Pig Islet Isolation Research Articles

Adult Pig Islet Isolation.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

Early Morning Workshop 1: Technical Aspects of Pig Islet Isolation

Early Morning Workshop 1: Technical Aspects of Pig Islet Isolation

The Amphiphilic Oxygen Carrier Perfluorohexyloctane Improves Oxygenation of Ischemic Pig Pancreases and Increases the Quality of Isolated Islets Compared to Perfluorodecalin

Introduction: Ischemia is one of the most critical variables for success or failure of islet isolation and islet transplantation. Prolonged cold storage is associated with reduced yield and decreased viability of isolated islets. Technical equipment for continuous vascular oxygen supply during cold storage of retrieved organs is expensive and requires trained personnel for operation. In the present study we assessed the efficiency of the amphiphilic compound perfluorohexyloctane (F6H8) to provide oxygen during static cold storage of pancreases retrieved from adult non-heartbeating pigs for subsequent islet isolation. Methods: Pancreases (n=10) were explanted after 15 min of warm ischemia and dissected ex-situ into the connecting and spleenic lobe. After intraductal perfusion with cold UW-solution the pancreatic segments were immersed in oxygen-precharged F6H8 or perfluorodecalin (PFD) and stored on ice for 9-10 hours until processing for islet isolation and purification. After culture at 37°C for 2-3 days islets were subjected to extensive quality assessment. All data are presented as mean ± SEM and were analysed by Wilcoxon test. Results: Pancreatic segments stored in oxygen-precharged F6H8 had a significantly higher intrapancreatic pO2 compared to preoxygenated PFD (10.1±3.9 vs. 1.6±1.1 mm Hg, p< 0.05). This finding corresponds to the significantly increased ATP content in islets isolated from segments stored in F6H8 (290±60 vs 203±31 pg/ng DNA, p< 0.01). Although freshly isolated islet yield per gram trimmed tissue did not significantly differ between F6H8 and PFD (3020±330 vs. 2940±270 IEQ/g) significantly more islets were recovered after culture when isolated from F6H8-stored lobes (57±6 vs. 39±6%, p< 0.01). Lower viability (76±3 vs. 82±3%, p< 0.05) measured in islets after PFD storage correlated with their inability to downregulate insulin release after switching from high (20 mmol/L) to basal (2 mmol/L) glucose concentration during static glucose incubation (Fig. 1).[Figure 1]Figure 1. Insulin release of islets isolated from ischemic porcine pancreatic segments after 9-10 hours of cold storage in oxygenprecharged PFD or F6H8. Islets were sequentially stimulated by 2 (white bars), 20 (grey bars) and again 2 mmol/L of glucose. *p< 0.05, **p< 0.01 as indicated. Conclusions: In the case that pig races, suitable for successful islet isolation, are locally not available, pancreas preservation for subsequent islet isolation can be significantly improved utilizing oxygen-precharged F6H8 for organ shipment. The same applies if pathogen-free pig pancreases have to be processed in institutions highly experienced in pig islet isolation.

Histomorphometrical Analysis of Porcine Islets of Langerhans

Xenotransplantation of pig islets of Langerhans may be an efficient method for treatment of type I diabetic patients. Considering the shortage of human pancreas donors, pigs offer readily available islet source. Unfortunately, their morphologic characteristics makes isolation of pig islets of Langerhans extremely difficult. The selection of a suitable donor may favorably affect the success of isolation. Therefore, we examined pig breeds and weights for their influence histomorphometric features of Langerhans islets. Pancreatic tissue biopsy specimens were obtained from Polish Large White pigs (n = 5), Polish Landrace pigs (n = 8), Pietrain pigs (n = 4), and marked-weight pigs of 100 kg (n = 8) or more than 200 kg (n = 10). Biopsy specimens were fixed and stored in 4% formaldehyde. Tissue sections were stained with polyclonal guinea pig anti-insulin antibody. The number of islets per 1 cm 2 was counted in each section. Mean diameters and areas of the islets were measured to calculate the percentage volume density. Differences were observed among the groups of pigs weighing 200 kg and 100 kg in islet mean diameter and area. The number of islets/cm 2 was higher among Pietrain than Polish Landrace pigs (425.45 ± 111.7 versus 315.33 ± 36.59 islet/cm 2, P < .05). Furthermore, Pietrain pigs showed the largest volume density (1.44%). Histological analysis revealed that Pietrain pigs of 100 kg and market-weight pigs of 200 kg were the best donors for islets of Langerhans.

