Susan K. Tam

Factors influencing alginate gel biocompatibility

Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

Direct effect of alginate purification on the survival of islets immobilized in alginate-based microcapsules

Alginate purification has been shown to decrease the host immune response to implanted alginate-based microcapsules, but the direct effect of contaminants on islet cell survival remains unknown. Wistar rat islets were immobilized in calcium alginate beads made with crude vs. purified alginate and then incubated in CMRL culture medium. Islet survival was evaluated at 1, 4, 7, 14 and 27 days post-encapsulation. Islet viability was investigated using a dual staining assay (propidium iodide and orange acridine). The islet cell necrosis and the proportion of apoptotic cells were quantified under optical microscopy and with a TUNEL assay, respectively. Islets immobilized in purified alginate were more viable, and had fewer necrotic centers, a smaller area of central necrosis and a lower number of apoptotic cells. At day 14 and 27 post-encapsulation, respectively, 48% and 23% of islets were viable with purified alginate vs. 18% and 8% with crude alginate (p<0.05). At day 14, the surface area of central necrosis and the number of necrotic islets were more important with the impure alginate (65% vs. 45% and 73% vs. 53%, respectively; p<0.05). We conclude that alginate purification improves the survival of islets that are immobilized in alginate-based microcapsules. These findings indicate that caution should be taken in the interpretation of in vivo experiments, as the results could be explained by either a direct effect on islet survival or a modification of the host reaction, or both. Moreover, it suggests that the effect on islet viability should be assessed during the development of biomaterials for cell encapsulation.

Adsorption of human immunoglobulin to implantable alginate-poly-L-lysine microcapsules: effect of microcapsule composition.

Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the immune response towards the implants. Here, we investigate the adsorption of immunoglobulins (IgG, IgM, and IgA) onto the surface of APA microcapsules in vitro after their exposure to human serum and peritoneal fluid. Immunoglobulins (Ig) are considered to be opsonizing proteins, thus they tend to mediate inflammation when adsorbed to foreign surfaces. Ig adsorption was monitored using direct immunofluorescence. The amount of Ig adsorbed to the microcapsule surface was not significantly influenced by the guluronic acid content nor the purity level of the alginate, although microcapsules of intermediate-G purified alginate corresponded with the lowest adsorption levels. Ig adsorption was negligible when the poly-L-lysine membrane was omitted, suggesting that positive charges at the microcapsule surface are responsible for binding Ig.

Role of protein contaminants in the immunogenicity of alginates.

Alginate is widely used for cell microencapsulation and transplantation. There is a lack of standardization of alginate purity and composition. In a previous study, we compared different alginate purification methods and concluded that polyphenol and endotoxin contaminants were eliminated efficiently but residual protein contaminants persisted with all of the methods under evaluation. The objective of this study was to test the hypothesis that residual proteins play a role in the immunogenicity of certain alginate preparations. Using preparative size exclusion chromatography (SEC) and a large scale purification protocol that was derived from the findings obtained with SEC, we substantially decreased the protein content of alginate preparations. When implanted into mouse peritoneum, barium alginate beads made of alginates that were purified using SEC or the derived large scale protocol induced significantly less pericapsular cell adhesion than those made with control alginates. In conclusions, these results suggest that removing residual protein contamination may decrease the immunogenicity of certain alginate preparations. The measurement of proteins could be used as a screening method for evaluating alginate preparations.

Advances and Barriers in Mammalian Cell Encapsulation for Treatment of Diabetes

Mammalian cell encapsulation is under investigation for the treatment of a wide variety of diseases, since it allows for transplantation of endocrine cells in the absence of undesired immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. In spite of the simplicity of the concept, progress in the field of immuno-isolation has been hampered. During the past two decades, three major approaches of encapsulation have been studied. These include (i) intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt, (ii) extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites, and (iii) extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of the three approaches are discussed and compared in view of applicability in clinical islet transplantation. At present, microcapsules, due to their spatial characteristics, offer better diffusion capacity than mac- rocapsules. During the past five years, important advances have been made in the knowledge of the characteristics and requirements capsules have to meet in order to provide optimal biocompatibility and survival of the enveloped tissue. Novel insight shows that islet-cells themselves and not the capsule materials should be held responsible for loss of a significant portion of the immuno-isolated islet cells and, thus, failure of the grafts on the long term. New approaches in which newly discovered inflammatory responses are silenced bring the technology of transplantation of immunoiso- lated cells close to clinical application.

