Porcine Islet Isolation Research Articles

Procedure of Adult Porcine Islet Isolation.

Islet transplantation is a useful therapeutic choice for severe diabetes mellitus; however, limited donor supplies have interfered with the use of this treatment. Therefore, the establishment of alternative donor sources and engineered tissue, which enables to produce appropriate insulin for controlling blood glucose, is an important challenge. The adult pig is a promising and feasible donor source and materials for the engineered tissue for the clinical setting among various candidates. The recent progress of gene-editing technology contributes to possible clinical porcine xenotransplantation, including porcine islet xenotransplantation. For the success of future clinical porcine islet xenotransplantation, establishing an islet isolation technique for acquiring adequate, good-quality porcine islets is equally important to use a gene-edited pig. However, the characteristics of porcine islets are different from other species; therefore, establishing a suitable technique for porcine islets is challenging. Impact statement Recent technological progress promotes the feasibility of xenotransplantation, including islet xenotransplantation, for clinical setting. Adult pig is a promising and feasible donor source for islet xenotransplantation and engineered tissue, which enables to control blood glucose in recipients. It is important to acquire porcine islets in good qualities for the promotion, however, establishing a technique for adult porcine islet isolation is important but challenging because of the vulnerability of adult porcine islets. Deciding the proper timing of stopping pancreatic digestion is one of the important factors for obtaining adult porcine islets in good quality.

Pancreas Preservation with a Neutrophil Elastase Inhibitor, Alvelestat, Contributes to Improvement of Porcine Islet Isolation and Transplantation.

For pancreatic islet transplantation, pancreas procurement, preservation, and islet isolation destroy cellular and non-cellular components and activate components such as resident neutrophils, which play an important role in the impairment of islet survival. It has been reported that inhibitors of neutrophil elastase (NE), such as sivelestat and α1-antitrypsin, could contribute to improvement of islet isolation and transplantation. In this study, we investigated whether pancreatic preservation with alvelestat, a novel NE inhibitor, improves porcine islet yield and function. Porcine pancreata were preserved with or without 5 μM alvelestat for 18 h, and islet isolation was performed. The islet yields before and after purification were significantly higher in the alvelestat (+) group than in the alvelestat (−) group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 55% and 5% of diabetic mice in the alvelestat (+) and alvelestat (−) groups, respectively. These results suggest that pancreas preservation with alvelestat improves islet yield and graft function and could thus serve as a novel clinical strategy for improving the outcome of islet transplantation.

Pancreas preservation with amphotericin B deteriorates islet yield for porcine islet isolation

Amphotericin B is a crucial agent in the management of serious systemic fungal infections. It is also known to be cytotoxic. In this study, we evaluated the effect of amphotericin B added to the preservation solution on islet yield during islet isolation. Porcine pancreata were preserved in the preservation solution with or without amphotericin B (0.25μg/mL) for approximately 18hours at 4°C, and then islet isolation was performed. An optimized number (1750IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The culture of isolated islets and acinar tissue with amphotericin B was also evaluated. The islet yield before and after purification in the amphotericin B (-) group was significantly higher than that in the amphotericin B (+) group. After islet transplantation into diabetic mice, blood glucose levels reached the normoglycemic range, with 50% and 0% of that of the diabetic mice in the amphotericin B (-) and amphotericin B (+) groups, respectively. In the culture study, amphotericin B was found to be cytotoxic to porcine islets and acinar tissue. Amphotericin B added to the preservation solution deteriorates islet yield during porcine islet isolation. Thus, the use of amphotericin B should be considered carefully for the preservation of the pancreas for islet isolation and islet culture before islet transplantation.

Pancreas Preservation in Modified Histidine-lactobionate Solution Is Superior to That in University of Wisconsin Solution for Porcine Islet Isolation.

We previously reported that modified extracellular-type trehalose-containing Kyoto (MK) solution, which contains a trypsin inhibitor (ulinastatin), significantly improved the islet yield compared with University of Wisconsin (UW) preservation, which is the gold standard for organ preservation for islet isolation. In this study, we evaluated the efficiency of a modified histidine-lactobionate (MHL) solution in addition to UW or MK solution. The MHL solution has a high sodium-low potassium composition with low viscosity compared with the UW solution. Moreover, similar to MK solution, MHL solution also contains ulinastatin. Porcine pancreata were preserved in UW, MK, or MHL solution, followed by islet isolation. An optimized number (1500 IE) of isolated islets from each group were then transplanted into streptozotocin-induced diabetic mice. The islet yield before and after purification was significantly higher in the MHL group than in the UW group. On the contrary, the islet yield before and after purification was not significantly different between the MHL and MK groups. Preserving the porcine pancreata in MHL solution improved the outcome of islet transplantation in streptozotocin-induced diabetic mice compared with that in UW solution. Pancreas preservation with MHL solution preserves islet function better than UW solution. The effect of MHL solution is similar to that of MK solution, suggesting that MHL solution can be used as an alternative to MK solution for pancreatic islet transplantation.

