Introduction: Alginate encapsulated islets have the potential to treat Type I diabetes in humans and provide long-term protection from immune-mediated graft damage. Alginate pore size is a crucial parameter that aids in the protection of islets from host immune recognition, while allowing insulin, oxygen, and other micronutrients to diffuse through the capsules. The aim of this study is to determine the permeability of alginate microcapsules by using dextrans of specific sizes to identify capsules with optimal diffusion parameters.
Materials and Methods: Blank and islet-containing microcapsules were made with varying concentrations (1.5%, 2.5%, 3.5% w/v) of ultra-pure low viscosity alginates (high mannuronate or UP LVM, high guluronate or UP LVG) using a microcapsule generator (Nisco Engineering AG). Microcapsules were incubated at 37oC in either 5mM CaCl2 (Ca5) or young porcine islet (YPI) media for 24-hours before permeability analysis using a dextran diffusion assay. Three fluorescently-tagged dextrans [cascade blue labeled 10kDa dextran, fluorescein isothyocyanate (FITC) labeled 70kDa, 150kDa, or 250kDa dextran, tetramethylrhodamine isothiocyanate (TRITC) labeled 500kDa dextran] were sequentially added to 500µL of solution Ca5 or YPI following which the microcapsule samples were added. Images were obtained (n=10) using a Leica TCS SP8 confocal microscope at 1, 30, and 60 minutes. The images were analyzed using a MATLAB™ (v. 2014a) Graphical User Interface (GUI) that determined the percentage change in fluorescence intensity inside the capsules over time. All data is reported as mean±SEM. Statistical analysis was performed using a one-way ANOVA with a post hoc Tukey test and p<0.05 was considered statistically significant.
Results and Discussion: Dextrans of all sizes diffused into the capsules and reached equilibrium, approximately one hour after plating. In all the groups studied, 10kDa dextrans showed the greatest diffusion followed by 70kDa, 150kDa, 250kDa, and 500kDa dextrans, suggesting that alginate microcapsules are relatively impermeable to large molecules. The presence of islets within the capsules did not significantly affect permeability (10kDa dextran; 60 min; islet: 48±1% vs. blank: 48±0%). Factors that significantly reduced microcapsule permeability were: high mannuronate content (35±0%; UP LVM, 66±0%; UP LVG), increasing alginate concentration (31±0%; 2.5% UP LVM, 14±0%; 3.5% UP LVM, p<0.01, ANOVA), exposure to YPI media (65±0%; 5mM CaCl2, 38±0%; YPI) and incubation at 37oC (48±0%l; 37oC, 61±0%; 24oC). Interestingly, with increasing alginate concentration (3.5% w/v) UP LVG capsules demonstrated similar permeability profiles for 70-250kDa dextrans (~size of IgG) compared to UP LVM capsules, while allowing greater 10kDa diffusion (~ size of insulin).
Conclusion: The results of this study suggest that alginate composition, concentration and storage conditions (temperature & osmolarity) greatly influence permeability. From the results obtained, it is believed that UP LVM microcapsules made using high concentrations and incubated at physiological conditions (isosmolar fluid at 37oC) would afford significantly better protection from the humoral immune system. Future studies will evaluate alginate microcapsule permeability in capsules implanted in the peritoneal cavity and subcutaneous space ex vivo, to accurately evaluate the effect of physiological temperatures and body fluids on microcapsule permeability.