Final Report Summary - BARP+ (Development of a bioartificial pancreas for type I diabetes therapy)
Four to five million people in Europe and about 80 million worldwide suffer from type 1 diabetes (insulin-dependent), characterised by a deficiency in insulin secretion resulting in hyperglycaemia. Beside transplantation of the pancreas or of islets, the only form of therapy is the administration of insulin by daily multi-injections or implantable pumps. However, these methods fail to reproduce the physiological secretion essential to maintain the glycaemia equilibrium. Diabetes is a serious illness as two-thirds of the affected individuals die from coronary diseases compared to one-third of the normoglycaemic subjects. In addition, 50 % of acquired blindness and 25 % chronic renal failures requiring dialysis are attributable to diabetes.
The project aimed to develop a new prototype of bioartificial pancreas suitable for encapsulation of insulin-secreting tissue and for implantation into the human. The final goal of this new approach is the successful treatment of type 1 diabetes by the use of the most modern technologies.
A protocol allowing the assembly of cells of both cell lines into pancreatic microorgans in vitro was set up. When perifused in a specially designed chamber, such pseudo-islets exhibited a physiological insulin secretory responsiveness. The most appropriate model, in which the device insert created a minimum flow resistance and turbulence during perifusion, was selected as the chamber for all further experiments. It was observed that the insulin-secreting cells assembled as pseudo-islets exhibited functional characteristics superior to those of single cells. The comparison of the functionality of RINm5F-GK insulin secreting cells and MIN-GK insulin secreting cells proved that the insulin secretory responsiveness to glucose was clearly superior in the MIN-GK insulin secreting cell line. Transferred into the bioartificial pancreas device under tissue culture conditions adapted to allow these pseudo-islets to grow in size to fit optimally into the free space of the grid inside, it was easily possible to accommodate up to several hundred of such pseudoislets and to close the device. Thus, it was possible to study in vitro the dynamic of diffusion of glucose and insulin into devices with membranes of different materials and pore sizes, resulting in different permeabilities for the various biological molecules.
The general aim was to test islet function in the BARP+ device in vivo and to assess the mechanism of cellular immune reaction as well as the potential beneficial effects of simultaneous use of mesenchymal stem cells to reduce immunoreaction against transplanted cells. In vivo function of islet containing devices was evaluated after implantation in the peritoneal cavity of rats made diabetic with streptozotocin. For syngeneic- and allotransplantation, animals failed to reach normoglycemia. For xenogenic experiments, animals reached normoglycemia 6 hours after implantation. Physiological blood glucose levels were maintained for up to three days after implantation. Concomitant mesenchymal stem cells transplantation showed a beneficial effect on islet function, but for a short time period.
After transplantation in all models, islets were damaged and only a few cells in the islets remained intact. The mild cellular deposition and the absence of CD8+ indicated that devices could provide an effective immunoisolation of transplanted islets from the host immune system of the recipient preventing the rejection of the implanted material. Therefore the loss of islet function and viability was not related to cellular rejection but may derive from encapsulation in the device. Further development of new materials could improve islet survival and function in the BARP+ device.
The consortium delineated the components to create an optimal environment of encapsulation, evaluate the effect of fluorocarbons on the preservation of islets viability and investigate on the necessary nutrients to be present at various stages of the development of the bioartificial pancreas. Studies performed in the presence of extracellular matrix components demonstrated that collagen had no effect on the viability and functionality of islets. A protocol to prepare the extracellular matrix components to be used as encapsulation environment was set up.
Regarding the fluorocarbons effect, studies with mouse insulinoma MIN-6 beta cell lines capable to form pseudo-islets were performed. In the presence of increased concentrations of fluorocarbons emulsion, the cellular adhesion was evaluated by the measurement of adhering cells. Viability of pseudo-islets was assessed by Blue Trypan dying.
To confirm the effect of fluorocarbons, experiments were also performed on primary cultures of porcine islets. The data obtained enabled to conclude that fluorocarbons have a beneficial effect on tissue preservation and the present study introduced a new approach in pancreatic islets preservation in culture by preventing cell adhesion and leading to the increase of cell viability.
In addition, it was discovered that fluorocarbons exhibited a strong chaotropic effect with two major consequences beneficial to the system, i.e. the prohibition of islets aggregation and the positive effect on the formation of pseudo-islets from cell lines. This may open a new research avenue on the generation of the latter that can be used as surrogate cells in a non-hypoxic environment.
