Genetic modification to the donor pig candidates for organ xenotransplantation currently serves as the most promising option to bypass the great interspecies immunological barriers that lead to xenograft rejection. However, selecting the most optimum combination of genetic modification, which comprises the removal of xenoreactive porcine antigens and the expression xenoprotective genes, remains a challenge. Previously we have established a 3D endothelial cell culture grown under pulsatileflow and shear stress using a microfluidic system to mimic the physiological phenotype and function of the vasculatureconditions. Using this in vitro system, we assessed the functional effect of the genetic modification on transgenic pig aortic endothelial cells (EC) carrying functional knockout of GGTA1 and CMAH genes and expressing five human genes (CD46, CD55, CD59, HO-1, and A20). ECs were cultured in a 3D-round channel and subjected to pulsatile flow for 72h. To examine the effect of the genetic modification on complement activation and antibody binding in a xenotransplantation setting, 3D-EC culture was perfused with normal human serum. We observed a significant reduction of IgG binding on the cell surface of the transgenic EC after perfusion. Activation of the complement system by transgenic EC, measured by the deposition of C3b/c on EC was also decreased compared to wild-type pig EC. We are currently using the microfluidic system to investigate the functional effect of the genetic modification set on EC activation, glycocalyx shedding associated with vascular damage, and the preservation of a pro-fibrinolytic phenotype of the EC. In conclusion, in vitro EC culture under physiological flow can be used to assess the functional effects of multigene modification and to define which sets shall be proceed for animal experimentation, supporting the 3Rs (replace, reduce, refine) implementation in xenotransplantation research.