivalent [IEQ]/g, P = .003) and higher recovery rate (85.45% ± 3.49%, P = .0043) were obtained from the HTK/iodixanol group as compared to the HBSS/Ficoll group (1905 ± 323.2 IEQ/g, and 67.22% ± 4.77%, respectively). Similar results were obtained in Yucatan pigs with greater body weight. ConclusionDuctal preservation and iodixanol-based islet purification using HTK solution improved the yield of adult porcine islet isolation compared to the conventional method using HBSS and Ficoll. The results of this study support the feasibility of an adult porcine islet isolation protocol using HTK solution and iodixanol, which have the favorable physical properties.

An improved method for murine islet isolation and subcapsular kidney transplantation

Objective To present a series of islet transplantation in mice by improve the method of islet isolation and subcapsular kidney transplantation.Methods Islet isolation was completed by end of the common bile duct ligation,Perfusion of the pancreas by 3 ml collagenase V (2 g/L),filtration by 100 μm filter and purification by picking islets under microscopy.Islets' yield,purity,function of insulin secretion and effect of islet transplantation were evaluated compared with islets isolated by Ficoll density gradient separation method.Results Purification by filtration increased islet yield (P ＜ 0.01),and resulted in higher insulin secretion in vitro (P ＜ 0.01).Moreover,when syngeneic diabetic hosts were transplanted with a marginal islet mass,the number of islets purified by filtration which restore normoglycemia significantly was less than those isolated by Ficoll.Conclusion Islet filtration is a simple and rapid procedure for purification of islets that demonstrate improved functional mass.The islet transplantation model consisted by Islet filtration and subcapsular kidney transplantation might be widely used in the research of islet transplantation. Key words: Islet isolation; Subcapsular kidney transplantation; Filtration

The experiment study of islet isolation and purification from mice pancreas

Objective To investigate the stable and efficient method of islet isolation and purification from mouse pancreas.Methods Collagenase is injected into the common bile duct (means perfusion method) and the pancreatic pieces are enzymatically digested in a collagenase solution (means pieces method).Mouse islets were isolated by the two approaches of collagenase digestion and purificated by iodixanol and Ficoll density gradient centrifugation.The production and purity of the islets were checked by Dithizone immunofluorescence staining.The vitality of the islets was detected by inducing insulin secretion through glucose.Results Comparing the two approaches collagenase digestion,islet yield was significantly higher in the experiments of perfusion method,298.00 ± 15.58 versus 36.00 ± 3.89 islets (P ＜ 0.01).Comparing the Ficoll density gradient centrifugation,iodixanol density gradient centrifugation provided with purity ＞ 90％ (P ＞ 0.05) and greater vitality of islets,the insulin release (3.120 ± 0.046) versus (3.060 ± 0.426) μg/L (P ＞0.05) with 2.75 mmol/L concentration glucose and (11.36 ±0.82) versus (7.98 ±0.56) μg/L (P ＜0.05) with 16.50 mmol/L concentration glucose.Conclusion Collagenase perfusion method provided a greater number of islets and iodixanol purification obtained greater vitality of islets.Collagenase perfusion and iodixanol density gradient are desired method to a desired outcome of islet isolation in both quantity and quality from mice pancreas. Key words: Islet; Isolation; Purification

Evidence for Instant Blood-Mediated Inflammatory Reaction in Clinical Autologous Islet Transplantation

A nonspecific inflammatory and thrombotic reaction termed instant blood-mediated inflammatory reaction (IBMIR) has been reported when allogenic or xenogenic islets come into contact with blood. This reaction is known to cause significant loss of transplanted islets. We hypothesized that IBMIR occurs in patients undergoing total pancreatectomy followed by autologous islet transplantation (TP-AIT) and tested this hypothesis in 24 patients and in an in vitro model. Blood samples drawn during the peritransplant period showed a significant and rapid increase of thrombin-anti-thrombin III complex (TAT) and C-peptide during islet infusion, which persisted for up to 3 h, along with a decreased platelet count. A concomitant increase in levels of inflammatory proteins IL-6, IL-8 and interferon-inducible protein-10 was observed. An in vitro model composed of pure islets plus autologous blood also demonstrated significantly increased levels of TAT (p<0.05), C-peptide (p<0.05), tumor necrosis factor-alpha (p<0.05) and MCP-1 (p<0.05), as well as strong tissue factor expression in islets. Islet viability decreased significantly but was rescued by the presence of low-molecular-weight dextran sulfate. In conclusion, AIT-induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin independence rate in TP-AIT patients.