Feasibility of immunotherapy by ice-free cryopreservation of engineered human all

描述（由申请人提供）：自体再生医学组织治疗费用非常高，因为当制造单个组织时，所有费用都落在单个患者身上。同种异体组织更便宜，因为可以批量生产，而且费用由许多患者共同承担。同种异体组织通常通过脱细胞去除细胞相关抗原。脱细胞使用刺激性化学物质和冷冻方法，可能会破坏组织基质。我们在体内异体羊模型和体外用无冰冷冻保存的猪组织和人应答细胞的异种模型的初步数据表明，公司开发的无冰冷冻保存方法可以改变受体的免疫反应。这项SBIR提案的主要目标是进行可行性研究，通过一组体外试验来确定当与异体人外周血单个核细胞结合使用时，人体组织的无冰冷冻保存是否也会导致很少或没有免疫反应。这一目标将在两个具体目标和相关假设中实现。人体组织将是由我们的合作者开发的用于治疗应用的组织工程血管移植物。在第一个目标中，细胞移植物将是无冰冷冻保存和清洗程序，以优化冷冻保护剂的去除将被开发。内皮细胞在经处理的人组织工程血管移植物上的附着和增殖将是评估清洗是否充分的标准。这是至关重要的，无冰组织是无毒的受体细胞整合到受体。纳升渗透法也将用于定量残余冷冻保护剂浓度。在第二个目标中，免疫原性的评估将通过细胞增殖和细胞因子释放试验进行。新鲜未经处理、去细胞化和无冰冷冻保存的人工程血管将使用异体人外周血单个核细胞作为体外反应进行比较。预期的结果是，与去细胞对照相比，无冰冷冻保存的组织将具有同等或更低的免疫原性。该结果将在随后的II期SBIR提案中进行体内移植研究。材料属性的保留也将在第二阶段得到确认。这项研究将对外科手术修复产生深远的临床影响，为各种手术应用特别是病变动脉置换提供前所未有的低成本非免疫原性组织异体移植物。我们的商业化战略包括向开发特定人类异体组织疗法的公司独家和非独家授权无冰冷冻保存方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.