ABSTRACT: Transplantation of Cryopreserved Thymus Advances in immunosuppression have improved patient outcomes after transplantation, but also place patients at risk of life-threatening infection and end-organ failure. Development of immune tolerance to the donor that eliminates the need for continuing immunosuppression would likely improve patient survival. Our pivotal prior work showed that cultured thymus tissue implantation (CTTI) into athymic humans can establish an immune system that is tolerant to antigens present on the donor thymus, while maintaining tolerance to recipient native tissue. We have extended these findings to reprogram the immune system of immunocompetent rats to recognize a transplanted donor heart as “self” using CTTI combined with transplantation of heart from the same donor. In August 2021, our team performed the first human CTTI/heart co-transplantation under eIND. The recipient is currently robustly producing naïve T cells within the implanted donor thymus and testing for tolerance is pending. However, a major hurdle to translating this approach more widely is that CTTI using freshly cultured thymus must occur between 12-21 days post- thymus/heart harvest. Development of strategies to preserve the reconstitution potential of donor thymus slices beyond 12-21 days would allow CTTI to be delayed until recipients are clinically stable and could eventually extend the benefits of CTTI to all recipients via banked, donor-matched thymus. Our team has developed methods for cryopreservation and thawing of cultured thymus tissue from both pigs and humans that appear to maintain its viability, while continuing to meet CTTI quality criteria. This proposal aims to determine the ability of cryopreserved cultured thymus tissue implantation (cCTTI) to re-establish thymus function and naïve T cell numbers in thymectomized and conditioned recipient pigs. These studies will leverage our unparalleled expertise as the only institution performing clinical CTTI in the Western hemisphere to provide critical proof of concept for using cryopreserved thymus to induce immune tolerance in the setting of human thymus-heart co-transplantation.