OBJECTIVE: To validate the use of a microelectronic real-time cell analyzer system (RTCA) we developed a complement-mediated antibody cytotoxicity assay to investigate the compatibility of a graft and a recipient in pig-to-baboon xenotransplantation. MATERIALS AND METHODS: Fibroblasts isolated from the skin of five hCD55, hCD59, and hCD46 transgenic pigs (TP) were cultured in 96 microelectronic well plates for 17 hours. Then, we added to each microwell 20 μL of normal sera from nine healthy adult olive baboons (Papio anubis)-three males and six females. The evolution of the cell culture was assessed every 3 minutes during the pretreatment period, at 11 hours postaddition, and every 30 minutes from 12 to 96 hours. Simultaneously, we performed a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fibroblasts from wild-type (WT) pigs were used as positive controls and microwells without serum addition from each TP as negative controls. The RTCA results were expressed as a normalized cellular index (NCI). RESULTS: Differences were observed between the five TP fibroblasts and the WT fibroblasts, with greater cytotoxicity on WT cells. Among TP, a higher cytolytic level was observed in males than females. The MTT results correlated with NCI at different times, with the minimum NCI and with the time to for NCI recovery before serum addition. The correlation was lower than that previously reported in environmental toxicity assays. CONCLUSIONS: RTCA allows a long-term assessment of the immunocytotoxic effect of baboon sera on pig cells, providing a suitable tool to perform compatibility tests for xenotransplantation.
OBJECTIVE: To validate the use of a microelectronic real-time cell analyzer system (RTCA) we developed a complement-mediated antibody cytotoxicity assay to investigate the compatibility of a graft and a recipient in pig-to-baboon xenotransplantation. MATERIALS AND METHODS: Fibroblasts isolated from the skin of five hCD55, hCD59, and hCD46 transgenic pigs (TP) were cultured in 96 microelectronic well plates for 17 hours. Then, we added to each microwell 20 μL of normal sera from nine healthy adult olive baboons (Papio anubis)-three males and six females. The evolution of the cell culture was assessed every 3 minutes during the pretreatment period, at 11 hours postaddition, and every 30 minutes from 12 to 96 hours. Simultaneously, we performed a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fibroblasts from wild-type (WT) pigs were used as positive controls and microwells without serum addition from each TP as negative controls. The RTCA results were expressed as a normalized cellular index (NCI). RESULTS: Differences were observed between the five TP fibroblasts and the WT fibroblasts, with greater cytotoxicity on WT cells. Among TP, a higher cytolytic level was observed in males than females. The MTT results correlated with NCI at different times, with the minimum NCI and with the time to for NCI recovery before serum addition. The correlation was lower than that previously reported in environmental toxicity assays. CONCLUSIONS: RTCA allows a long-term assessment of the immunocytotoxic effect of baboon sera on pig cells, providing a suitable tool to perform compatibility tests for xenotransplantation.
Authors: Andrew G MacLean; Edith Walker; Gautam K Sahu; Gail Skowron; Preston Marx; Dorothee von Laer; Richard P Junghans; Stephen E Braun Journal: J Med Primatol Date: 2014-08-20 Impact factor: 0.667