A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of trans planted pig organs. The purpose of this study was to establish a suitable i n vitro method that would allow for screening and comparison of various age nts and methods potentially useful in the prevention of hyperacute rejectio n. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG084 72), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytot oxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conve ntional (51)Chromium (C-51(r))-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotox icity assay performed in Terasaki trays. Using the microcytotoxicity assay, a I-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studie d, PK15 cells were the most sensitive to cytotoxic injury, followed by AG c ells and the primary endothelial culture. Good correlation between the Cr-5 1-release and the two-color fluorescence method was documented. There was g ood agreement between the results obtained using the 2-chambered slide meth od and the microcytotoxicity assay, as there was between the 1- and the 2-s tep assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluoresce nce assay is suitable for the rapid and inexpensive screening of therapeuti c interventions that might be useful in the prevention of hyperacute xenogr aft rejection, and that PK15 cells are suitable for use in this assay. (C) 1999 Elsevier Science B.V. All rights reserved.