Optimization of Nano-encapsulation on Neonatal Porcine Islet-like Cell Clusters Using Polymersomes.
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ABSTRACT:
SUBMITTER: Lee SH
PROVIDER: S-EPMC8012476 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The transplantation of pancreatic islets can prevent severe long-term complications in diabetes mellitus type 1 patients. With respect to a shortage of donor organs, the transplantation of xenogeneic islets is highly attractive. To avoid rejection, islets can be encapsulated in immuno-protective hydrogel-macrocapsules, whereby 3D bioprinted structures with macropores allow for a high surface-to-volume ratio and reduced diffusion distances. In the present study, we applied 3D bioprinting to encapsulate the potentially clinically applicable neonatal porcine islet-like cell clusters (NICC) in alginate-methylcellulose. The material was additionally supplemented with bovine serum albumin or the human blood plasma derivatives platelet lysate and fresh frozen plasma. NICC were analysed for viability, proliferation, the presence of hormones, and the release of insulin in reaction to glucose stimulation. Bioprinted NICC are homogeneously distributed, remain morphologically intact, and show a comparable viability and proliferation to control NICC. The number of insulin-positive cells is comparable between the groups and over time. The amount of insulin release increases over time and is released in response to glucose stimulation over 4 weeks. In summary, we show the successful bioprinting of NICC and could demonstrate functionality over the long-term in vitro. Supplementation resulted in a trend for higher viability, but no additional benefit on functionality was observed.
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Project description:Neonatal porcine islet-like cell clusters (NPICCs) are a promising source for islet cell transplantation. Excellent islet quality is important to achieve a cure for type 1 diabetes. We investigated formation of cell clusters from dispersed NPICCs on microwell cell culture plates, evaluated the composition of re-aggregated porcine islets (REPIs) and compared in vivo function by transplantation into diabetic NOD-SCID IL2rγ-/- (NSG) mice with native NPICCs. Dissociation of NPICCs into single cells and re-aggregation resulted in the formation of uniform REPI clusters. A higher prevalence of normoglycemia was observed in diabetic NSG mice after transplantation with a limited number (n = 1500) of REPIs (85.7%) versus NPICCs (n = 1500) (33.3%) (p < 0.05). Transplanted REPIs and NPICCs displayed a similar architecture of endocrine and endothelial cells. Intraperitoneal glucose tolerance tests revealed an improved beta cell function after transplantation of 1500 REPIs (AUC glucose 0-120 min 6260 ± 305.3) as compared to transplantation of 3000 native NPICCs (AUC glucose 0-120 min 8073 ± 536.2) (p < 0.01). Re-aggregation of single cells from dissociated NPICCs generates cell clusters with excellent functionality and improved in vivo function as compared to native NPICCs.
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Project description:Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin- and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived ? cells early post-Tx. A higher percentage of NPCC-derived ? cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new ? cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new ? cell post-Tx.
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Project description:The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-?1,3-galactose (?Gal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from ?Gal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.
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Project description:Islet transplantation is an effective treatment for type 1 diabetes (T1D). However, a shortage of donors and the need for immunosuppressants are major issues. The ideal solution is to develop a source of insulin-secreting cells and an immunoprotective method. No bioartificial pancreas (BAP) devices currently meet all of the functions of long-term glycemic control, islet survival, immunoprotection, discordant xenotransplantation feasibility, and biocompatibility. We developed a device in which porcine islets were encapsulated in a highly stable and permeable hydrogel and a biocompatible immunoisolation membrane. Discordant xenotransplantation of the device into diabetic mice improved glycemic control for more than 200 days. Glycemic control was also improved in new diabetic mice "relay-transplanted" with the device after its retrieval. The easily retrieved devices exhibited almost no adhesion or fibrosis and showed sustained insulin secretion even after the two xenotransplantations. This device has the potential to be a useful BAP for T1D.