Various media required specifically for preparation, encapsulation, storage and transportation of the islets and recommended for the various species or surrogate, i.e. human, pig, rat, cell lines, dispersed cells, were prepared.
Minicells, devices of small size, and permeation cells to test the diffusion through the PVP or cellulose coated polycarbonate membranes were produced. Devices of a capacity of 100 000 pancreatic islets were developed for in vivo tests in the pig. The second-generation device implied the improvement of the chamber. For that purpose, two moulds for silicone and one mould for thermoplastic were manufactured to modify the frame of the device, include inlets and outlets with integrated system of septa to drain the old material and fill with fresh cells through silicone tubing according to various modes, i.e. sucking, gravity, pressure or any combined solution. Several prototypes were built and tested.
The membrane improvement followed two axes: i) new treatments of the polycarbonate film; and ii) conception, testing and development of intelligent multilayers membranes. Different ion track-etched prototype membranes were produced and characterised. After treatments and sterilisation, surfaces were analysed and compared to the untreated ones. Polycarbonate membranes treated with PVP or cellulose offered the best results in terms of glucose diffusion and prohibited immunoglobulin diffusion. After sterilisation, the diffusion of glucose through the PVP or cellulose treated polycarbonate membrane was increased as compared to the crude membrane and the protection against immunoglobulin crossing maintained.
Thus both treatments and sterilisation proved effective and not to alter the membranes characteristics and properties. Furthermore, silicone slides used for the device manufacture were treated to make them hydrophilic to improve their surface properties. Finally, in the second-generation device, all silicone was hidden underneath the biocompatible membrane.
Formation of multilayers on flat surface to incorporate cells and islets within the polyelectrolyte films was undertaken. With polyelectrolytes of biochemical nature, the study of the interactions between beta-cells and multilayer modified substrates showed that a three-layer system would be an appropriate treatment to obtain adhering aggregated beta-cells. Such surface modification would be convenient for pancreatic islets to reduce or prohibit the cellular multiplication of undesired adhering cells like fibroblasts. The non-toxicity of the polymers - poly-alginate and poly-L-lysine, was established by viability tests performed as before. The confirmation of these results was placed under investigation with pancreatic islets.
Several combinations of polyelectrolyte pairs of chemical nature, number of layers and mediums were prepared to encapsulate pancreatic islets and/or surrogate cells. To analyse their effects on encapsulated human, pig and rat pancreatic islets, tests were performed regarding the cytotoxicity, long-term viability after encapsulation and cell response to an external signal, glucose or amino acids.
The project aimed to develop a new prototype of bioartificial pancreas suitable for encapsulation of insulin-secreting tissue and for implantation into the human. The final goal of this new approach is the successful treatment of type 1 diabetes by the use of the most modern technologies.
A protocol allowing the assembly of cells of both cell lines into pancreatic microorgans in vitro was set up. When perifused in a specially designed chamber, such pseudo-islets exhibited a physiological insulin secretory responsiveness. The most appropriate model, in which the device insert created a minimum flow resistance and turbulence during perifusion, was selected as the chamber for all further experiments. It was observed that the insulin-secreting cells assembled as pseudo-islets exhibited functional characteristics superior to those of single cells. The comparison of the functionality of RINm5F-GK insulin secreting cells and MIN-GK insulin secreting cells proved that the insulin secretory responsiveness to glucose was clearly superior in the MIN-GK insulin secreting cell line. Transferred into the bioartificial pancreas device under tissue culture conditions adapted to allow these pseudo-islets to grow in size to fit optimally into the free space of the grid inside, it was easily possible to accommodate up to several hundred of such pseudoislets and to close the device. Thus, it was possible to study in vitro the dynamic of diffusion of glucose and insulin into devices with membranes of different materials and pore sizes, resulting in different permeabilities for the various biological molecules.
The general aim was to test islet function in the BARP+ device in vivo and to assess the mechanism of cellular immune reaction as well as the potential beneficial effects of simultaneous use of mesenchymal stem cells to reduce immunoreaction against transplanted cells. In vivo function of islet containing devices was evaluated after implantation in the peritoneal cavity of rats made diabetic with streptozotocin. For syngeneic- and allotransplantation, animals failed to reach normoglycemia. For xenogenic experiments, animals reached normoglycemia 6 hours after implantation. Physiological blood glucose levels were maintained for up to three days after implantation. Concomitant mesenchymal stem cells transplantation showed a beneficial effect on islet function, but for a short time period.
