Monitoring cytotoxicity against pig cells after transplantation using two-color fluorescence viability assay

• Published in: Transplantation proceedings Vol. 35; no. 5; pp. 2047 - 2048
• Main Authors: Díaz, T.M, Manez, R, Moscoso, I, Lopez, E, Centeno, A, Ortega, D, Domenech, N
• Format: Journal Article Conference Proceeding
• Language: English
• Published: New York, NY Elsevier Inc 01.08.2003; Elsevier Science
• Subjects: Animals; Animals, Genetically Modified; Biological and medical sciences; CD55 Antigens - genetics; CD55 Antigens - immunology; Cell Survival; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Heart Transplantation - immunology; Heart Transplantation - pathology; Papio; Propidium; Swine; Tissue, organ and graft immunology; Heart; Performance evaluation; Flow cytometry; Fluorescence; Homograft; Cytotoxicity; Transplantation; Xenogeneic system; Heterotransplantation; Two color diagram; Kidney; Surgery; Graft; Serum; Heteroantibody; Ungulata; Humoral immunity; Immune response; Exploration; Pig; Vertebrata; Mammalia; Surveillance; Animal; Artiodactyla; Technique; Viability
• ISSN: 0041-1345; 1873-2623
• DOI: 10.1016/S0041-1345(03)00708-5

Acute humoral xenograft rejection (AHXR) is now the major hurdle for the long-term survival of pig organs transplanted into nonhuman primates. Mechanisms involved in this rejection are not well understood, albeit that it has been proposed to require the participation of antibodies with specificities other than αGal. In this study, we evaluated a two-color fluorescence method, fluorescein dicetate (FAD)/propodium iodide (PI), to stain live versus dead cells, respectively, to monitor complement-mediated antibody cytotoxicity in hDAF pig-to-baboon xenotrasplants. FDA/PI flow cytometry assays showed a high correlation (rho Spearman = .736; P = .003) with the cytotoxic activities of baboon serum antibodies against PK15 cells, using either endogenous or exogenous complement. Average serum cytotoxicity against AOC40 was higher (59.82 ± 17.90) compared with PK15 (33.69 ± 13.05) and L35 (37.64 ± 12.77) cells, albeit the difference did not reach statistical significance. Incubation of serum samples with low–molecular weight heparin reduced serum cytotoxicity against PK15 cells in dose-dependent fashion. Therefore, FDA-PI two-color fluorescence is a suitable method to study antibody-mediated cytotoxicity by endogenous or exogenous complement for various pig cells.