title: Assessment of the reproducibility of alginate encapsulation of pancreatic islets using the MTT colorimetric assay.
abstract: Radioisotope diffusion experiments demonstrate that alginate/polyaminoacid encapsulation can prevent antibody and cytotoxic cell contact in vitro. The unpredictable outcome of xenotransplantation of encapsulated islets may reflect incomplete encapsulation. We have assessed a cytotoxic/MTT (tetrazolium) assay to test antibody permeability of capsules. Samples of free porcine islet tissue, and islet tissue encapsulated in alginate/poly-L-lysine/alginate microspheres were incubated with fresh autologous pig serum or normal human serum overnight. Cell metabolism was assessed by the MTT assay. Data from eight experiments (10 replicates/experiment) were analyzed using the Mann-Whitney U-test. Values were deemed significant when p < 0.05. Free islets cultured in human serum showed a significant reduction in metabolism when compared with islets cultured in pig serum: percentage reduction 52 +/- 23% (mean +/- SD). The differences in formazan production were significant in all experiments (p < 0.05). Alginate encapsulation of islets or removal of xenoreactive antibodies in human serum by adsorption was shown to prevent the effects of complement-mediated lysis. However, in one of the eight experiments, there was a significant reduction in islet metabolism after exposure of encapsulated porcine islets to human serum. In conclusion, it has been shown that alginate encapsulation can prevent complement-mediated lysis. However, the encapsulation process employed was imperfect and did not prevent complement-mediated lysis of porcine islets in all experiments. The cytotoxicity/MTT assay allows investigation of the permeability of capsules to serum antibodies and could be performed to determine the viability of the islets and the integrity of microcapsules prior to transplantation.
authors.affiliation: Department of General Surgery, Worcester Royal Infirmary, UK.
authors.forename: K A
authors.initials: KA
authors.lastname: Heald
authors.forename: T R
authors.initials: TR
authors.lastname: Jay
authors.forename: R
authors.initials: R
authors.lastname: Downing
mesh_terms: D000464:Alginates; D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D003124:Colorimetry; D015941:Complement Hemolytic Activity Assay; D006084:Graft Rejection / Q000517:prevention & control; D006801:Humans; D007515:Islets of Langerhans / Q000276:immunology* / Q000502:physiology; D016381:Islets of Langerhans Transplantation; D008567:Membranes, Artificial; D011107:Polylysine / Q000031:analogs & derivatives*; D013552:Swine / Q000097:blood / Q000502:physiology
chemical_list: D000464:Alginates; D008567:Membranes, Artificial; C051546:alginate-polylysine-alginate; D011107:Polylysine