Nicolas Lembert

Encapsulation of islets in rough surface, hydroxymethylated polysulfone capillaries stimulates VEGF release and promotes vascularization after transplantation.

The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.

Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets.

The determination of islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. The common microscopical analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 μm. However, islets are of irregular form, which makes this determination user dependent, and the analysis is irreproducible once the original sample is discarded. This routine technique of islet quantification was compared with the analysis of areal density measurements. It was assumed that the entire area occupied by islets can be expressed in IEQ without sizing and counting individual islets. Porcine islets were isolated by continuous digestion/filtration and purified by gradient centrifugation. Purified islets were stained with dithizone and were repeatedly pictured under the microscope with random area selection. A total of 51 pictures was taken from 11 different purifications and stained islets were detected by digital image analysis. The correlation coefficient (r) between both analyses was 0.977 with an underestimation of islet yield by areal density detection (slope: 0.75 ± 0.03). Areal density analysis per picture took about 1 min, which is about 10 times faster than the traditional method without increasing the method error (CV 2.1% vs. 2.7%). In summary, areal density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single step analysis without computer programming and is valuable for online determinations of islet yield preceding transplantation.

Methyl pyruvate initiates membrane depolarization and insulin release by metabolic factors other than ATP.

The role of mitochondria in stimulus-secretion coupling of pancreatic beta-cells was examined using methyl pyruvate (MP). MP stimulated insulin secretion in the absence of glucose, with maximal effect at 5 mM. K+ (30 mM) alone, or in combination with diazoxide (100 microM), failed to enhance MP-induced secretion. Diazoxide (100 microM) inhibited MP-induced insulin secretion. MP depolarized the beta-cell in a concentration-dependent manner (5-20 mM). The sustained depolarization induced by 20 mM MP was not influenced by 100 microM diazoxide, but the continuous spiking activity was suppressed by 500 microM diazoxide. Pyruvate failed to initiate insulin release (5-20 mM) or to depolarize the membrane potential. ATP production in isolated beta-cell mitochondria was detected as accumulation of ATP in the medium during incubation in the presence of malate or glutamate in combination with pyruvate or MP. There was no difference in ATP production induced by pyruvate/malate or MP/malate in isolated beta-cell mitochondria. ATP production by MP/glutamate was higher than that induced by pyruvate/glutamate, but it was much lower than that induced by alpha-ketoisocaproate/glutamate. Pyruvate (5 mM) or MP (5 mM) had no effect on the ATP/ADP ratio in whole islets, whereas glucose (20 mM) significantly increased the whole islet ATP/ADP ratio. It is concluded that MP-induced beta-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production. Instead MP may exert a direct extramitochondrial effect, or it may stimulate beta-cell mitochondria to produce coupling factors different from ATP to initiate insulin release.

Oxidative stress increases potassium efflux from pancreatic islets by depletion of intracellular calcium stores

Oxidative stress to B-cells is thought to be of relevance in declining B-cell function and in the process of B-cell destruction. In other tissues including heart, brain and liver, oxidative stress has been shown to elevate the intracellular free calcium concentration and to provoke potassium efflux. We studied the effect of oxidative stress on Ca2+ and K+ (Rb+) outflow from pancreatic islets using the thiol oxidants DIP and BuOOH. Both compounds reversibly increased 86Rb+ efflux in the presence of 3 and 16.7 mmol/l glucose. Stimulation of 86Rb+ efflux was also evident in the absence of calcium. DIP evoked release of 45Ca2+ from the pancreatic islets both in the presence or absence of extracellular calcium. Employing inhibitors of the calcium-activated potassium channel (KCa) and the high conductance K+-channel (BKCa), the effect of DIP on 86Rb+ efflux was slightly diminished. Tolbutamide had no effect on 86Rb+ efflux in the presence of DIP. On the other hand thapsigargin, a blocker of the Ca2+-ATPase of the endoplasmic reticulum, completely suppressed the DIP-mediated 86Rb+ outflow. The data suggest that thiol oxidant-induced potassium efflux from pancreatic islets is mainly mediated through liberation of intracellular calcium and subsequent stimulation of calcium-activated potassium efflux.

In vitro test of new biomaterials for the development of a bioartificial pancreas.

Abstract: The implantation of macroencapsulated islets has the potential to restore endogenous insulin secretion in type 1 diabetics, with no need for lifetime immunosuppression. To match the physiological fluctuations of blood glucose concentrations with appropriate insulin release, the macroencapsulation material must combine immunoprotection with optimal diffusion properties for glucose and insulin. The impact of chemical modifications of polysulphone (PSU) capillary polymers with a cutoff of 50 kD on glucose‐induced insulin secretion of macroencapsulated rat islets was studied in perifusion experiments. The insulin release of free‐floating islets showed the typical rapid response to glucose stimulation. Total insulin release (AUC between minute 30 and 120 of perifusion) reached 117 ± 22 ng/ml. Blending PSU with polyvinylpyrrolidone or sodium‐dodecyl‐sulfate was not suitable for islet macroencapsulation, since glucose‐induced insulin release was absent or disturbed. Hydroxy‐methylation (CH2OH) of PSU improved the secretory behavior of macroencapsulated islets depending on the degree of PSU substitution (DS 0.8, AUC 62 ± 15 ng/ml; DS 1.8, 111 ± 24 ng/ml). In highly substituted PSU‐capillaries the kinetics of glucose‐induced insulin release was very similar to that observed in free‐floating islets. Two consecutive glucose stimulations potentiated insulin release of free‐floating islets during the second period of stimulation. Furthermore, freshly isolated macroencapsulated islets responded with more efficient insulin secretion after the initial priming. In conclusion, in vitro membrane screening identified highly substituted hydroxy‐methylated PSU as the material of choice for islet encapsulation in a bioartificial pancreas.

