Islet Purification Research Articles

Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research.

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.

235.4: New pancreatic islet purification method in pancreatic islet isolation applying photoimmunotherapy

235.4: New pancreatic islet purification method in pancreatic islet isolation applying photoimmunotherapy

235.5: SEPAX -SEFIA a repurposed closed system for human islet purification and processing in cGMP environment

235.5: SEPAX -SEFIA a repurposed closed system for human islet purification and processing in cGMP environment

Continuous vs. discontinuous purification of isolated human islets: functional and morphological comparison.

The COBE 2991 cell processor, commonly used for pancreatic islet isolation, is no longer distributed in Europe, leading to a search for alternative purification procedures with equivalent efficacy. The aim of this study was to evaluate the efficacy of an alternative method based on the discontinuous purification of islets. The conventional isolation procedure using a standard continuous islet purification with COBE 2991 of n = 4 human pancreas was compared to n = 8 procedures using a discontinuous purification with a "bottle" method from donors of similar characteristics. Islet equivalents, purity, and dynamic glucose-stimulated insulin secretion were evaluated. A similar islet yield was obtained using continuous vs. discontinuous purification methods (76,292.5 ± 40,550.44 vs. 79,625 ± 41,484.46 islet equivalents, p = 0.89). Islets from both groups had similar purity (78.75% ± 19.73% vs. 55% ± 18.16%, p = 0.08) and functionality both in terms of stimulation index (3.31 ± 0.83 vs. 5.58 ± 3.38, p = 0.22) and insulin secretion (1.26 ± 0.83 vs. 1.53 ± 1.40 mean AUC, p = 0.73). Moreover, the size of the islets was significantly larger in the discontinuous vs. continuous purification group (19.2% ± 10.3% vs. 45.4% ± 18.8% of islets less than 100 µm, p = 0.0097 and 23.7% ± 5.3% vs. 15.6% ± 5.8% of 200-250 µm islet size, p = 0.03). Compared to the conventional purification procedure, discontinuous purification with a bottle method shows similar results with regard to isolation yield and islet secretory function. Furthermore, this alternative technique allows for obtaining larger islets.

241-OR: Preservation of Islet Graft Function and Blood Glucose Control after Total Pancreatectomy with Islet Autotransplantation in Patients with Chronic Pancreatitis and Intractable Pain—Nine-Year Follow-up

Background: Total pancreatectomy is a last resort treatment for patients with chronic pancreatitis and intractable abdominal pain, however its utility has been hampered by the uncertain endocrine outcomes of islet transplantation and risk of postsurgical diabetes. Here, we present our 9-year clinical results after total pancreatectomy and islet transplantation. Materials and Method: Forty-one patients with chronic or recurrent acute pancreatitis were treated with TPIAT over the last 9 years at the University of Chicago Medical Center. There were 15 male and 26 female with a median age of 33 (6-65) and with median BMI of 25 (17-39). Five (12%) patients were already diabetic prior to surgery. Islet isolation was performed at a local laboratory compliant with current Good Manufacture Practice regulations. Islet purification was implemented in 4 (10%) cases to reduce islet pellet volume below 20mL for intraportal infusion. Results: All patients required opioids for pain control prior to surgery. Most of the patients (93%) reported resolution of pancreatic-type pain by postoperative year one. The number of patients requiring opioids for different type or location of pain declined over time from 13 (31%) at 1 year to 5 (12%) at 5 years after TPIAT. During TPIAT patients received on average 211,000±111,000 islet equivalents (IEQ), 2,500±1,600,). The majority of patients (95%) maintained endocrine beta cell function during follow up (serum c-peptide >0.5ng/ml). Over 40% of patients maintained long-term insulin independence: 44% (15/34) at 1-year and 45% (5/11) at 5-year follow-up. Conclusions: TPIAT successfully provided pancreatic pain resolution of pancreatic pain and preservation of the endocrine beta cell function in majority of the patients with no mortality. Islet autotransplantation prevented diabetes in over 40% of patients. Disclosure W.Lin: None. L.Wang: None. M.Tibudan: None. R.Barth: None. J.Fung: None. P.Witkowski: Advisory Panel; Vertex Pharmaceuticals Incorporated, Novartis. M.Ogledzinski: None. S.Gondek: None. K.Milejczyk: None. B.Juengel: None. L.Potter: None. P.J.Bachul: None. L.Basto: None. L.Perea: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK020595)

