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

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 alphaGal. 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.