Xeno-immunogenicity of ice-free cryopreserved porcine leaflets.

Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.