Characteristics of research-focused human islet preparations from organ donors with type 2 diabetes

ABSTRACT Type 2 diabetes mellitus (T2D) affects 463 million individuals worldwide. β-cell dysfunction and relatively inadequate β-cell mass has been implicated in the pathogenesis of T2D. Primary human islets from T2D patients can reveal the islet dysfunction and the underlying mechanisms and thus have become valued resources for diabetes research. Our center (Human Islet Resource Center, China) has prepared a number of batches of human islets from T2D organ donors. The present study aims to characterize islet isolation processes, islet yields, and qualities of T2D pancreases by comparing with non-diabetic (ND) ones. Overall, 24 T2D and 80 ND pancreases were obtained with informed research consents. The digestion time, islet purity, yield, size distribution, islet morphology score, viability, and function in each islet preparation were analyzed. We found that at digestion stage, T2D pancreases need significantly longer digestion duration and have worse digestion rates and lower gross islet yields. At purification stage, T2D pancreases have poorer purity, purification rate, morphology score, and islet yields after purification. Functional evaluation by GSI assay showed that the human T2D islets have significantly lower glucose stimulated insulin secretion ability. In conclusion, the features of longer digestion duration, lower yields and quality, and impaired insulin secretion in T2D group are consistent with the pathological condition of this disease. Both islet yields and islet function evaluation results did not support human T2D islets as clinical transplantation resources. However, they could serve as good research models for T2D disease studies and promote the advancement of diabetes research.

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37°C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25mL of cold (4°C) human serum albumin and centrifuge twice at 150g for 3min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30min. Digested pancreatic tissue was centrifuged at 2200rpm for 5min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

ODP434 Maternally-Derived Pancreatic Extracellular Vesicle Encompassed miRNAs Influence Placental Development in Pregnancies Complicated by Gestational Diabetes

Abstract Pregnancies affected by gestational diabetes (GDM) commonly result in large-for-gestational-age (LGA) infants; these infants have an increased risk of developing cardiometabolic complications compared to appropriate-for-gestational-age (AGA) infants. The mechanisms responsible are unclear but GDM/LGA is associated with altered placental development/function. We have recently reported altered levels of extracellular vesicle (EV) encapsulated miRNAs, including miR-375 (a pancreatic-specific miRNA) in maternal serum prior to the onset of LGA. We propose that the EVs containing miR-375 are produced by maternal pancreas, and that increased miR-375 in maternal circulation contributes to LGA by influencing placental development/function. Human placental tissue was collected from GDM pregnancies that delivered AGA (n=14) or LGA infants (n=10). Levels of mature miR-375 and primary transcript (pri-miR-375) were measured by QPCR. Mature miR-375 was increased in GDM-LGA (4.74-fold; p<0. 01) compared to GDM AGA placentae. Mature miR-375, but not pri-miR-375, was detected in human placenta suggesting that miR-375 is not produced in the placenta but instead is transported, potentially via EVs in maternal circulation. Intact human pancreatic islets (20,000 IEQ/patient) were obtained through the Integrated Islet Distribution Program (IIDP) from female donors of reproductive age. Islets were cultured in EV-depleted media containing 5.5mM (normoglycemia) or 7mM (mild hyperglycaemia) glucose for 3 days. Islet purity and viability were confirmed using dithizone incorporation and trypan blue exclusion, respectively. Media was collected at 24-hour intervals, pooled from all time points, concentrated and EVs were isolated by size exclusion chromatography. Confirmation of the presence of EVs was confirmed by electron microscopy (typical cup-shape morphology of EVs), nanoparticle tracking analysis (NTA; mean size of 83.5-163.2nm; concentration of 1.28×109-1.82×1010 particles/ml), Western blotting (EV-enriched proteins). QPCR confirmed that mature miR-375 was present in islet-derived EVs and revealed that levels were altered following culture in 7mM glucose compared to 5.5mM. Islet-derived EVs were labelled with maleimide-488 and uptake into explants of normal human term placenta was confirmed using fluorescent microscopy=3). miR-375 was overexpressed in normal human placental explants (by 30-fold, p<0. 05; n=6) using specific mimics (100nM for 72 hours) and TMT mass spectrometry was performed to assess the impact of miR-375 overexpression on the placental proteome. Functional enrichment analysis (FEA) was performed on differentially expressed proteins (DEP; P<0. 05; -0.41≥log2foldchange≥0.58) using over representation analysis and Ingenuity Pathway Analysis. This revealed that DEGs were significantly enriched in pathways associated with placental growth, mitochondrial function and glucose metabolism. Our data demonstrates that miR-375 is increased in the placenta in GDM/LGA and that this influences placental development/function. miR-375 does not appear to be produced in the placenta but instead, is likely released in EVs from maternal pancreas under conditions of hyperglycaemia and transported to the placenta, via maternal circulation. This provides insight into the potential mechanisms contributing to LGA in pregnancies complicated by GDM. Presentation: No date and time listed

Is ABO Compatibility Important in Islet Transplantation?

