A. Horcher

Production of purified alginates suitable for use in immunoisolated transplantation.

Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats.

Alginate coating of islets of Langerhans: in vitro studies on a new method for microencapsulation for immuno-isolated transplantation.

Immuno-isolated transplantation offers the attractive prospect of being able to transplant xenogeneic islets without immunosuppression. This study introduces a completely new method of coating single islets using a homogenous alginate membrane approximately 10 μm thick. During glucose challenge (perifusion and static incubation) encapsulated islets show the same pattern and quantity of insulin release as non-encapsulated controls. This encapsulation method markedly reduces the amount of transplanted material by reducing the size of the capsule. It is suggested that encapsulated islets may be transplanted into sites such as the renal capsule or omentum or even by intraportal injection into the liver.

BIOCOMPATIBILITY AND IMMUNOLOGY IN THE ENCAPSULATION OF ISLETS OF LANGERHANS (BIOARTIFICIAL PANCREAS)

Successful transplantation of encapsulated islets (bioartificial pancreas) would circumvent problems of islet availability and rejection in the treatment of insulin-dependent diabetes with biological organ replacement. Alginates are widely used as a hydrogel matrix or membrane for immunoprotected transplantation. A major problem in the use of diffusion-based devices is the biocompatibility of the material used. The foreign body reaction after implantation of empty microcapsules into different compartments in rats, dogs and pigs is evaluated in this article. However, biocompatibility of the bioartificial pancreas has three different aspects: reaction of the entrapped islet to the encapsulation technique and material; reaction of the recipient against the incorporated device (= foreign body reaction); and finally the reaction of the recipient against the encapsulated islet (= immunology of bioartificial pancreas). It is obvious from different experiments that even if foreign body reactions (reactions against material) are almost abolished the recipient may react against material released from the encapsulated islet. In conclusion, transplantation of encapsulated islets induces various morphological reactions (i.e. inflammation and fibrosis) as a result of a variety of donor and recipient related factors. Therefore, the use of an adequate animal model that reflects the human situation is essential for progress in the development of a bioartificial pancreas.

Barium-cross-linked alginate beads: a simple, one-step method for successful immunoisolated transplantation of islets of Langerhans

Transplantation of xenogeneic islets in immunoisolating membranes may solve the problems of the availability of human donor organs and long-term immunosuppression. Alginates are widely used for microencapsulation of isolated islets. This study presents data of a new method in which alginate was cross-linked with barium ions. In the perifusion experiment microencapsulated rat islets showed a biphasic insulin release with a short delay of the first phase. During static glucose challenge the insulin release ranged from 40% to 70% compared to free floating controls. In 3 of 11 diabetic mice, transplantation of 800 barium-alginate-bead encapsulated rat islets resulted in a non-fasting normoglycaemia at least up to 70 days. In the same model, transplantation of 3000 encapsulated porcine islets resulted in 6 of 10 recipients in normoglycaemia up to day 70, while 3 animals were still normoglycaemic on day 100. On day 21 after transplantation of encapsulated rat and porcine islets and intraperitoneal glucose tolerance test revealed rapid glucose assimilation in both groups. Histological examination demonstrated well-preserved islets at the end of the experiments. Immunohistological B-cell staining revealed the absence of recruitment of β-cells in the recipients own pancreas. The barium-alginate microencapsulation method represents a simple one-step method for effective immunoisolated transplantation of large-scale islet preparations.

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4±30.1×106) compared with empty capsules (41.4±19.7×106) and non-encapsulated porcine islets (18.1±3.3×106). The percentage of CD 3 positive T-lymphocytes rose to 44.5±11.5% in case of microencapsulated xenografts compared with 19.2±8.2% for non-encapsulated xenografts and 4.9±2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2±8.9% vs 15.2±7.0% for free islets and 4.9±1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Alginate-based Microcapsules for Immunoprotected Islet Transplantationa

Islet transplantation is a promising therapeutic approach for the treatment of insulin-dependent diabetes mellitus. Nevertheless, its broader clinical use is hampered by the shortage of human organ donors as well as the need for a permanent immunosuppressive drug therapy in order to avoid rejection. Microencapsulation shall help to overcome this problem by creating an immunoprotected transplantation site. Biocompatibility of the encapsulation material and the possible immuno-interaction of the grafted tissue and the host immune system need to be examined very carefully. In transplantation experiments, we could show that the long-term function of the graft is dependent on the species of the islet donor, indicating that there has to be a recognition of the encapsulated islet despite the encapsulation membrane. This could be confirmed by in vitro data in the mixed lymphocyte islet culture (MLIC). Moreover, morphological studies of the tissue reaction toward encapsulated syngeneic vs. allogeneic vs. xenogeneic encapsulated islets reveal that the greater the difference between donor and recipient species the greater the amount of fibrous tissue formation. Thus, for the outcome of transplantation experiments, not only the material-related biocompatibility but as well the reaction towards the whole device (consisting of the capsule plus the encapsulated tissue) are crucial. Therefore, immunoprotection does not only comprise the protection of the grafted tissue from the host immune effector mechanisms but as well the inhibition of the recognition of the graft by the host immune system.

Transplantation of free and microencapsulated islets in rats: evidence for the requirement of an increased islet mass for transplantation into the peritoneal site.

Microencapsulation of islets of Langerhans may avoid the necessity of a permanent immunosuppressive drug therapy and opens up new perspectives for xenotransplantation in the treatment of insulin dependent diabetes. In a mouse model we recently showed long-term normoglycemia after microencapsulated xenotransplantation. Since the acceptance of mice to any kind of foreign material is quite high we assume that the rat model better reflects the situation of higher mammalians or even humans. Due to the volume of the transplanted material (i.e. islets + alginate-capsule) only the peritoneal cavity can be used up to now. The quantity of islets necessary to normalize the non-fasting blood glucose level was much higher than expected and free transplants needed even a higher amount of islets than encapsulated ones (3000 encapsulated vs. 2 x 3000 non-encapsulated). Transplantation beneath the kidney capsule was successful with only 1200-1500 islets per rat proving the metabolic potency of the islets. Implantation of empty capsules did not alter the diabetic state. We conclude that the alginate matrix may act as a “spacer” creating a distance between the consuments of a lacking substrate esp. oxygen in an unfavourable environment and perhaps protect it from unspecific mediators released during the postoperative period. Our findings underline the necessity for smaller capsules that would enable us to use other transplantation sites.

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12–14 days culture at 22°C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.

Lymphoid activation by micro-and macroencapsulated islets during mixed lymphocyte islet culture

We have recently demonstrated long-lasting normoglycaemia after transplantation of barium alginate microencapsulated rat and porcine islets. Nevertheless the transplantation results obtained with different microencapsulation techniques have been controversial. Little is known about possible immune interactions between host and encapsulated islet. This study demonstrates in vitro stimulation of lymphoid cells by encapsulated islets that is similar to that of unencapsulated islets. This stimulation was reduced by a 4-day culture with unencapsulated islets only. After macroencapsulation of islets in hollow fibres a stimulatory effect was also observed, but this was less pronounced than after microencapsulation. Empty microcapsules as well as macrocapsules induced lymphoid proliferation as a result of mitogenic impurities in the encapsulation materials themselves. In the same donor-recipient combination in which we have shown successful transplantation, we found activation of the sensibilization arm of the immune system. This suggests that microencapsulation results in protection of the transplanted islets from the action of the effector arm. This lymphoid activation could be triggered by the mitogeniticity of the encapsulation material itself. In the case of alginates these mitogenic factors could not be abolished by culture (i.e. dialysis).