A new in vitro model for the study of pig-to-human vascular hyperacute rejection

Studies on vascular hyperacute xenograft rejection (HAR) are usually conduc ted in vitro on cultured endothelial cells (EC) exposed to human serum, in complex whole organ perfusion models using heparinized blood or in vivo mod els. Here we describe a new model allowing perfusion of pig vessels with hu man whole blood without anticoagulants. Segments of the porcine iliac arter y were connected to circular polyvinyl chloride (PVC) tubing, whose inner s urface was conjugated with immobilized heparin. The vessels were perfused w ith 7 to 8 ml of fresh, non-anticoagulated human blood by rocking of the tu bing device for 5, 15 or 60 min in an incubator at 37 degreesC. Human iliac arteries (n=4) were perfused with fresh human ABO-compatible blood as cont rols. Perfusion of human vessels resulted in changes in the blood and plasm a parameters similar to those in the PVC control loop. Overall, perfusion o f the porcine vessels generated high levels of C3a, sC5b-9 and thrombin-ant i-thrombin (TAT). Platelet consumption was near total (97.2 +/- 1.2%; "high " responders) in six of 13 vessels perfused and only moderate (55.8 +/- 9.9 %; "low" responders) in the remaining seven vessels. The "high" responder v essel group showed a significantly higher platelet reduction, neutrophil lo ss and monocyte consumption and higher C3a and TAT factor at 60 min compare d with the human vessels. The "low" responder porcine vessel group also gen erated significantly higher TAT levels at 60 min compared with the human ve ssels, but lower levels compared with the "high" responder porcine vessel g roup. Immunohistochemical examination of perfused porcine vessels revealed binding of human IgM, IgG, IgA, Clq, C3, fibrin and platelets at 5 min. The binding of these proteins was even stronger at 15 and 60 min, and at 60 mi n C9 could also be detected. Addition of soluble complement receptor 1 (sCR 1) to the blood resulted in a significant reduction in C3a and sC5b-9 (P=0. 046 and P=0.046, respectively). However, sCR1 did not reduce Clq, C3c or C5 staining, but did abolish C9 binding to the endothelium. In conclusion, in vitro perfusion of porcine vessel segments with nonheparinized, fresh huma n blood triggered events characterizing HAR. The small quantity of blood an d xenogenic tissue that is needed makes this model ideal for investigations of the mechanisms and treatments of rejections associated with xenogeneic pig-to-human xenotransplantation.