Quality criteria for porcine islet cells suitable for xenotransplantation

Introduction: Intact functional pancreatic islets can be successfully isolated from various domestic pig breeds, including some minipig strains. Nevertheless, the technically demanding pig islet isolation procedure needs further improvements. Few efforts have been made to determine the underlying mechanisms for the greatly varying islet yields in many domestic pig breeds. This problem also relates to Landrace Pigs that are often used as donor animals to insert different human genes into the pig genome in order to overcome the various immunological reactions in response to transplantation of a porcine xenograft.Methods: To obtain a clearer picture on the efficiency of islet yields, we evaluated the number of islets, their morphology and intensity of insulin staining in pancreata of various brain‐dead pigs of crossbred and pure bred strains. We performed histochemistry with frozen tissue sections and anti‐pig‐insulin antibody and applied an evaluation procedure, based upon &gt;1.600 microscopically screened pancreata, &gt;800 isolations, and sub‐sequent functional in vivo and in vitro assays.Results: In first series 40 pancreata (tail end of the splenic lobe) of both sexes of the following donor animals were investigated: Piétrain [PI] × German Landrace Pig [GL] (n = 5), German Pure Bred Pig [GPB] x GL (n = 3), PI x GL/GPB (n = 9), GL (n = 12), GPB (n = 3), PI (n = 7), and Duroc (n = 1). Pigs were 6‐7 months old, and were fed and kept under identical conditions. Only three of 40 pancreata (7.5%) displayed islets of sufficient quality for subsequent isolation [data not shown], i.e. sufficient numbers of mostly rounded islets of ≥200 μm in diameter, displaying intensive and evenly distributed insulin staining. Each of the 3 “good” donor organs belonged to a different pig breed. This test was repeated a second time, using 50 pancreata from brain‐dead pigs (both sexes) collected in a Bavarian pig breeding institution: PI × GL (n = 16), PI × GL/GPB (n = 16), GL × GPB (n = 5), GL (n = 12), GPB (n = 2), PI (n = 1). Age, keeping and feeding conditions were identical with conditions of the first trial. Overall, four of 50 pancreata (8%) were determined to be suitable for subsequent islet isolation [not shown here], according to our microscopic evaluation criteria. Again, those four “good” pancreata belonged to four different pig breeds, i.e., a correlation between a particular pig breed and “good” islets could not be detected. In both tests, pancreata that displayed islets with large insulin‐free areas, pancreata with insufficient islet numbers, and islets that displayed very weak insulin staining, as compared to positive control islets, were determined as being unsuitable for islet isolation.Conclusions: Only 7.5–8% of domestic pig pancreata (n = 90) appear to be suitable for islet isolation; in previous years this number was 35–50%. The reasons for this continuous decline in suitable donor organs are presently unknown. None of the crossbreeds and pure breeds tested here displayed a genetic background that correlated with either “good” or “bad“ islets; sufficient animal numbers from pure bred pig strains could clarify this important point. A high versus low caloric diet could help to elucidate the possible influence of nutrients on islet development. From preliminary findings, we hypothesise that a certain amount of carbohydrate feeding may be beneficial in stimulating islet morphology and also islet function.

Parameters for successful pig islet isolation as determined using 68 specific‐pathogen‐free miniature pigs

Islet cell transplantation is a novel therapeutic modality for the cure of diabetes. Pig islet cells are an attractive substitute for human islet cells; however, they are known to be particularly difficult to isolate because of a weak islet capsule and a tendency to be fragmented during enzymatic digestion. Therefore, parameters favoring successful pig islet isolation were investigated using specific-pathogen-free (SPF) miniature pigs. Sixty-eight SPF miniature pigs were used for islet isolation. Birth weight, body weight, age, sex, pregnancy history, and the fasting blood glucose levels of each pig were determined. Each pig's general condition was assessed with regard to feeding status and physical activity. Pancreas procurement was performed by one surgical team. Anesthesia duration, operation duration, procedure quality, and perfusate type were recorded. After pancreatectomy, a biopsy was performed for islet density analysis. Decapsulation, cannulation duration, degree of distension, and cold ischemic time were assessed. During islet isolation, pancreas weight, digestion time, and digested tissue proportion were recorded. Isolation results were evaluated by total islet equivalents (IEQ), islet equivalents per gram of pancreas (IEQ/g), isolation index, islet recovery rate, purity, and visual grade. To identify the predictors of higher islet isolation yield, we performed binary logistic regression analysis with significant (P < 0.05) variables from the univariate analysis. The pigs were categorized into high (n = 34) and low yield (n = 34) groups according to the median IEQ/g or total IEQ values. Body weight and age were significantly different between the two groups. Being male or a positive history of pregnancy in females was factors favoring successful islet isolation. General condition assessments failed to estimate islet isolation results. Long anesthesia duration, which might have caused ischemic injury to the pancreas, negatively affected islet isolation results. Decapsulation, cannulation duration, and subsequent pancreas distension were significantly important in successful islet isolation. Inter-lot variability of Liberase was not observed because of screening processes performed before purchase. Isolation index and islet recovery rate correlated well with islet yields. Multivariate analysis using total IEQ and IEQ/g as outcome variables indicated that age older than 2, being male and moderate distension by Liberase injection are major determinants influencing successful islet isolation.