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Project description:Polymersomes are attractive nanocarriers for hydrophilic and lipophilic drugs; they are more stable than liposomes, tunable, and relatively easy to prepare. The copolymer composition and molar mass are critical features that determine the physicochemical properties of the polymersomes including the rate of drug release. We used the triblock-copolymer, poly(2-methyl-2-oxazoline)-block-poly-(dimethysiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA), to form amphipathic polymersomes capable of loading proteins and small hydrophobic agents. The selected agents were unstable neurotrophins (nerve growth factor and brain-derived neurotrophic factor), a large protein CD109, and the fluorescent drug curcumin. We prepared, characterized, and tested polymersomes loaded with selected agents in 2D and 3D biological models. Curcumin-loaded and rhodamine-bound PMOXA-PDMS-PMOXA polymersomes were used to visualize them inside cells. N-Methyl-d-aspartate receptor (NMDAR) agonists and antagonists were also covalently attached to the surface of polymersomes for targeting neurons. Labeled and unlabeled polymersomes with or without loaded agents were characterized using dynamic light scattering (DLS), UV-vis fluorescence spectroscopy, and asymmetrical flow field-flow fractionation (AF4). Polymersomes were imaged and tested for biological activity in human and murine fibroblasts, murine macrophages, primary murine dorsal root ganglia, and murine hippocampal cultures. Polymersomes were rapidly internalized and there was a clear intracellular co-localization of the fluorescent drug (curcumin) with the fluorescent rhodamine-labeled polymersomes. Polymersomes containing CD109, a glycosylphosphatidylinositol-anchored protein, promoted cell migration in the model of wound healing. Nerve growth factor-loaded polymersomes effectively enhanced neurite outgrowth in dissociated and explanted dorsal root ganglia. Brain-derived neurotrophic factor increased dendritic spine density in serum-deprived hippocampal slice cultures. NMDAR agonist- and antagonist-functionalized polymersomes targeted selectively neurons over glial cells in mixed cultures. Collectively, the study reveals the successful incorporation into polymersomes of biologically active trophic factors and small hydrophilic agents that retain their biological activity in vitro, as demonstrated in selected central and peripheral tissue models.
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Project description:Porcine islet xenografts have the potential to provide an inexhaustible source of islets for β cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild-type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.
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Project description: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.
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Project description:Cell encapsulation is a widely used technique in the field of Tissue Engineering and Regenerative Medicine (TERM). However, for the particular case of liquefied compartmentalised systems, only a limited number of studies have been reported in the literature. We have been exploring a unique cell encapsulation system composed by liquefied and multilayered capsules. This system transfigured the concept of 3D scaffolds for TERM, and was already successfully applied for bone and cartilage regeneration. Due to a number of appealing features, we envisage that it can be applied in many other fields, including in advanced therapies or as disease models for drug discovery. In this review, we intend to highlight the advantages of this new system, while discussing the methodology, and sharing the protocol optimization and results. The different liquefied systems for cell encapsulation reported in the literature will be also discussed, considering the different encapsulation matrixes as core templates, the types of membranes, and the core liquefaction treatments.
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Project description:BackgroundNeonatal porcine pancreatic cell clusters (NPCCs) have been proposed as an alternative source of β cells for islet transplantation because of their low cost and growth potential after transplantation. However, the delayed glucose lowering effect due to the immaturity of NPCCs and immunologic rejection remain as a barrier to NPCC's clinical application. Here, we demonstrate accelerated differentiation and immune-tolerant NPCCs by in vitro chemical treatment and microencapsulation.MethodsNPCCs isolated from 3-day-old piglets were cultured in F-10 media and then microencapsulated with alginate on day 5. Differentiation of NPCCs is facilitated by media supplemented with activin receptor-like kinase 5 inhibitor II, triiodothyronine and exendin-4 for 2 weeks. Marginal number of microencapsulated NPCCs to cure diabetes with and without differentiation were transplanted into diabetic mice and observed for 8 weeks.ResultsThe proportion of insulin-positive cells and insulin mRNA levels of NPCCs were significantly increased in vitro in the differentiated group compared with the undifferentiated group. Blood glucose levels decreased eventually after transplantation of microencapsulated NPCCs in diabetic mice and normalized after 7 weeks in the differentiated group. In addition, the differentiated group showed nearly normal glucose tolerance at 8 weeks after transplantation. In contrast, neither blood glucose levels nor glucose tolerance were improved in the undifferentiated group. Retrieved graft in the differentiated group showed greater insulin response to high glucose compared with the undifferentiated group.Conclusionin vitro differentiation of microencapsulated immature NPCCs increased the proportion of insulin-positive cells and improved transplant efficacy in diabetic mice without immune rejection.
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