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.

Stem cells and immortalized cells have considerable therapeutic potential but present risks of malignant transformation. Cell microencapsulation allows transplantation without immunosuppression. We have developed a method for microencapsulating living cells within covalently cross-linked membranes that are chemically and mechanically extremely resistant. We provide herein direct evidence that these microcapsules can prevent malignant cell dissemination. When 20,000 or more nonencapsulated EL-4 thymoma cells were implanted intraperitoneally in mice, all recipients died with widespread metastasis within 26.3±1.0 days. All recipients of 250,000 EL-4 cells microencapsulated in covalently cross-linked membranes were living and disease-free, 150 days post-implantation. Encapsulation in standard microcapsules only slightly delayed the recipient death. Pancreatic islets transplanted using either type of microcapsule presented similar survival. We conclude that microencapsulation in covalently cross-linked membranes prevents malignant cell dissemination.

Alginate, a polysaccharide extracted from brown seaweed, is widely used for the microencapsulation of islets of Langerhans, allowing their transplantation without immunosuppression. This natural polymer is known to be largely contaminated. The implantation of islets encapsulated using unpurified alginate leads to the development of fibrotic cell overgrowth around the microcapsules and normalization of the blood glucose is restricted to a very short period if it is achieved at all. Several research groups have developed their own purification method and obtained relatively good results. No comparative evaluation of the efficiencies of these methods has been published. We conducted an evaluative study of five different alginate preparations: a pharmaceutical-grade alginate in its raw state, the same alginate after purification according to three different published methods, and a commercially available purified alginate. The results showed that all purification methods reduced the amounts of known contaminants, that is, polyphenols, endotoxins, and proteins, although with varying efficiencies. Increased viscosity of alginate solutions was observed after purification of the alginates. Despite a general efficiency in decreasing contamination levels, all of the purified alginates contained relatively high residual amounts of protein contaminants. Because proteins may be immunogenic, these residual proteins may have a role in persisting microcapsule immunogenicity.

Alginate-poly-L-lysine-alginate (APA) microcapsules are currently being investigated as a means to immuno-isolate transplanted cells, but their biocompatibility is limited. In this study, we verified the hypothesis that poly-L-lysine (PLL), which is immunogenic when unbound, is exposed at the APA microcapsule surface. To do so, we analysed the microcapsule membrane at the micrometric/nanometric scale using attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. The results indicate that PLL and alginate molecules interact within the membrane. PLL exists in considerable amounts near the surface, contributing to the majority of the carbon within the outermost 100 Angstroms of the membrane. PLL was also detected at the true surface (the outermost monolayer) of the microcapsules. The exposure of PLL does not appear to result from defects in the outer alginate coating. This physicochemical model of APA microcapsules could explain their immunogenicity and will play an important role in the optimization of the microcapsule design.

Alginate is frequently used for cell encapsulation, but its biocompatibility is neither optimal nor reproducible. Purifying the alginate is critical for achieving a suitable biocompatibility. However, published purification methods vary in efficiency and may induce changes in polymer biofunctionality. Applying X-ray photoelectron spectroscopy, we showed that commercial alginates, purified by in-house and industrial methods, contained elemental impurities that contributed 0.41–1.73% of their atomic composition. Residual contaminants were identified to be proteins (nitrogen/COOH), endotoxins (phosphorus), and fucoidans (sulphur). Studies using attenuated total reflectance Fourier transform infrared spectroscopy suggested that trace contamination did not alter the alginate molecular structure. Alginate hydrophilicity increased by 19–40% after purification, in correlation with a reduction in protein and polyphenol content. Solution viscosity of the alginate increased by 28–108% after purification, in correlation with a reduction in protein content. These results demonstrate that commercial alginates contain potentially immunogenic contaminants that are not completely eliminated by current purification methods. Moreover, these contaminants alter the functional properties of the alginate in a manner that may compromise biocompatibility: Hydrophilicity may affect protein adsorption and solution viscosity influences the morphology of alginate-based microcapsules. These findings highlight the need to improve and better control alginate purity to ensure a reproducible biofunctionality and optimal biocompatibility of alginate and microcapsules.

The characteristics of the microcapsule surface, which interacts directly with the host macrophages, may have a role in the biocompatibility of alginate-poly-L-lysine (PLL)-alginate (APA) microcapsule. The objectives of the study were: 1) to develop and validate a simple, rapid, and sensitive in vitro method for assessing microcapsule biocompatibility, based on microcapsule coincubation with macrophages and measurement, by reverse transcriptase-polymerase chain reaction, of cytokine mRNA expression, and 2) to evaluate the effect of alginate purification and PLL coating on macrophage activation. The mRNA expression of tumor necrosis factor-alpha and interleukin-1beta was significantly higher when macrophages were coincubated with beads made with nonpurified compared with purified alginate (p<0.01, p<0.05, respectively) and negative control (p<0.001) or with APA microcapsules compared with non-PLL-coated alginate beads and negative control (p<0.001). The mRNA expression of interleukin-6 differed significantly only when APA microcapsules were compared with a negative control (p<0.05). These results confirm that alginate purification improves microcapsule biocompatibility, and suggest that PLL is not completely covered and/or neutralized by the second alginate incubation and thus has a role in the host macrophage activation. The assay is sensitive to both alginate contaminants and microcapsule surface characteristics and may be a useful tool for the development of biocompatible microcapsules.

Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-l-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.