Autologous regenerative medicine tissue therapy costs are very high because when individual tissues are manufactured all the costs fall on a single patient. Allogeneic tissues are cheaper because large tissue batches can be made and the costs are shared by many patients. Allogeneic tissues are commonly treated to remove cell-associated antigens by decellularization. Decellularization employs harsh chemicals and freezing methods that may damage tissue matrices. Our preliminary data in an in vivo allogeneic sheep model and an in vitro xenogeneic model with ice-free cryopreserved porcine tissue and human responder cells indicates that an ice- free cryopreservation method developed by the Company modifies the recipient's immune reaction. The primary goal of this SBIR proposal is a feasibility study to determine whether ice-free cryopreservation of human tissue also results in little or no immune reaction when combined with allogeneic human peripheral blood mononuclear cells using a panel of in vitro assays. This goal will be pursued in two Specific Aims and associated Hypotheses. The human tissues will be a tissue engineered vascular graft being developed by our collaborators for therapeutic applications. In the first aim cellular grafts will be ice-free cryopreserved and washing procedures to optimize cryoprotectant removal will be developed. Endothelial cell attachment and proliferation on the treated human tissue engineered vascular grafts will be the criteria for assessment of washing adequacy. It is critical that the ice-free tissues be non-toxic to recipient cells for integration in to the recipient. Nanoliter osmometry will also be employed to quantify residual cryoprotectant concentrations. In the second aim evaluation of immunogenicity will be performed using cell proliferation and cytokine release assays. Fresh untreated, decellularized and ice-free cryopreserved human engineered blood vessels will be compared using allogeneic human peripheral blood mononuclear cells as responders in vitro. The anticipated outcome is that the ice-free cryopreserved tissues will be equivalent or less immunogenic compared with decellularized controls. This outcome will be followed by in vivo transplant studies in a subsequent Phase II SBIR proposal. Retention of materials properties will also be confirmed in Phase II. This research will have a far-reaching clinical impact on surgical repairs by providing unprecedented access to low cost non- immunogenic tissue allografts for a variety of surgical applications and diseased artery replacement in particular. Our commercialization strategy involves exclusive and non-exclusive licensing of ice-free cryopreservation methods to companies developing specific human allogeneic tissue-based therapies.
Natural and engineered allogeneic tissues potentially impact huge orthopedic, urinary, cardiac and vascular surgery applications. The potential worldwide market for vascular grafts alone is predicted to be $2,588M in 2013. The technology development in this proposal will minimize costs by reducing the manufacturing steps required for engineered human tissue-derived products to be non-immunogenic. This technology simultaneously provides a long-term tissue storage method, which has proven retention of extracellular matrix components and biomaterial properties compared with alternative preservation strategies, with the potential for little if any immune response in vivo after implantation in patients.