One of the current criteria for clinical islet allotransplantation is ABO blood group compatibility between donor and recipient.1 The distribution of ABH antigens in human pancreata was first examined >40 y ago, revealing expression in acinar and vascular cells but not in islets.2 This underlined the importance of ABO matching in whole pancreas transplantation, but the situation in islet transplantation is more complicated. Although islets in situ appear to be ABH-negative,2 islets prepared for transplant are invariably contaminated with exocrine tissue including acinar cells. Furthermore, it is not known whether the islet isolation and transplantation process itself influence ABH expression. It is therefore somewhat surprising that ABH expression in islet preparations before and after transplant has not been characterized until now. In this issue of Transplantation, Verhoeff et al3 present a detailed immunohistochemical and flow cytometric survey of ABH antigen levels in human pancreatic tissue, isolated human clinical-grade islets, and islets derived from human embryonic stem cells (hESCs). After confirming that ABH antigens are present on acinar cells but not on endocrine and ductal cells in the pancreas, the authors demonstrate that islet preparations consist primarily of ABH-negative endocrine cells but are significantly contaminated with ABH-positive acinar cells. When islets were transplanted into immunodeficient rats, the endocrine component was ABH-negative at 24 wk, whereas remnant acinar tissue stained positive. Pancreatic endocrine progenitors derived from hESCs were ABH-negative and remained so after in vivo maturation following transplantation into rats, although this was not unexpected given that the hESC donor was blood group O. The human-to-immunodeficient rat islet xenograft model used in the latter part of the study does have some limitations. First, the kidney subcapsular site does not accurately reflect the clinical scenario, in which islets are transplanted intraportally.1 Subcapsular delivery co-localizes the islets with acinar tissue fragments and potentially exposes the islets to “bystander” injury and inflammation when anti-ABH antibodies attack the acinar cells. In contrast, intraportal delivery disperses islets and acinar fragments widely throughout the liver microvasculature, which probably minimizes co-localization and thus reduces the likelihood of bystander effects. Furthermore, subcapsular but not intraportal delivery shields the graft from exposure to anti-ABH IgM antibodies. Although these differences are not relevant in the immunodeficient rat model, they may well be relevant in the clinical setting. Second, the immunodeficient rat model does not allow investigation of the importance of the in vivo anti-ABH immune response to islet grafts. I have worked with one of the authors (L.J.W.) to develop a mouse model of anti-A antibody-mediated rejection of transgenic blood group A-expressing cardiac grafts.4 However, these A-transgenic donor mice are unsuitable for examining anti-A–mediated islet graft rejection because expression of the antigen is endothelial cell-restricted and absent on islets. Humanized mouse models may be more appropriate, as suggested by the authors. The take-home message of this interesting paper is that ABO matching is indeed likely to be important in islet allotransplantation—as is the case for HLA matching5—but may become less so if islet purification efficiency improves sufficiently or if hESC-derived islets are used. On the former point, the authors speculate that the restriction of surface expression of ABH antigens to acinar tissue (starkly illustrated by Figure 6A in the paper) provides an opportunity to enrich the purity of A, B, or AB islets by some form of negative selection. Although practicality at scale may be an issue, this is an intriguing proposition that is worthy of further consideration.

PRISM: A Novel Human Islet Isolation Technique.

