Microencapsulation Of Pancreatic Islets Research Articles

Biological Assessments of Encapsulated Pancreatic β-Cells: Their Potential Transplantation in Diabetes

Microencapsulation of pancreatic islets has been considered as a promising method for cell transplantation and diabetes treatment. However, in vivo trials to date have been hampered by fibrotic overgrowth and very limited to no success, long-term. Future success requires suitable microencapsulating method and possibly a simplified and suitable formulation which will produce a microcapsule that provides an immunobarrier, maintain full β-cell functionality whilst also reducing the inflammatory processes that induce fibrosis. In multiple studies, we screened various formulations and microencapsulating methods, and obtained promising results using bile acid-based microcapsules containing β-cells, in terms of cell functions and insulin release. Thus, this study aimed to refine further the microencapsulating method using a simple alginate-poly-l-ornithine formulation and test the effect of adding a promising bile acid, ursodeoxycholic acid (UDCA), on cell functions. Using Buchi concentric nozzle, viable NIT-1 cells were microencapsulated using alginate-poly-l-ornithine, with or without UDCA at a ratio of 1:1.2 or 1:1.2:4. Screening for nozzle temperature and nozzle-gelation bath distance was carried out to form best microcapsules. Microcapsules were cultured for 48 h and examined for size and surface morphology, chemical profiling and β-cell viability. Culture supernatants were examined for insulin and inflammatory cytokines. When using 30 °C nozzle-temperature and 5 cm nozzle-gelation bath distance, in the presence of the bile acid, cell mitochondrial activities and insulin production were optimised. Under deployed microencapsulating method with nozzle-temperature of 30 °C and nozzle-gelation bath distance of 5 cm, the incorporation of the bile acid into the microcapsules resulted in enhanced β-cell survival, function and improved overall biocompatibility supporting potential applications in transplantation.

Separation of empty microcapsules after microencapsulation of porcine neonatal islets

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.

Extensive amyloid formation in transplanted microencapsulated mouse and human islets

Objective: Deposition of cell toxic islet amyloid is a frequent finding in type 2 diabetes and also in transplanted human islets, where it is a possible explanation for their long-term failure. One suggested reason for amyloid in transplanted islets is that their low vascular density results in a disturbed local clearance of islet amyloid polypeptide (IAPP). To test this hypothesis we analysed accumulation of amyloid in microencapsulated islets, which exemplify a non-vascularised islet graft. Methods: Isolated islets from human or transgenic mice expressing human IAPP were microencapsulated in alginate and cultured in vitro or transplanted under the kidney capsule of normoglycemic nude mice. The degree of amyloid was determined after Congo red staining and subcellular alterations were analysed with electron microscopy. Results: Insulin and IAPP secretion from transgenic mouse islets were markedly increased during stimulation with glucose after one week of culture, but encapsulated islets in general released less insulin. Amyloid was detected after both one and three weeks of culture in the transgenic mouse islets and the encapsulated islets were most affected. After transplantation, electron microscopy displayed both intra- and extracellular amyloid in microencapsulated as well as in non-encapsulated human and transgenic mouse islet grafts. However, amyloid was more frequent in the encapsulated grafts. Conclusion: Micro-encapsulation of pancreatic islets might serve as an important tool for studies of amyloid formation under enhanced circumstances.

Microencapsulation of Pancreatic Islets With Canine Ear Cartilage for Immunoisolation

Improving human islet transplantation is often limited by the shortage of donors and the side effects of immunosuppressive agents. If immunoisolation is properly used, it can overcome these obstacles. Because artificial materials are adopted in this technique, however, there are still multiple issues with biocompatibility and foreign body reactions. We developed a chondrocyte microencapsulated immunoisolated islet (CMI-islet) that allows living cells to act as the immunoisolating material. To manufacture CMI-islets for xenotransplantation, isolated rat pancreatic islets were placed on low cell-binding culture dishes. Subsequently, expanded canine auricular cartiage primary cells were seeded on these dishes at a high density and maintained in a suspended state via a shaking culture system. Morphological evaluations showed good islet viability and a clear progression of the islet- encapsulation events. When the cells were challenged with glucose, they were able to secrete sufficient insulin according to glucose concentrations. The CMI-islets responded better to the glucose challenge than did nude pancreatic islets and created better glucose-insulin feedback regulation. Moreover, insulin secretion into the culture medium was confirmed over a period of 100 days, showing the survival and secretory capacity of the CMI-islet cells. By microencapsulating pancreatic islets with recipient ear cartilage cells, long-term insulin secretion can be maintained and the response to glucose challenges improved. This new immunodelusion technology differs from other immunoisolation techniques in that the donor tissue is enclosed with the recipient's tissue, thus allowing the transplanted cells to be recognized as recipient cells. This microencapsulation method may lead to developing viable xenotransplantation techniques that do not use immunosuppressive drugs.