K-ATP channel independent effects of pinacidil on ATP production in isolated cardiomyocyte or pancreatic β-cell mitochondria

Evidence has been presented that mitochondria contain ATP sensitive potassium channels (mK-ATP channels), which may confer tissue protection upon activation. It is, however, not known whether activation of mK-ATP channels has a direct effect on mitochondrial ATP production. This study was performed to define the effect of pinacidil (PIN) on ATP production by oxidative phosphorylation in isolated cardiomyocyte or pancreatic beta-cell mitochondria. Cardiomyocyte mitochondria produced seven times more ATP than beta-cell mitochondria in the presence of pyruvate/malate. PIN inhibited pyruvate/malate-induced mitochondrial ATP production with half maximal effect at 360 microM in both cell types. The inclusion of 5-hydroxydecanoate (5-HD) did not prevent this inhibition. Succinate induced a similar ATP production in cardiomyocyte or beta-cell mitochondria. In beta-cell mitochondria succinate-induced ATP production was inhibited by PIN with half maximal effects at 500 microM PIN. However, in cardiomyocyte mitochondria PIN stimulated succinate-induced ATP production 3-fold with half maximal effect at 100 microM and maximal effect at 200 microM. This PIN-dependent stimulation was mimicked by rotenone. The inclusion of 5-HD could not prevent these PIN effects. In conclusion, PIN may inhibit complex 1 of the respiratory chain without indications of opening mK-ATP channels. In cardiomyocytes with metabolically inhibited succinate dehydrogenase this results in a stimulation of ATP production conferring tissue protection. In beta-cells without a metabolically inhibited succinate dehydrogenase, there is no stimulation by PIN and tissue protection by PIN is not to be expected.

A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities.

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 ± 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were than purified by adding a gradient (bottom loading). Using this closed system 1098 ± 489 IEQ per gram pancreas were purified with a total cell viability of 67 ± 10% and a β-cell viability of 41 ± 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of β-cells was still 56 ± 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-α to 40 ± 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

Kapillarmembranen aus modifizierten Polysulfonen

The transplantation of islets of Langerhans would be the optimal treatment of insulin dependent diabetes. For this purpose capillary membranes are developed which show good diffusion characteristics for glucose, essential nutrient and insulin but at the same time protect the xenogenic transplant from immune rejection of the host. Modified udel® and radel* polysulfones are used as membrane materials which show, because of the introduced hydrophilic groups, lower protein adsorption compared to the source material. By phase inversion technique capillary membranes with different membrane structures are produced and the diffusion characteristics of insulin are observed. Capillaries made of hydroxymethylated polysulfone, having a degree of substitution of 1.7 and a finger-like structure, prove to be ideal relative to insulin due to their almost immediate and very good diffusion properties for the further development of a bioartifical pancreas. A c B O m a t e r i a l i e n 2 (2/3), 2001 Anja Enderle: Kapillarmembranen aus modifizierten Polysulfonen ORIGINAL· ARBEITEN

PK11195, ein spezifischer Ligand des peripheren Benzodiazepinrezeptors (PBR) schützt Langerhans’sche Inseln des Schweins vor zytokininduziertem Zelltod

Cytokines play an important role in the inhibition of function of transplanted islets. Exposure of islets to IL1β and TNF-α can reduce insulin secretion and favour islet cell apoptosis. This may contribute to the progressive loss of function of the islet graft as observed in clinical trials. Our studies with mouse islets have recently shown, that mouse islets express PBR and that PBR ligands protect isolated beta-cell mitochondria more effectively from cytokine induced dysfunction than cyclosporine A. In the present study, this anti-apoptotic effect of PBR-ligands was tested with porcine islets. Pancreata were procured from young marked weight pigs. Islets were isolated according to the continuous digestion filtration method and purified on a ficoll gradient using the COBE2991 cell processor. Organs and islet fractions were characterized according to insulin content/mg protein, insulin release, amylase activity/mg protein and vitality. In order to study cytoprotection by PBR ligands, islets where cultured for 24 h (8 °C, HAMS-F12) in the presence of cytokines alone or in combination of cytokines with PBR ligands PK11195 or Ro5-4864. In six consecutive preparations, 116875 ± 17759 IEQ were purified (1290 ± 118 IEQ/g pancreas). The total insulin content of the isolated islet fraction was 92 ± 31U. The purity was 98 % and 4/6 preparations showed glucose dependent insulin secretion. The viability of freshly isolated islet cells was 76 ± 10 %. Exposure of islets to cytokines reduced the fraction of vital b-cells from 45 ± 17 % to 33 ± 10 %. Co-incubation of islets with Ro5-4864 was without protective effect. However, co-incubation with PK11195 protected islet cells from cytokine induced cell death thus the observed fractional b-cell survival after co-culture was 45 ± 26 %. In conclusion, islet preparations from pigs compared well with human islet preparations with respect to number of IEQ, function and purity. Pig islets may thus constitute a suitable model system. PK11195 protects β-cells from pig islets very effectively from cytokine induced cell death. This effect will be studied in a transplantation model.