Successful pancreatic islet isolations are a key requirement for islet transplantation in selected patients with type 1 diabetes. However, islet isolation is a technically complex, time-consuming, and manual process. Optimization and simplification of the islet isolation procedure could increase islet yield and quality, require fewer operators, and thus reduce cost. We developed a new, closed system of tissue collection, washing, buffer change, and islet purification termed PancReatic Islet Separation Method (PRISM). In the developmental phase, pump and centrifuge speed was tested using microspheres with a similar size, shape, and density as digested pancreatic tissue. After optimization, PRISM was used to isolate islets from 10 human pancreases. Islet equivalents viability (fluorescein diacetate/propidium iodide), morphology, and dynamic glucose-stimulated insulin secretion were evaluated. PRISM could be performed by 1 operator in 1 flow cabinet. A similar islet yield was obtained using PRISM compared to the traditional islet isolation method (431 234 ± 292 833 versus 285 276 ± 197 392 islet equivalents, P = 0.105). PRISM islets had similar morphology and functionality. PRISM is a novel islet isolation technique that can significantly improve islet isolation efficiency using fewer operators.

Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species

Gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O 2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O 2) and hypoxia (1%O 2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions, respectively. GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group ( P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C ( P<0.05). GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.

P.124: Withaferin A Prevents Inflammatory Phenotype in Cultured Islets

Introduction: Islets undergo enormous ischemic, and mechanical stress throughout clinical isolation steps which affect the quality of processed islets available for transplantation. Isolated islets produce pro-inflammatory cytokines and chemokines that are further exacerbated during culture. Transplantation of islets with a strong pro-inflammatory phenotype may induce the activation of IBMIR and affect overall transplant outcomes. Strategies to improve the outcome of isolation and protection of islets from pro-inflammatory gene activation is an unmet requirement. Here, we sought to investigate the protective/anti-inflammatory effect of Withaferin A: a plant-derived inhibitor of Nuclear Factor Kappa B (NFκB). Methods: A bisected split lobe isolation technique was adopted where the head and tail were isolated subsequently with a control or WA-treated (2µM) medium during isolation. WA was added in the isolation solutions including preservation media, Collagenase, Recombination Media, Wash Buffer, and also in islet culture media. The islets isolated from (+/-) WA treatment during isolation was analyzed for the viability, function, and intracellular pro-inflammatory cytokine expression. Western blots were performed to study the activation of transcription factors and stress-regulated genes post-culture. Exosomes were isolated and the presence of pro-inflammatory mediators was examined. Results: Islet damage was reduced in the WA-treated group as shown by miRNA 375 release. No significant difference in the isolation outcomes such as yield, purity, and viability of islets between WA and the control group was seen. The purity of islets post-culture was better maintained in the WA treated group, compared to the control group (Figure 1). No difference in viability and insulin secretion was observed post-culture in the two groups. The western blotting of WA-cultured islets (at 24 and 48H) showed a significant reduction in the activation of p65 NFκB (P-s536) and P-IκBα (P-s32). Interestingly, WA treatment also synergistically reduced the activation of PERK (P-T982) and IRE1 mediated ER stress signals compared to the non-WA treated cultured islets. There was a significant reduction in the intracellular mRNA expression (Tissue factor, MCP1, and IL-8) and subsequent exosomal content of pro-inflammatory mediators (IP-10, IL-6, and IL-2) in the WA treated cultured islets (24 and 48H) compared to non-WA treated islets. Conclusion: Withaferin A treatment prevented islet damage during isolation and also prevented the generation of a proinflammatory phenotype in islets post-culture. The study proposes a novel protective strategy to control islet inflammation during isolation and peritransplant steps which may improve the functional outcomes of transplantation.

P.100: Impact of Islet Purity on Short- and Long-term Graft Function in Islet Allotransplantation

P.100: Impact of Islet Purity on Short- and Long-term Graft Function in Islet Allotransplantation

Engineering a macroporous oxygen-generating scaffold for enhancing islet cell transplantation within an extrahepatic site