Pancreas preservation in extracellular-type p38 inhibitor-containing solution improves islet yield for porcine islet isolation.

For islet transplantation, pancreas preservation and islet isolation activate p38, which is a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). In this study, we evaluated an extracellular-type p38 inhibitor-containing (EP) solution with University of Wisconsin (UW) solution, the gold standard for organ preservation. The EP solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EP solution contains a recently developed p38 inhibitor (11R-p38I110 ) from our laboratory. Porcine pancreata were preserved in UW, EP, or EP-P solution (EP solution without 11R-p38I110 ), and then islet isolation was performed. An optimized number (1500 IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The islet yield before and after purification was significantly higher in the EP group than in the UW group. The islet yield before and after purification was not significantly different between the EP and EP-P groups; however, the EP solution prevented a reduction in the number of islets during culture. Western blot analysis showed that p38 activation was attenuated by EP solution. For islet transplantation into streptozotocin-induced diabetic mice, pancreas preservation in EP solution improved the outcome of islet transplantation. Pancreas preservation with EP solution preserved islet function better than with UW solution. The advantages of EP solution over UW solution may include the inhibition of p38 activity as well as the composition of the solution.

Comparison of Islet Characterization from Use of Standard Crude Collagenase to GMP-Grade Collagenase Enzyme Blends in Preweaned Porcine Islet Isolations

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution.

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

Cost and Scalability Analysis of Porcine Islet Isolation for Islet Transplantation: Comparison of Juvenile, Neonatal and Adult Pigs

The limited availability of human islets has led to the examination of porcine islets as a source of clinically suitable tissue for transplantation in patients with diabetes mellitus. Islets from porcine donors are commonly used in both in vitro and in vivo experiments studying diabetes mellitus. However, there are significant differences in quality and quantity of islet yield depending on donor pig age, as well as substantial differences in the costs of pancreas procurement in adult versus neonatal and juvenile pigs. In this study, we compared the total cost per islet of juvenile pig pancreata with that of neonatal and adult pigs. Although adult porcine pancreata yield, on average, more than five times the amount of islets than do juvenile and neonatal pancreata, we found that the high price of adult pigs led to the cost per islet being more than twice that of juvenile and neonatal islets (US $0.09 vs $0.04 and $0.02, respectively). In addition, neonatal and juvenile islets are advantageous in their scalability and retention of viability after culture. Our findings indicate that isolating neonatal and juvenile porcine islets is more cost-effective and scalable than isolating adult porcine islets.

A Novel Preservation Solution Containing a JNK Inhibitory Peptide Efficiently Improves Islet Yield for Porcine Islet Isolation.

For islet transplantation, pancreas preservation in University of Wisconsin (UW) solution is associated with disadvantages, such as collagenase inhibition, resulting in poor islet yield and islets with poor viability. In this study, we evaluated a novel preservation solution, the extracellular-type c-Jun N-terminal kinase (JNK) inhibitor-containing (EJ) solution. The EJ solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EJ solution contains a recently developed JNK inhibitor from our laboratory. We first compared the performance of EJ solution with that of UW solution. Islet yield before and after purification was significantly higher in the EJ group than in the UW group. Second, we compared the performance of EJ solution with that of EJ solution without the JNK inhibitor (EJ-J solution). After pancreas preservation in EJ solution, JNK activity was maintained at a relatively low level during islet isolation. Islet yield before and after purification was significantly higher in the EJ group than in the EJ-J group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 61.5% and 7.7% of diabetic mice in the EJ and EJ-J groups, respectively. Moreover, EJ solution exhibited reduced inhibition of collagenase digestion compared with UW solution. Advantages of EJ solution over UW solution were inhibition of JNK activity and reduced collagenase inhibition. EJ solution may therefore be more suitable for islet isolation than UW solution.

Recent progress in porcine islet isolation, culture and engraftment strategies for xenotransplantation.

