B.J de Haan

Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets

Summary Graft failure of alginate-polylysine microencapsulated islets is often interpreted as the consequence of a non-specific foreign body reaction against the microcapsules, initiated by impurities present in crude alginate. The aim of the present study was to investigate if purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Alginate was purified by filtration, extraction and precipitation. Microcapsules prepared from crude or purified alginate were implanted in the peritoneal cavity of normoglycaemic AO-rats and retrieved at 1, 2, 3, 6, 9, and 12 months after implantation. With crude alginate, all capsules were overgrown within 1 month after implantation. With purified alginate, however, the portion of capsules overgrown was usually less than 10 %, even at 12 months after implantation. All recipients of islet allografts in capsules prepared of purified alginate became normoglycaemic within 5 days after implantation, but hyperglycaemia reoccurred after 6 to 20 weeks. With intravenous and oral glucose tolerance test, all recipients had impaired glucose tolerance and insulin responses were virtually absent. After graft failure, capsules were retrieved (80–100 %) by peritoneal lavage. Histologically, the percentage of overgrown capsules was usually less than 10 % and maximally 31 %. This small portion cannot explain the occurrence of graft failure. The immunoprotective properties of the capsules were confirmed by similar if not identical survival times of encapsulated islet allo- and isografts. Our results show that purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Nevertheless, graft survival was still limited, most probably as a consequence of a lack of blood supply to the encapsulated islets. [Diabetologia (1997) 40: 262–270]

Efficacy of a prevascularized expanded polytetrafluoroethylene solid support system as a transplantation site for pancreatic islets.

An intraperitoneally located and prevascularized expanded polytetrafluoroethylene solid support is potentially a suitable transplantation site for encapsulated pancreatic islets, because it allows for both the implantation of a large volume islet graft in the immediate vicinity of blood vessels, and its complete removal. The present study investigates the efficacy of such solid supports for the implantation of nonencapsulated islet isografts in streptozotocin diabetic rat recipients. These solid supports were always coated with acidic fibroblast growth factor, because we found that this growth factor enhances the neovascularization. The success rates of 5-microl (group A) and 10-microl (group B) islet isografts in solid supports were compared with the success rates of 5-microl (group C) and 10-microl (group D) islet isografts implanted in the unmodified peritoneal cavity. Four of seven rats in group A and all seven rats in group B became normoglycemic for at least 6 months. Only two of eight rats in group C and four of eleven rats in group D showed normoglycemia. The normoglycemia lasted for at least 6 months in zero of two animals in group C and in three of four animals in group D. Because of the low success rates in groups C and D, intravenous and oral glucose testing were restricted to the successful recipients in groups A and B. Glucose tolerance was found to be proportional to the grafted islet volume but, expectedly, in both groups the glucose tolerance and the insulin responses were somewhat lower than in controls. Thus, the prevascularized expanded polytetrafluoroethylene solid support, rather than the unmodified peritoneal cavity, is an efficacious transplantation site, potentially suitable for encapsulated islets.

Association between macrophage activation and function of micro-encapsulated rat islets

Aims/hypothesisSurvival of microencapsulated islet grafts is limited, even when inflammatory reactions against the capsules are restricted to a small portion of less than 10%.MethodsThis study investigates both in vivo in rat recipients and in vitro whether cellular overgrowth on this minority of the capsules contributes to limitations in the functional survival of the 90% of the encapsulated islets which remain free of any cellular overgrowth.ResultsIn successful rat recipients of an allogenic microencapsulated islet graft we found that the vast majority of cells in the capsular overgrowth were activated ED-1 and ED-2 positive macrophages which were found in numbers of approximately 1500 per capsule. Co-culture of encapsulated islets with 1500 (nr8383) rat-macrophages per capsule showed that the activation of macrophages was caused by islet-derived bioactive factors since TNF-α and IL-1β secretion by macrophages was induced by islet-containing capsules and not by empty capsules. This activation of macrophages was associated with a decrease in function of the encapsulated islets as evidenced by a quantitatively reduced (35%) insulin response in static incubation and a slower response in perifusion.Conclusion/interpretationPresent research aims to design strategies for the temporary inhibition of macrophage activation since macrophages are predominantly present in the first two months after implantation. These strategies will serve as a pertinent basis for future clinical application of microencapsulated islets.

Kinetics of Intraperitoneally Infused Insulin In Rats: Functional Implications for the Bioartificial Pancreas

Intraperitoneal transplantation of encapsulated islets can restore normoglycemia in diabetic recipients but not normal glucose tolerance nor normal insulin responses to a physiological stimulus. This study investigates whether the intraperitoneal implantation site as such contributes to the interference with optimal transport kinetics between the islets and the bloodstream. Insulin was infused into the peritoneal cavity of conscious and freely moving rats in doses of 20, 40, and 80 pmol.l-1.min-1 during 15 min, to mimic the gradual release of insulin from an encapsulated, i.e., a nonvascularized, islet graft. With 20 pmol.l-1.min-1, we observed virtually no rise of insulin levels, and it took 30 min until glucose levels had dropped significantly. With 40 and 80 pmol.l-1.min-1 insulin infusions, there was a dose-dependent rise of insulin and decrease of glucose levels. When compared with intraportal infusions with the same insulin dosages, however, they were strongly delayed and reduced as well as prolonged. Similar results were obtained when inulin instead of insulin was intraperitoneally infused, with indicates that the transport of insulin from the peritoneal cavity to the bloodstream is mainly by passive diffusion. With a view on the clinical efficacy of the bioartificial pancreas, our findings indicate that we should focus on finding or creating a transplantation site that, more than the unmodified peritoneal cavity, permits close contact between the bloodstream and the encapsulated islet tissue.

Rat islet isolation yield and function are donor strain dependent

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.