Understanding the biocompatibility of microcapsules designed for islet cell transplantation

| 233 (score 1.02 ± 0.03). This difference in biocompatibility was significant (p < 0.001). Yet, alginate hydrophilicity was similar, as water droplets on dry films had contact angles of 38.7 ± 1.7° and 39.0 ± 1.3° for Alg I and Alg II, respectively. When PLL was added to the capsule membrane, the immune response was more severe (p < 0.01), as cell adhesion scores increased to 1.11 ± 0.11 for Alg I and to 1.55 ± 0.07 for Alg II. Similarly, the cell adhesion score increased to 0.49 ± 0.06 when PLO was added to Alg I capsules, indicating a non-significant increase in capsule immunogenicity. A comparison of contact angles of water on dry films containing polycations was not possible due to irregular surfaces and complete wetting. We are currently measuring the contact angles of air bubbles on hydrated films. conclusions: The guluronic acid content of alginate has a significant effect on its biocompatibility in vivo. Incorporating polycations into the membranes of alginate microcapsules lowers their biocompatibility. So far, these results cannot be explained by the hydrophilicity of the samples. Transplantation islet and pancreas No conflict of interest

Improving the function of microencapsulated islets using co-encapsulation with pancreatic duct cells

Islet transplantation has normalized the blood glucose levels in patients with type 1 diabetes. However, this treatment requires the use of potentially harmful immunosuppressive drugs. An alternative to immunosuppression is the use of semi-permeable microcapsules that allow the diffusion of glucose, nutrients and oxygen but protect the transplanted cells against immune reaction. Another important problem is the limited survival of encapsulated and free islet cells. Pancreatic duct cells have been shown to play an important role in pancreas morphogenesis. The co-incubation of pancreatic duct cells and islets has decreased cell apoptosis and necrosis in the immediate post-isolation period. We have previously shown that incubating islets with IGF-II, the principal peptide that is secreted by duct cells, diminishes apoptosis and necrosis of encapsulated islets and allows the restoration of euglycaemia in diabetic mice using fewer islets. The objective of this study is to investigate the effect of co-encapsulating islets with pancreatic duct cells on islet cell survival. Pancreatic duct cells were isolated from C57BL/6 mice and their identity was confirmed by immunohistochemistry using antibodies against cytokeratin 7 (CK7) and IGF-II. The presence of known contaminants (fibroblasts and mesenchymals cells) was investigated by flow cytometry using their common marker: vimentin. Islets encapsulated alone or co-encapsulated with two different concentrations of duct cells (20 or 100 cells per capsule) were maintained in culture and evaluated at days 1, 7, 14 and 27 postencapsulation. Islet viability was evaluated using fluorescent dyes (acridine orange and propidium iodide), the percentage of necrotic islets by inverted-microscope analysis and their function by an MTS test. Duct cells expressed IGF-II and CK7. The cell preparation was pure at >85%. Islets cell viability was between 60-80% for islets encapsulated alone or with duct cells with no significant difference. This coencapsulation did not have a significant impact on the islets necrosis which remained consistently between 30-40%. Co-encapsulating islets with 100 duct cells had a significant impact on islet function (vs. islets encapsulated alone). This was demonstrated by the MTS test, which showed an absorbance at 492 nm of 0.32 ± 0.06 for co-encapsulated islets (vs 0.09 ± 0.01; p<0.005) at day 1 and 0.38 ± 0.12 (vs 0.10 ± 0.02; p<0.05) at day 27. The present study demonstrated that co-encapsulation with duct cells had a moderate effect on islet function, but failed to show a positive effect on islet cell survival. Considering the concordant results of previous studies, the present results were unexpected. Further studies are needed to explain these results and will involve in vivo transplantation in diabetic animals.