Xenotransplantation of porcine islets is a realistic option to restore β-cell function in type 1 diabetic patients. Among other factors, such as islet donor age (fetal, neonatal and adult) and genotype (wild type and genetically modified), choice of the transplantation site, and immune protection of the islets, efficient strategies for islet isolation, culture and engraftment are critical for the success of islet xenotransplantation. Neonatal porcine islets (NPIs) are immature at isolation and need to be matured in vitro or in vivo before they become fully functional. Recent developments include a scalable protocol for isolation of clinically relevant batches of NPIs and a stepwise differentiation protocol for directed maturation of NPIs. In addition, different sources of mesenchymal stem cells were shown to support survival and functional maturation of NPIs in vitro and in various transplantation models in vivo. A plethora of different culture media and supplements have been tested; however, a unique best culture system for NPIs is still missing. New insights, for example from single-cell analyses of islets or from stem cell differentiation toward β cells may help to optimize culture of porcine islets for xenotransplantation in an evidence-based manner.

Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

ABSTRACTSince the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.

A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution.

Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.

Methods for Microencapsulated Porcine Islet Production.

Allogeneic islet transplantation has become a viable option for the treatment of unstable type 1 diabetes; however, donor shortage and the necessity for immunosuppressive drugs are two major drawbacks of this treatment. Microencapsulated porcine islets can solve both of these drawbacks, and clinical trials have been conducted. Previous clinical trials demonstrated the safety and marginal efficacy of this treatment; therefore, it is necessary to improve its efficacy. The production of microencapsulated porcine islets consists of pancreas procurement, islet isolation, and microencapsulation. In this chapter, we describe our current methods for porcine pancreas procurement, islet isolation, and microencapsulation.

Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function.

BackgroundHuman allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure.MethodsPorcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue.ResultsReducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 ± 7% of tissue digested and 1088 ± 299 islet equivalents per gram of pancreas vs 47 ± 11% and 1080 ± 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration.ConclusionsCurrent protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application.

Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application.

One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes. For culture, BSA is replaced with human serum albumin and supplemented with Z-VAD-FMK general caspase inhibitor and a protease inhibitor cocktail. The caspase inhibitor was added to the media for only the first 90 min of culture. NPIs isolated using the scalable protocol had significantly more cellular insulin recovered (56.9 ± 1.4 µg) when compared to the standard protocol (15.0 ± 0.5 µg; p < 0.05). Compared to our standard protocol, recovery of β-cells (6.0 × 10(6) ± 0.2 vs. 10.0 × 10(6) ± 0.4; p < 0.05) and islet equivalents (35,135 ± 186 vs. 41,810 ± 226; p < 0.05) was significantly higher using the scalable protocol. During a static glucose stimulation assay, the SI of islets isolated by the standard protocol were significantly lower than the scale-up protocol (4.3 ± 0.2 vs. 5.5 ± 0.1; p < 0.05). Mice transplanted with NPIs using the scalable protocol had significantly lower blood glucose levels than the mice that receiving NPIs from the standard protocol (p < 0.01) and responded significantly better to a glucose tolerance test. Based on the above findings, this improved simpler scalable protocol is a significantly more efficient means for preparing therapeutic quantities of clinical grade NPIs.

Pancreatic L-Glutamine Administration Protects Pig Islets From Cold Ischemic Injury and Increases Resistance Toward Inflammatory Mediators.

The isolation and transplantation of porcine islets represent a future option for the treatment of type 1 diabetic patients. Stringent product release criteria and limited availability of transgenic and specific pathogen-free pigs will essentially require processing of explanted pig pancreata in specialized, possibly remote isolation facilities, whereby pancreata are exposed to cold ischemia due to prolonged tissue transit time. In the present study we investigated whether pancreas oxygenation can be efficiently combined with an antioxidant strategy utilizing intraductal L-glutamine administration. Pig pancreata were intraductally perfused after retrieval and after cold storage in oxygen-precharged perfluorohexyloctane utilizing University of Wisconsin solution supplemented with (n = 16) or without (n = 14) 5 mmol/L L-glutamine. After isolation purified islets were subjected to extensive quality assessment. Islet recovery postpurification was significantly higher in glutamine-treated pancreata (77.0 ± 3.3% vs. 60.3 ± 6.0%, p < 0.05). Glutamine administration increased intraislet content of reduced glutathione (117.8 ± 16.5 vs. 15.9 ± 2.8 ng/ng protein, p < 0.001) associated with increased islet recovery after culture (65.8 ± 12.1% vs. 40.3 ± 11.7%, p < 0.05), enhanced glucose stimulation index (1.82 ± 0.16 vs. 1.38 ± 0.10, p < 0.05), and improved posttransplant function in diabetic nude mice (p < 0.05). Furthermore, intraductally administered glutamine increased pig islet resistance toward reactive oxygen species, nitric oxide, and high-dose proinflammatory cytokines. The present study demonstrates that quality and function of pig islets exposed to warm and cold ischemia can significantly be improved using intraductal l-glutamine administration. As the efficiency of the intraductal route may be inferior compared to intravascular administration further studies should aim on assessment of l-glutamine as supplement for pancreas perfusion during organ procurement.