Long-term graft function of cryostored alginate encapsulated rat islets

Microencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of encapsulated allogenic islets. Recently, we reported a gentle cryopreservation protocol for rat islets encapsulated in an alginate-based microcapsule system. Here, we report for the first time long-term transplantation data of these cryopreserved microencapsulated islets. We detected a stable graft function for more than 12 month (experiments still continuing) after transplantation of 2500 cryopreserved microencapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Moreover, the glucose clearance rate during an IPGTT was well preserved up to 56 weeks after transplantation. In addition, hyperglycemic blood glucose levels after removal of rat islet grafts 12 and 56 weeks after transplantation confirmed the efficacy of the encapsulated islets. Finally, the retrieved encapsulated rat islets responded well with a 7-fold increase of insulin secretion to a glucose stimulus (12 and 56 weeks). In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function for a very long time.

The effects of microencapsulation on pancreatic islet osmotically induced volumetric response.

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 microm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22 degrees C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37 degrees C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 +/- 1.04% (mean +/- SEM) and 70.11 +/- 1.05%, and in 2 M EG to 72.89 +/- 1.93% and 69.33 +/- 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 +/- 0.91% and 67.67 +/- 1.91% for canine and 62.22 +/- 0.66.% or 65.89 +/- 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 +/- 1.47% of their original volume in 2 M and 80.33 +/- 0.89% in 3 M DMSO, and 87.33 +/- 1.86% in 2 M and 82.80 +/- 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 +/- 2.13% of their original volume in 2 M and 78.22 +/- 0.95% in 3 M DMSO, and 85.44 +/- 1.92% in 2 M and 78.11 +/- 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.

Microencapsulation of pancreatic islets: Some relevant factors

The following paragraphs by no means intend to present a review of the present status of microencapsulation of pancreatic islets, and the reader is kindly referred to a much more comprehensive overview (7). Here, some arguments are given in favour of microencapsulation as a promising approach for eventual clinical application of islet transplantation in young diabetics. This is pertinent, since islet transplantation has preferably to be applied at an early stage of diabetes in order to prevent secondary complications of the disease. The use of immunosuppression in young patients is not desirable, and immunoprotection of islets by a semi-permeable membrane seems to be a reasonable and effective alternative. The presented data are encouraging, but further studies are required for achieving clinical applicability. Such studies should be directed to experiments in large animals, to the development of techniques for isolating islets on a large scale, and to the improvement of the biocompatibility of the biomaterials used for microencapsulation in order to diminish, or even prevent, cellular overgrowth of the islet containing microcapsules.

Microencapsulation of pancreatic islets: A technique and its application to culture and transplantation

Hamster pancreatic islets were encapsulated by a biocompatible membrane composed of the molecular sequence of alginate-polylysine-alginate. The encapsulated islets released insulin into the culture medium in response to secretagogues in short-term incubation. In long-term culture, the encapsulated islets maintained their insulin-releasing capacity for 28 days at a level similar to that of the unencapsulated islets. No overgrowth of fibroblastic cells was observed inside the capsule even after 70 days of culture. Further, the encapsulated hamster islets were xenotransplanted to streptozotocin-induced diabetic rats intraperitoneally. Some of the encapsulated islets, which were recovered from a recipient 27 days after transplantation, were found to be viable, although prolonged normalization of fasting plasma glucose levels of the recipients could not been achieved. On the contrary, the unencapsulated islets were replaced by massive connective tissue elements and insulin-positive B cells were hardly detected within the grafts 22 days after transplantation. The results of this study seem to confirm the potential of the application of the encapsulating technique to primary culture of parenchymal cells and to transplantation of pancreatic islets.