HYPOTHERMIC AND CRYOPRESERVATION OF HUMAN BLOOD VESSELS

BioLife Technologies(BLT)成立于1998年3月31日。BLT的主要任务是开发改进的低温和冷冻保存解决方案，旨在保持细胞、组织和器官处于接近暂停生命的状态。心脏停搏液、器官移植和选定的非监管市场已成为发展的目标。这一阶段的SBIR建议使用四种心血管细胞株作为体外工具，以帮助开发未来的HTS解决方案，该解决方案针对人类血管保存进行了优化。BLT的低温解决方案HypoThermosol(R)(HTS)系列在保护肾脏、心脏和皮肤细胞方面比十年前由杜邦-默克公司生产的Viaspan(R)更好。Viaspan(注册商标)目前在保存解决方案业务中占有最大的市场份额，但Viaspan(注册商标)的设计并不是基于现代分子生物学研究。BLT启动了一项激进的计划，以确定延长低温和低温保存期间细胞死亡的分子基础，以便了解这些事件可以导致新一代低温解决方案。由于BLT的早期成功，HTS目前正被用于胰岛移植的I期临床试验。这项提案中提出的DNA凝胶显示，细胞在恢复常温(37摄氏度)两天后，冷储存过长或冷冻保存后因细胞凋亡(程序性细胞死亡)而死亡。HTS补充了细胞凋亡抑制剂，阻止了这一过程，改善了HTS的性能。BLT目前有一项正在申请的专利，该专利将允许BLT独家设计未来的HTS解决方案，从而抑制细胞凋亡。这一第一阶段项目的具体目标是：(A)确定保存引起的细胞损伤对血管细胞的凋亡和坏死的相对贡献；(B)确定哪些凋亡抑制剂在提高HTS疗效方面最有效；(C)确定坏死/凋亡、线粒体细胞色素C泄漏和线粒体跨膜能力丧失之间的关系(如果有的话)；以及(D)确定延长保存时间是否会缩短端粒，从而加速细胞的过早衰老。第二阶段的研究将致力于HTS保存完整的人体血管--这项工作将与我们的合作伙伴CryoLife Inc.共同完成。建议的商业应用：BioLife正在开发新一代保存解决方案，该解决方案将改善用于临床心血管应用的人类血管的长期存储。这些解决方案还将促进目前正在开发的未来组织工程血管的保存。

BioLife Technologies (BLT) was incorporated on March 31, 1998. BLT's primary mission is to develop improved hypothermic and cryopreservation solutions designed to maintain cells, tissues and organs in a near state of suspended animation. Cardioplegia, organ transplant and selected non- regulated markets have been targeted for development. This Phase I SBIR proposes to use four cardiovascular cell strains as in vitro tools to help develop a future HTS solution that is optimized for human blood vessel preservation. BLT's hypothermic solutions, the HypoThermosol(R) (HTS) series, are better at cold-protecting kidney, heart and skin cells than is ViaSpan(R) - a product produced by DuPont-Merck ten years ago. ViaSpan(R) currently commands the largest market share in the preservation solution business, but the design of ViaSpan(R) is not based on modern molecular biology investigations. BLT has launched an aggressive program to determine the molecular basis of cell death during extended hypothermic and cryopreservation storage so that knowledge of these events can lead to a new generation of hypothermic solutions. As a result of BLT's early success, HTS is currently being used in a pancreatic islet transplantation Phase I clinical trial. DNA gels presented in this proposal demonstrate that cells cold stored for too long or cryopreserved die by apoptosis (programmed cell death) two days after they are returned to normothermic (37 degrees C) temperatures. HTS supplemented with apoptosis inhibitors blocks this process and improves HTS's performance. BLT currently has a patent pending that will allow BLT the exclusive right to formulate future HTS solutions by design so that apoptosis is inhibited. The Specific Aims of this Phase I project are to (a) determine the relative contributions of apoptosis and necrosis as a consequence of preservation-induced cellular injury to blood vessel cells; (b) determine which apoptosis inhibitors are the most effective at increasing HTS efficacy; (c) determine the relationship, if any, between necrosis/apoptosis, cytochrome C leakage from mitochondria, and loss of mitochondrial transmembrane potential; and (d) determine if extended preservation shortens telomeres and as a consequence precipitates premature cell senescence. Phase II studies will be devoted to HTS preservation of intact human blood vessels - work to be accomplished with our collaborator, CryoLife Inc. PROPOSED COMMERCIAL APPLICATIONS: BioLife is developing a new generation of preservation solutions that will improve the long term storage of human blood vessels used for clinical cardiovascular applications. The solutions will also facilitate preservation of future, tissue engineered blood vessels currently under development.