After decellularization of porcine heart valves: no non‐Gal antigeneic epitopes detectable by non conditioned human sera

Abstract

BackgroundPatients that undergo heart valve replacement surgery with glutaraldehyde fixated porcine grafts do not face acute rejection as recipients of native porcine organs and tissues do, however, grafts calcify over time and fail after 10–20 years in adults and 3–4 years in children. Responsible for hyperacute and acute rejection of unfixated tissue are preformed antibodies that recognize xenoantigens. Major targets are carbohydrates like the αGal epitope or Neu5Gc linked to proteins as well as to lipids on cells and extracellular matrix. To overcome the limited availability of decellularized allogeneic heart valve grafts, which show the ability to remodel and to grow by ingrowth of autologous cells, a characteristics lacking in glutaraldehyde fixated heart valve grafts, we want to develop a porcine derived decellularized matrix that is well tolerated in humans.MethodsThe generation of such optimal porcine heart valves matrices is dependent on a successful elimination of bound xenoantigens. To quantify the amount of xenoantigens present on decellularized matrix, we established an inhibition ELISA by exposing human IgG to crushed matrix and measuring unbound IgG in an ELISA set up. High levels of measured IgG means low levels of xenoantigens present on the decelllularized matrix. To enhance the specificity of IgG tested in the ELISA, human sera were perfused through porcine kidneys, followed by intensive washing and elution of bound xenoantibodies. Total amount of IgG and αGal specific antibodies was monitored to control the purification steps. Inhibition ELISA was performed with matrices derived from Landrace pigs, GalT‐KO pigs and humans. Binding of total IgG as well as αGal specific antibodies was assayed.ResultsThe amount of total IgG binding correlates with the amount of αGal specific antibodies binding to decellularized porcine matrix indicating the presence of remaining αGal epitopes on the porcine matrix after decellularization. However, decellularized GalT‐KO pig and decellularized human matrix showed no binding of αGal specific antibodies and comparable amounts of total IgG binding.ConclusionOur results indicate that only αGal epitopes present on decellularized porcine heart valve matrixes are recognized by preformed xenoantibodies while no other xenoantigens are detected. Therefore, further evaluation focusing on induction of immunogeneic reactions has to be conducted in an in vivo model like the humanized mouse utilizing decellularized heart valve material derived from GalT‐KO pigs.