Jean-Pierre Hallé

Biocompatibility and physicochemical characteristics of alginate-polycation microcapsules.

There is a need for better understanding of the biocompatibility of alginate-polycation microcapsules based on their physicochemical characteristics. Microcapsules composed of alginate with 44% (IntG) or 71% (HiG) guluronate, gelled with calcium (Ca) or barium (Ba) and coated with poly-L-lysine (PLL) or poly-l-ornithine (PLO), followed by IntG alginate were compared. For microcapsules with an IntG(Ca) gel core, using PLO instead of PLL resulted in less immune cell adhesion after 2 days in C57BL/6J mice. The PLO microcapsules were also characterized by greater hydrophilicity and superior resistance to swelling and damage under osmotic stress. For microcapsules with a PLL membrane, replacing the IntG(Ca) gel core with IntG(Ba) or HiG(Ca) gel resulted in stronger immune responses (p<0.05). This was explained by poor penetration of PLL into the gel, as demonstrated by Fourier transform infrared spectroscopy analyses and membrane rupturing during osmotic swelling. X-ray photoelectron spectroscopy analyses show that all microcapsules had the same amount of polycation at their surface. Moreover, alginate coatings had non-significant effects on the biocompatibility and physicochemical properties of the microcapsules. Thus, alginate-polycation interactions for membrane formation are more important for biocompatibility than either the quantity of polycation at the surface or the alginate coating.

Studies on small (<350 μm) alginate‐poly‐L‐lysine microcapsules. III. Biocompatibility of smaller versus standard microcapsules

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

Studies on small (< 300 microns) microcapsules: II--Parameters governing the production of alginate beads by high voltage electrostatic pulses.

The size of microcapsules is a critical parameter in the immunoisolation of islets of Langerhans by microencapsulation. The use of smaller capsules decreases the total implant volume and improves insulin kinetics and oxygen supply. A high voltage electrostatic pulse system was used for the production of small (<300 μm) alginate beads, the first step of the encapsulation technique. However, islets often protruded from capsules that were too small, further emphasizing the need for a method to control bead size. A study of 7 parameters [electrostatic pulse amplitude (A), duration (D) and wavelength (λ), pump flow rate (P), needle gauge, alginate viscosity and distance between electrodes] showed that P (r = 0.981, p = 0.003) and λ (r = 0.988, p = 0.0002) were the principal determinants of bead size. To detect potential interactions between parameters, 270 combinations of different levels of A, D, λ, and P were studied. A multivariate regression analysis of these data confirmed that P and λ are the prime determinants of bead size, and showed that a 2-parameter (P, λ) model could be used to precisely predict bead size (R2 = 0.84), while keeping the application simple. The precision of the predictive model is only slightly improved by the use of additional parameters. The reliability of the data used to elaborate this model was demonstrated (p = 0.6226) by comparing them with a second data set obtained under the same conditions. A third set of experiments confirmed the applicability of the model. This work has major implications on the preclinical application of microencapsulation since it showed that it is possible to predetermine the bead size.

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.

Studies on smaller (approximately 315 microM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules.

The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700–1200 μm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (~315 μm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 μm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 ± 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 μm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-μm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 ± 8 mmHg and remained elevated after 4 h (12 ± 3 mmHg), but returned to normal (8 ± 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. Conclusion: intrahepatic implantations of ~315-μm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Protection of islets of Langerhans from antibodies by microencapsulation with alginate poly-l-lysine membranes.

Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microen-capsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.

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.

Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress

A quantitative method has been developed for the evaluation of biomicrocapsule resistance to mechanical stress. Fluorescein isothiocyanate-labelled dextran (M.W. 2 x 10(6)) was microencapsulated in alginate-poly-L-lysine membranes. Microcapsules of 302.0 +/- 3.2 microns were mixed with 3 mm glass beads and continuously agitated for 0 to 144 h. The percentage of broken capsules was calculated by measuring the fluorescence in the supernatant and in the residual intact capsules after the latter were dissolved. The fluorescence method was validated by comparison with a manual method (handpicking under a stereomicroscope). The highest percentage of broken capsules was obtained with a ratio of 225 +/- 25 glass beads per 1000 microcapsules. The percentage of broken capsules increased linearly from 7.3% at 12 h to 48.3% at 72 h of continuous agitation. The applicability of the method was evaluated by studying microcapsules of potentially different levels of resistance. The results confirmed that capsule resistance is improved by increasing poly-L-lysine concentrations and incubation times. Microcapsules made with guluronic acid-rich alginate were stronger than those made with mannuronic acid-rich alginate. In conclusion, this is a simple, precise and sensitive method for the quantification of biomicrocapsule resistance to mechanical stress.

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.