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

描述(由申请人提供)：自体再生药物组织疗法的成本非常高，因为当单个组织被制造时，所有的成本都落在一个患者身上。同种异体组织更便宜，因为可以生产大批量的组织，而且成本由许多患者分担。同种异体组织通常通过去细胞来去除细胞相关抗原。脱细胞使用严酷的化学物质和冷冻方法，可能会损害组织基质。我们在体内同种异体绵羊模型和体外异种模型中的初步数据表明，该公司开发的一种无冰冷冻保存方法可以改变受者的免疫反应。这项SBIR建议的主要目标是一项可行性研究，以确定人体组织的无冰冷冻保存在与同种异体人外周血单核细胞结合时，是否也会产生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。人体组织将是我们的合作者正在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，细胞移植物将被无冰冷冻保存，并将开发出优化冷冻保护剂去除的洗涤程序。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成为评估洗涤充分性的标准。至关重要的是，无冰组织对受体细胞无毒，以便整合到受体体内。纳升渗透压法也将被用来量化剩余的低温保护剂浓度。在第二个目标中，将使用细胞增殖和细胞因子释放试验来评估免疫原性。新鲜的未经处理的、脱细胞的和无冰的冷冻保存的人类工程血管将在体外以同种异体人外周血单核细胞作为响应物进行比较。预期的结果是，与去细胞对照相比，无冰冷冻保存的组织具有同等或更低的免疫原性。这一结果之后将在随后的第二阶段SBIR方案中进行体内移植研究。材料性能的保留也将在第二阶段得到证实。这项研究将为各种外科应用，特别是患病的动脉置换提供前所未有的低成本非免疫原性组织同种异体移植，对外科修复产生深远的临床影响。我们的商业化战略包括向开发特定的人类同种异体组织疗法的公司独家和非独家许可无冰冷冻保存方法。 公共卫生相关性：天然和工程异体组织可能会影响巨大的整形外科、泌尿外科、心脏和血管外科应用。2013年，仅血管移植物的全球潜在市场预计就将达到25.88亿美元。这项提案中的技术开发将通过减少非免疫原性的工程化人类组织衍生产品所需的制造步骤来最大限度地降低成本。这项技术同时提供了一种长期的组织保存方法，与其他保存策略相比，这种方法证明了细胞外基质成分和生物材料特性的保留，植入患者体内后几乎没有免疫反应的可能性。

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.