After transplantation in all models, islets were damaged and only a few cells in the islets remained intact. The mild cellular deposition and the absence of CD8+ indicated that devices could provide an effective immunoisolation of transplanted islets from the host immune system of the recipient preventing the rejection of the implanted material. Therefore the loss of islet function and viability was not related to cellular rejection but may derive from encapsulation in the device. Further development of new materials could improve islet survival and function in the BARP+ device.
The consortium delineated the components to create an optimal environment of encapsulation, evaluate the effect of fluorocarbons on the preservation of islets viability and investigate on the necessary nutrients to be present at various stages of the development of the bioartificial pancreas. Studies performed in the presence of extracellular matrix components demonstrated that collagen had no effect on the viability and functionality of islets. A protocol to prepare the extracellular matrix components to be used as encapsulation environment was set up.
Regarding the fluorocarbons effect, studies with mouse insulinoma MIN-6 beta cell lines capable to form pseudo-islets were performed. In the presence of increased concentrations of fluorocarbons emulsion, the cellular adhesion was evaluated by the measurement of adhering cells. Viability of pseudo-islets was assessed by Blue Trypan dying.
To confirm the effect of fluorocarbons, experiments were also performed on primary cultures of porcine islets. The data obtained enabled to conclude that fluorocarbons have a beneficial effect on tissue preservation and the present study introduced a new approach in pancreatic islets preservation in culture by preventing cell adhesion and leading to the increase of cell viability.
In addition, it was discovered that fluorocarbons exhibited a strong chaotropic effect with two major consequences beneficial to the system, i.e. the prohibition of islets aggregation and the positive effect on the formation of pseudo-islets from cell lines. This may open a new research avenue on the generation of the latter that can be used as surrogate cells in a non-hypoxic environment.
Various media required specifically for preparation, encapsulation, storage and transportation of the islets and recommended for the various species or surrogate, i.e. human, pig, rat, cell lines, dispersed cells, were prepared.
Minicells, devices of small size, and permeation cells to test the diffusion through the PVP or cellulose coated polycarbonate membranes were produced. Devices of a capacity of 100 000 pancreatic islets were developed for in vivo tests in the pig. The second-generation device implied the improvement of the chamber. For that purpose, two moulds for silicone and one mould for thermoplastic were manufactured to modify the frame of the device, include inlets and outlets with integrated system of septa to drain the old material and fill with fresh cells through silicone tubing according to various modes, i.e. sucking, gravity, pressure or any combined solution. Several prototypes were built and tested.
The membrane improvement followed two axes: i) new treatments of the polycarbonate film; and ii) conception, testing and development of intelligent multilayers membranes. Different ion track-etched prototype membranes were produced and characterised. After treatments and sterilisation, surfaces were analysed and compared to the untreated ones. Polycarbonate membranes treated with PVP or cellulose offered the best results in terms of glucose diffusion and prohibited immunoglobulin diffusion. After sterilisation, the diffusion of glucose through the PVP or cellulose treated polycarbonate membrane was increased as compared to the crude membrane and the protection against immunoglobulin crossing maintained.
Thus both treatments and sterilisation proved effective and not to alter the membranes characteristics and properties. Furthermore, silicone slides used for the device manufacture were treated to make them hydrophilic to improve their surface properties. Finally, in the second-generation device, all silicone was hidden underneath the biocompatible membrane.
Formation of multilayers on flat surface to incorporate cells and islets within the polyelectrolyte films was undertaken. With polyelectrolytes of biochemical nature, the study of the interactions between beta-cells and multilayer modified substrates showed that a three-layer system would be an appropriate treatment to obtain adhering aggregated beta-cells. Such surface modification would be convenient for pancreatic islets to reduce or prohibit the cellular multiplication of undesired adhering cells like fibroblasts. The non-toxicity of the polymers - poly-alginate and poly-L-lysine, was established by viability tests performed as before. The confirmation of these results was placed under investigation with pancreatic islets.
Several combinations of polyelectrolyte pairs of chemical nature, number of layers and mediums were prepared to encapsulate pancreatic islets and/or surrogate cells. To analyse their effects on encapsulated human, pig and rat pancreatic islets, tests were performed regarding the cytotoxicity, long-term viability after encapsulation and cell response to an external signal, glucose or amino acids.