Insufficient oxygenation is a serious issue arising within cell-based implants, as the hypoxic period between implantation and vascularization of the graft is largely unavoidable. In situ oxygen supplementation at the implant site should significantly mitigate hypoxia-induced cell death and dysfunction, as well as improve transplant efficacy, particularly for highly metabolically active cells such as pancreatic islets. One promising approach is the use of an oxygen generating material created through the encapsulation of calcium peroxide within polydimethylsiloxane (PDMS), termed OxySite. In this study, OxySite microbeads were incorporated within a macroporous PDMS scaffold to create a single, streamlined, oxygen generating macroporous scaffold. The resulting OxySite scaffold generated sufficient local oxygenation for up to 20 days, with nontoxic levels of reaction intermediates or by-products. The benefit of local oxygen release on transplant efficacy was investigated in a diabetic Lewis rat syngeneic transplantation model using a clinically relevant islet dosage (10,000 IEQ/kg BW) with different isolation purities (80%, 90%, and 99%). Impure islet preparations containing pancreatic non-islet cells, which are common in the clinical setting, permit examination of the effect of increased overall oxygen demand. Our transplantation outcomes showed that elevating the oxygen demand of the graft with decreasing isolation purity resulted in decreased graft efficacy for control implants, while the integration of OxySite significantly mitigated this impact and resulted in improved graft outcomes. Results highlight the superior clinical translational potential of these off-the-shelf OxySite scaffolds, where islet purity and the overall oxygen demands of implants are increased and highly variable. The oxygen-generating porous scaffold further provides a broad platform for enhancing the survival and efficacy of cellular implants for numerous other applications. Statement of significanceHypoxia is a serious issue within tissue engineered implants. To address this challenge, we developed a distinct macroporous scaffold platform containing oxygen-generating microbeads. This oxygen-generating scaffold showed the potential to support clinically relevant cell dosages for islet transplantation, leading to improved treatment efficacy. This platform can also be used to mitigate hypoxia for other biomedical applications.

Failure mode and effect analysis in human islet isolation: from the theoretical to the practical risk

ABSTRACT This study aimed to assess the global mapping risk of human islet isolation, using a failure mode and effect analysis (FMEA), and highlight the impact of quality assurance procedures on the risk level of criticality. Risks were scored using the risk priority number (RPN) scoring method. The risk level of criticality was made based on RPN and led to risk classification (low to critical). A raw risk analysis and a risk control analysis (with control means and quality assurance performance) were undertaken. The process of human islet isolation was divided into 11 steps, and 230 risks were identified. Analysis of the highest RPN of each of the 11 steps showed that the 4 highest risks were related to the pancreas digestion and islet purification stages. After implementation of reduction measures and controls, critical and severe risks were reduced by 3-fold and by 2-fold, respectively, so that 90% of risks could be considered as low to moderate. FMEA has proven to be a powerful approach for the identification of weaknesses in the islet isolation processes. The results demonstrated the importance of staff qualification and continuous training and supported the contribution of the quality assurance system to risk reduction.

Engineering an Macroporous Oxygen-Generating Scaffold for Enhancing Islet Cell Transplantation within an Extrahepatic Site

Insufficient oxygenation is a serious issue arising within cell-based implants, as the hypoxic period between implantation and vascularization of the graft is largely unavoidable. In situ oxygen supplementation at the implant site should significantly mitigate hypoxia-induced cell death and dysfunction, as well as improve the transplant efficacy, particularly for highly metabolically active cells such as pancreatic islets. One promising approach is the use of an oxygen generating material created through the encapsulation of calcium peroxide within polydimethylsiloxane (PDMS), termed OxySite. In this study, OxySite microbeads were incorporated within a macroporous PDMS scaffold to create a single, streamlined, oxygen generating macroporous scaffold. The resulting OxySite scaffold generated sufficient local oxygenation for up to 20 days, with nontoxic levels of reaction intermediates or by-products. The benefit of local oxygen release on transplant efficacy was investigated in a diabetic Lewis rat syngeneic transplantation model containing clinically relevant islet isolation purities (80%, 90%, and 99%). Transplantation outcomes showed that elevating the oxygen demand of the graft with decreasing isolation purity resulted in decreased graft efficacy for control implants, while the integration of OxySite significantly mitigated this impact and resulted in significantly improved graft outcomes. Results highlight the superior clinical translational potential of these off-the-shelf OxySite scaffolds, where islet purity and the overall oxygen demands of implants are increased and highly variable. The oxygen-generating porous scaffold further provides a broad platform for enhancing the survival and efficacy of cellular implants for numerous other applications.

Pancreatic Islet Purification from Large Mammals and Humans Using a COBE 2991 Cell Processor versus Large Plastic Bottles.

The islet purification step in clinical islet isolation is important for minimizing the risks associated with intraportal infusion. Continuous density gradient with a COBE 2991 cell processor is commonly used for clinical islet purification. However, the high shear force involved in the purification method using the COBE 2991 cell processor causes mechanical damage to the islets. We and other groups have shown human/porcine islet purification using large cylindrical plastic bottles. Shear stress can be minimized or eliminated using large cylindrical plastic bottles because the bottles do not have a narrow segment and no centrifugation is required during tissue loading and the collection processes of islet purification. This review describes current advances in islet purification from large mammals and humans using a COBE 2991 cell processor versus large cylindrical plastic bottles.

