de Bart Haan

Factors Influencing Insulin Secretion From Encapsulated Islets

Adequate regulation of glucose levels by a microencapsulated pancreatic islet graft requires a minute-to-minute regulation of blood glucose. To design such a transplant, it is mandatory to have sufficient insight in factors influencing the kinetics of insulin secretion by encapsulated islets. The present study investigates factors influencing the glucose-induced insulin response of encapsulated islets in vitro. We applied static incubations and did the following observations. (i) Small islets (90–120 μm) showed a similar instead of a lower glucose-induced insulin response, suggesting that inclusion of only small islets, which are associated with lower protrusion and failing rates, has no consequences for the functional performance of the graft. (ii) A capsule diameter of 800 μm showed identical rather than lower glucose-induced insulin responses as smaller, 500-μm capsules. (iii) Capsule membranes constructed with a conventional permeability interfered with diffusion of insulin, as illustrated by a lower response of islets in capsules with a 10-min poly-L-lysine (PLL) membrane than islets in capsules with a 5-min PLL membrane. (iv) Irrespective of the tested porosity, the capsules provided sufficient immunoprotection because the 10-min PLL membranes did block diffusion of the cytokines IL-1β (17 kDa) and TNF-α (70 kDa) while the 5-min PLL membranes interfered with the diffusion of the vast majority of the cytokines. We conclude that capsules containing small islets (90–120 μm) and a membrane with a lower permeability than routinely applied is preferred in order to obtain a graft with adequate glucose-induced insulin responses.

Pancreatic Beta-Cell Purification by Altering FAD and NAD(P)H Metabolism

Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate (NAD(P)H) autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(P)H fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(P)H contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(P)H fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

Impaired glucose tolerance in recipients of an intraperitoneally implanted microencapsulated islet allograft is caused by the slow diffusion of insulin through the peritoneal membrane.

I NTRAPERITONEAL transplantation of microencapsulated islets has been reported to restore normoglycemia both in chemically induced and in autoimmune diabetic animal models,‘-5 and recently also in man.6 But a previous report from our laboratory showed that glucose tolerance remains disturbed in spite of normoglycemia, and that there is no increase of plasma insulin levels in response to a physiologic stimulus such as the intake of a meal5 This may be the consequence of several factors such as a gradual decrease of the number of viable and functioning islets after transplantation as a consequence of fibrotic overgrowth of the capsules, or a slow release of insulin in response to a glycemic stimulus as a consequence of overly large capsule diameters.’ The transplantation site as such may be another factor. Previous studies on the kinetics of intraperitoneal insulin absorption report a short delay between the intraperitoneal administration of insulin and rise of plasma insulin levels. *-‘i However, these studies were often performed with insulin bolus injections in unphysiologically high doses which may well be associated with a more rapid absorption of insulin than should be expected with a gradual release of insulin from encapsulated pancreatic islets. Therefore, the present study investigates whether the intraperitoneal implantation site as such interferes with optimal transport kinetics between the islets and the bloodstream.

An artificial transplantation site for pancreatic islets

MICROENCAPSULATED islets still have their functional limitations. Transplantation near blood vessels may improve the function. An intraperitoneally located and prevascularized expanded polytetrafluouroethylene (ePTFE) solid support is potentially a suitable transplantation site for encapsulated pancreatic islets because it allows both for the implantation of a large volume islet graft in the immediate vicinity of blood vessels and for its complete removal. ePTFE seems a suitable material for the construction of solid supports as it is biocompatible and can be coated to induce vascularization. The present study investigates the efficacy of an ePTFE solid support for the implantation of nonencapsulated islet isografts in streptozotocin-induced diabetic rat recipients.