One of the main issues that hamper xenotransplantation to be clinically feasible is humoral rejection. Xenoreactive antibodies, both natural or elicited, contribute significantly to rejection of xenotransplants by activation of complement and interactions with a variety of effector cells. Sugars – namely Gal-α1,3-Gal, Neu5Gc and β4GalNT2 – expressed on the surface of endothelial cells are thought to play an important role. In this study, we tested the potential xenoprotective role of human complement regulators – hCD46, hCD55, hCD59 – combined with A20 (NF-κB inhibitor and anti-apoptotic protein) and HO-1 (oxidative stress-response protein) using a microfluidic model where porcine kidney fibroblasts (PKF) grown on artificial 3D microvessels were perfused with normal human serum (NHS). Cell lines lacking the expression of Gal-α1,3-Gal (GGTA1 KO), Neu5Gc (CMAH KO) and β4GalNT2 (β4GalNT2 KO) sugars were also tested in order to assess the effects on complement activation and immunoglobulin binding.Methods
PKF WT and transgenic were seeded on artificial microvessels with diameters of 550 μm fabricated in PDMS. The cells were cultured overnight under static condition to allow them to reach confluence. A peristaltic pump was then connected and the cells were perfused for 2 hours in a close loop with 5mL of 1:10 diluted NHS. Control samples were perfused with medium without NHS. The channels were washed with PBS, fixed and stained to detect complement deposition (C3b/c and C4b/c) and immunoglobulin binding (IgG and IgM). Microfluidic chips were analyzed under laser scanning confocal microscope. Five pictures/microchannel were acquired and the fluorescence intensity was quantified and plotted in a graph.Results
Data showed that the absence of sugars from the cell surface of transgenic fibroblasts resulted in a significant decrease of antibody binding and antibody dependent complement deposition. Furthermore, the addition of 5 transgenes (hCD46, hCD55, hCD59, A20 and HO-1) to the GGTA1 KO background significantly improved the protection from complement deposition as shown by the immunofluorescence pictures and by the quantification. Experiments with PKF with triple KO together with the expression of the 5 trangenes are currently ongoing.Conclusions
This study shows that immunoglobulin binding and complement deposition on transgenic PKF are strongly reduced when xenoantigens (GGTA1, CMAH, β4GalNT2) are removed from the cell surface. The addition of the 5 human transgenes further enhances the protection of the graft from complement. Transgenic pigs carrying such genetic modification might represent suitable donors for future pig to baboon preclinical xenotransplantation experiments. Furthermore, the study reveals the potential of our in vitro microfluidic system in screening the effects of different transgenes prior to the production of the respective pigs for in vivo experiments.