Current Assessment of Clinical Pancreatic Islet Allotransplantation

Clinical islet allotransplantation represents a minimally invasive, efficacious alternative to pancreas transplantation for restoring glycemic control and insulin independence in select patients with type 1 diabetes that is complicated by intractable impaired hypoglycemia awareness and/or severe hypoglycemic events refractory to stabilization by other means. Over the last decade, islet transplantation outcomes have steadily improved in part due to refinements in the selection of optimal donors, islet isolation techniques, safer engraftment methods, and effective immunomodulatory and anti-inflammatory therapies. Insulin independence rates at five years post-transplantation at select centers have reached parity with pancreas alone transplantation, and marked progress has been achieved in islet transplantation outcomes using single-donor pancreas. However, widespread application of the procedure is still hindered due to a limited supply of donor pancreases, inadequate engraftment, and the harmful side effects of chronic immunosuppression. Strategies to address some of these challenges involve the use of alternative sources of beta cells or islets, extrahepatic sites of implantation, encapsulation of islets and novel therapies to induce tolerance. While several countries have now transitioned islet transplantation from experimental status to a funded clinical cure for patients with brittle type 1 diabetes that cannot be stabilized by more conventional means, in the US it still awaits regulatory approval and a financial mechanism for sustainable reimbursement. This review details the history and the current status of clinical islet allotransplantation while summarizing improvements that have been made in techniques involving isolation, purification, culture and assessment of human islets as well as the islet transplantation process itself. Furthermore, it discusses the limitations encountered that prevent its widespread application, strategies that address those limitations, and last but not least, clinical trials being conducted that will help position islet transplantation as a mainstay treatment for the cure of type 1 diabetes.

Microencapsulation of cellular aggregates composed of differentiated insulin and glucagon-producing cells from human mesenchymal stem cells derived from adipose tissue

BackgroundIn type I diabetes mellitus (T1DM) pancreatic β cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies.AimsWe postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy.MethodshASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed.ResultsThe results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of ~ 80 µm, and 65% of the aggregates reached 4000 µm2 at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba2+, yielding average diameters of ~ 300 µm. Interestingly, Ba2+-microencapsulated aggregates respond to high external glucose with insulin secretion.ConclusionsThe IPC/GPC differentiation process from hASC, followed by the generation of cellular aggregates that are later microencapsulated, could represent a possible treatment for T1DM.

1690-P: In Vitro Differentiation of Pancreas-Derived Mesenchymal Stem Cells into Insulin-Producing Beta Cells

Differentiation of insulin-producing cells (IPCs) from stem cells has been widely studied as an alternative source for cell transplantation therapy in diabetes. Mesenchymal stem cells (MSCs) are a useful therapeutic option for islet transplants due to their immunomodulatory properties and differentiation potential. In this study, we introduce a method for in vitro differentiation of pancreatic MSCs into IPCs. Research-grade pancreas from cadaveric donors was used to isolate islets, and the COBE remnant after islet purification was used for the generation of pancreatic MSCs. The remnant tissue was cultured in RPMI medium and plastic to isolate tissue-resident MSCs. Surface marker expression analysis and differentiation assays were performed to characteristically confirm that the cells obtained were in fact MSCs. Osteogenic, adipogenic and chondrogenic differentiation of MSCs were induced by StemPro® differentiation kits. The protocol for the differentiation of IPCs from stem cells was adopted from Pagliuca et al. 2014. Similarly, we have treated the pancreatic MSCs with molecules and growth factors at different stages of development to produce IPCs. The insulin secretion from differentiated IPCs also analyzed at all stages. The culture of remnant tissue after islet isolation resulted in plastic-adherent cells of fibroblast-like morphology after 3 passages. Flow cytometry analysis showed homogeneous expression of markers (CD90, CD73, CD29, CD105) and negative expression of CD14, CD34, and CD45 as classically characterized by cultured MSCs. Interestingly, there was a significant increase in insulin levels up to 5 and 100 folds in the media at developmental stages S5 (P= 0.0686) and S6 (P &amp;lt;0.0001) respectively. Our results reveal that MSCs can be directly differentiated into IPCs. Human pancreas-derived MSCs may serve as an alternative source of insulin-producing beta cells that can be transplanted to type 1 diabetes patients. Disclosure J. Kalivarathan: None. P. Saravanan: None. M.F. Levy: None. M.A. Kanak: None.