Use of the Continuous Glucose Monitoring System in Goettingen Minipigs, With a Special Focus on the Evaluation of Insulin-Dependent Diabetes

Objectives Adult pig islet isolation has greatly improved in the past few years. Islet grafts may now be tested in large animals. Continuous Glucose Monitoring System (CGMS) was applied to diabetic Goettingen Minipigs (GMP) to improve the management of hyperglycemia and hypoglycemia and their welfare before transplantation. Methods GMP (25–35 kg) received a minipig diet once daily. Diabetes was induced by streptozotocin (STZ; 150 mg/kg intravenous [IV]; n = 5) or by surgical pancreatectomy (PGMP; n = 3). Interstitial glucose concentration (IGC) was monitored continuously with an implanted sensor; CGMS was calibrated using conventional blood glucose tests 3–4 times per day; CGMS data were fed into the monitor memory and analyzed using CGMS software. Results Glucose sensors were handled accurately. Diabetes occurred 2–3 days after STZ or immediately after pancreatectomy with basal C-peptide secretion of <0.4 ng/mL (measured using intravenous glucose tolerance test) and prompt loss of body weight. Insulin substitution was necessary to keep the GMP in good condition for up to 5–6 months, with stable body weight and normal behavior. Some GMP became hypoglycemic, which was only documented by CGMS, but not by conventional glucose assays. Tight glucose control and substitution of exocrine enzymes (Creon 25,000 E/d) reduced morbidity of the PGMP, which was then comparable with that of STZ-GMP. Conclusions The CGMS, developed for humans, is equally suitable for the 2 GMP diabetes models. Close-meshed glucose monitoring and insulin treatment improved the general condition of the diabetic GMP, ie, the islet graft recipients, and will thus greatly add to posttransplantation success.

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig‐to‐primate models

In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin-producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre-clinical results and data in the pig-to-primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K+/Na+ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15,000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P < 0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short-term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm2 (r = 0.708, P < 0.01) or with the percentage of large islets (r = 0.680, P < 0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm2 and more than 42% of large islets (>100 microm) thus enabled more than 120,000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C-peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post-transplantation, however, loss of islet function was associated with graft destruction and immune reaction. Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.

Successful Pancreas Preservation by a Perfluorocarbon-Based One-Layer Method For Subsequent Pig Islet Isolation

The oxygenation of human pancreas by the two-layer method (TLM) during cold storage was recently established for clinical islet transplantation. Simplification of TLM would facilitate the application of perfluorocarbon (PFC) as a regularly used preservation solution for subsequent islet transplantation. The present study examined whether PFC can be used in a one-layer method (OLM) for long-term pancreas preservation before isolation of adult pig islets. Resected pancreases were intraductally flushed with cold University of Wisconsin solution and immediately processed (n=6) or subjected to 7-hour storage by OLM (n=8) or TLM (n=10). Subsequently, pancreases were intraductally distended with collagenase NB-8 supplemented with neutral protease. Isolation and purification were performed as previously described. Compared with unstored pancreases (3,670+/-740 islet equivalents [IEQ]) purified islet yield in TLM-stored organs (2,080+/-290 IEQ, P<0.05) was significantly decreased in contrast with OLM-preserved pancreases (3,110+/-520 IEQ, NS). No differences were observed between groups regarding purity (>90%), trypan-blue exclusion (>95%), adenosine triphosphate content, and mitochondrial viability of islets. Stimulation index during static glucose incubation (20 vs. 2.8 mm) was decreased after storage by TLM (1.81+/-0.20, P<0.05) but not by OLM (2.27+/-0.57) if compared with unstored pancreases (2.47+/-0.36). However, transplantation into diabetic nude mice resulted in sustained normoglycemia of recipients of either group until nephrectomy of graft-bearing kidneys was performed. This study demonstrates that PFC alone can be used in a one-layer procedure for successful pig-pancreas preservation. This simplification can facilitate the broad application of PFC as pancreas preservation solution without reducing its benefits demonstrated by TLM.