Fighting Diabetes: Lessons from Xenotransplantation and Nanomedicine

Increasing incidence of diabetes and shortage of specific beta cells, hormonal switches like that of delta and PP cells of the islets for transplantation, have forced the scientific community to look for alternative sources through xenotransplantation and nanomedicine. The Edmonton protocol of islet transplantation has shown proof of principle of long term survival of islets in type I diabetic patients, leading to insulin prick free life. Copious volume of literature exists on the use of mammalian islets, especially of porcine origin for diabetes reversal in humans with follow-up studies upto 10 yrs. There is an obvious lack of pre-clinical results and data in the pig-to-primate model. The difficulty is in reproducing regularly the successful porcine islet isolation. Although some of the parameters have been taken, making xenotransplantation an attractive and viable alternative therapy. However, scarcity of islets is the main hurdle in the success of islet transplantation programs. Since the islet cell receptor and the insulin molecule have remained conserved throughout the evolution of vertebrates, we reviewed islet studies from other vertebrates especially, jawless fish, cartilaginous as well as bony fishes and chick islets. The similarities of chick B islets with human islets in terms of Streptozotocin insensitivity and retention of glucose responsiveness by new born chick islets tempted us to hypothesize the use of fish and chick islets as alternative sources for transplantation to reverse experimental diabetes. Since ontogeny recapitulates phylogeny, the islets recovered from lower vertebrates are likely to be less immunogenic and may open possibility of using them without immunosuppression. Complementing xenotransplantation, nanotechnology offers an excellent module for addressing the diabetes problem from detection and treatment points of view. This review attempts to throw some light on both these approaches for an effective management and cure of diabetes.

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation.

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

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.

Pretreatment of Donor Pigs With a Diet Rich in Soybean Oil Increases the Yield of Isolated Islets

IntroductionThe pig is considered the donor species of choice for islet xenotransplantation. However, isolation of porcine islets is difficult, particularly from young pigs. Early life exposure to a high-fat diet (HFD) reportedly encourages islet β-cell expansion in neonatal rodents and improves islet viability in culture from pretreated weanling pigs. In this study, we examined the influence of young donor pretreatment with a soybean oil–enriched HFD on porcine islet mass and yield after islet isolation. Materials and MethodsPostweaning and between days 70 and 250, pigs were fed either a standard diet (control group; n = 5) or an HFD (experimental group; n = 6). Biochemical blood parameters and acute C-peptide response to intravenous glucose were monitored before pancreas procurement. The study was blinded to objectively evaluate the influence of treated diet. After procurement, pancreas biopsy samples were taken from control and pretreated donor pigs to assess islet number by using a dithizone scoring method and histologic islet area fraction determination. Control and HFD donor pig islets were isolated by using our standard isolation protocol to determine islet yield. Islet isolation characteristics and islet quality were assessed in both groups, and the results were compared. ResultsThere were no significant differences in the donor characteristics (age, body weight, glucose disposal rate, acute C-peptide response to intravenous glucose, cholesterol, and aspartate aminotransferase) except fasting blood glucose level between the control and treatment groups (84 ± 6 vs 99 ± 12 mg/dL; P = .0317). The stimulated insulin and C-peptide levels between groups were similar. However, the dithizone score was slightly higher in the treatment group compared with the control group (95.4 ± 38.5 vs 62.6 ± 23.9; P = .1208). Digestion time, digested pancreas weight, pellet volume, and the fragility index were similar in both groups. However, the average islet count (islet equivalent number/g pancreas) at the digest level was significantly higher in the HFD group than in the control group (1578 ± 994 vs 738 ± 202; P = .0344). The functional viability of 2- and 7 day-cultured islets, as assessed by using oxygen consumption rate corrected for DNA, was similar in both groups. ConclusionsPretreatment of pigs with HFD enriched with soybean oil could potentially be used to improve the islet mass in donor pigs. Further studies are needed to confirm and optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.