LONG-TERM PRESERVATION OF THE PIG PANCREAS BY THE ONE-LAYER METHOD FOR SUCCESSFUL ISLET ISOLATION

P33 Aims: The oxygenation of the human pancreas during cold storage was recently established for clinical islet transplantation by introduction of the two-layer method (TLM). Nevertheless, simplification of TLM would facilitate its application as a broadly used pancreas preservation method for subsequent islet transplantation. Preliminary data about vascularized pancreas transplantation in rats and dogs indicate that successful preservation by a one-layer method (OLM) omitting UW solution (UWS) as the second layer is also possible. The purpose of the present study was therefore to examine the efficiency of OLM for pig islet isolation after 7 hours of cold ischemia. Methods: After resection adult pig pancreata were intraductally flushed with 2 mL/g of cold UWS. Subsequently, pancreata were either continuously oxygenated during cold ischemia by the TLM (n=10) or OLM (n=8), or processed promptly (n=6). Immediately before digestion-filtration pancreata were intraductally distended with cold UWS supplemented with Collagenase NB 8 and Neutral Protease NB (SERVA). Isolated islets were isolated and purified as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin and ATP content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: No significant differences were found regarding islet purity (>90%) and viability (>95%). Compared to freshly procured pancreata (429200±86700 IEQ) postpurification islet yield was significantly decreased (P<0.05) after prolonged cold storage by TLM (238000±26600 IEQ). In contrast, islet yield was significantly higher (P<0.05) in cold-stored organs using OLM (338600±42100 IEQ, NS vs fresh). Metabolic islet integrity was not different between unstored pancreata and organs preserved by OLM or TLM as indicated by the intracellular insulin content (710±130 vs 830±120 vs 750±70 μU/IEQ), formazan production (0.7±0.1 vs 0.6±0.1 vs 0.7±0.1 OD/1000 IEQ) and ATP content (390±70 vs 370±80 vs 410±170 pg/IEQ). In contrast, glucose stimulation index was significantly decreased in islets isolated after TLM-storage (1.8±0.2, P<0.05) but not after OLM-storage (2.3±0.6, NS) compared to freshly isolated islets (2.5±0.4). Transplantation into diabetic nude mice demonstrated islet graft function in all experimental groups. Conclusions: The present study demonstrates clearly that viable pig islets can be successfully isolated from long-term stored pig pancreata utilizing perfluorochemicals as a single layer for oxygenation of pancreatic tissue during cold storage. This methodological simplification of the TLM might be of relevance for broad application in clinical islet transplantation.

Isolation of adult porcine islets of Langerhans.

While human islet allotransplantation is limited due to the shortage of donors, pig islet xenotransplantation appears to be a possible alternative. Enzymatic digestion is the method most commonly used for adult pig islet isolation, but since the description of the semiautomated isolation technique in the late 1980s, yields of pig islet isolations have not increased significantly. This review article is intended to collate published information on adult pig islet isolation to provide the reader with a clear overview of the relevant issues in this field, and to detail the current state of the art.

Improved outcome of pig islet isolation by pefabloc inhibition of trypsin

Improved outcome of pig islet isolation by pefabloc inhibition of trypsin

Islet Isolation from the porcine pancreas, using a continuous digestion method

Abstract: This paper describes a method for pig islet isolation with the following original features: first, we set up a reproducible, rapid, sterile, and simple method for pancreas procurement and preparation with double duct incannulation. Second, we demonstrated that UW solution can be used for pancreas digestion; third, we designed an original method for pancreas digestion, derived from the method originally described by Ricordi, except that a double digestion circuit made it possible to digest continuously, within one step, the pancreas, which makes the method operator‐independent; finally we designed a simple method for islet purification based on a two‐layer Histopaque/ UW density gradient and the use of the COBE 2991 cell processor.