Feasibility of Unfrozen Sub-zero Storage of Mammalian Tissues with Insect Anti-Fr

描述(由申请人提供)：长期目标是开发出比目前临床上可能使用的新型昆虫抗冻蛋白更好、更长时间的肝移植低温保存条件。低温损伤会导致肝脏保存过程中的血管功能障碍，因此在这一阶段的SBIR方案中将采用血管组织模型。从兔颈静脉和颈动脉提取的血管环将被使用，因为它们将允许解剖低温储存对血管形态和功能活性的影响，而且这些研究可以用我们目前的制造方法可以生产的少量昆虫来源的抗冻蛋白来进行。在第一阶段SBIR提案的经济限制下，扩大抗冻蛋白生产以达到肝脏所需的数量是不可能的。使用低温作为主要手段来抑制与不含抗冻蛋白的对照，以增加低温储存的时间。测试温度-10℃是基于昆虫抗冻肽产生的更大的非结合冰点下降而选择的，与之前描述的来自其他来源的其他抗冻蛋白相比，这些抗冻肽的活性几乎高出一个数量级。昆虫提取的抗冻蛋白将在-10℃下抑制冰核，允许哺乳动物组织以低得多的内源代谢活动水平储存。这些储存条件应该会导致组织和器官的低温储存期更长。在这个第一阶段的SBIR建议中，我们的顾问John Duman教授从关于热滞后的研究中挑选了昆虫衍生的抗冻蛋白配方，并将在随后的第二阶段SBIR建议中使用兔静脉和动脉组织模型以及一组评估组织病理学和生理学肝脏的分析方法对昆虫衍生的抗冻蛋白配方和对照进行比较。公共健康相关性：用于移植的肝脏目前在4℃的化学定义的溶液中使用静态冷藏储存，储存时间长达6小时。然而，静态冷藏的应用已被证明不足以满足不可逆性肝功能衰竭患者的器官需求，因为静态冷藏不能以最佳的方式保存边缘肝脏足够长的时间，以便进行器官评估。这些限制应该通过使用较低的、低于零度的低温(-10℃)来克服，以进一步减少新陈代谢，并结合昆虫来源的抗冻蛋白来抑制冰核，从而增加器官移植的可用性。

DESCRIPTION (provided by applicant): The long term goal is the development of better, longer hypothermic storage conditions for liver transplants than currently possible in clinical practice using novel insect antifreeze proteins. Hypothermic injury results in vascular dysfunction during liver storage, therefore vascular tissue models will be employed in this Phase I SBIR proposal. Blood vessel rings, derived from rabbit jugular veins and carotid arteries, will be employed because they will permit dissection of hypothermic storage effects upon the morphology and functional viability of vessels and because these studies can be performed with the small quantities of insect-derived antifreeze proteins that can be produced with our current manufacturing methods. Scale up of antifreeze protein production to achieve the quantities required for livers is not possible within the economic limitations of a Phase I SBIR proposal. The use of hypothermia as the principal means to suppress metared with controls without antifreeze proteins in order to increase the duration of hypothermic storage. The test temperature, -10¿C, was selected on the basis of the greater non-colligative freezing point depression produced by the insect antifreeze peptides that provide almost an order of magnitude greater activity compared with other previously described antifreeze proteins derived from other sources. The insect-derived antifreeze proteins will inhibit ice nucleation at -10¿C permitting mammalian tissue storage at much lower levels of endogenous metabolic activity. These storage conditions should lead to longer hypothermic storage periods for tissues and organs. In this Phase I SBIR proposal insect-derived antifreeze protein formulations, selected from research studies on thermal hysteresis by our consultant, Professor John Duman, and controls will be compared employing rabbit venous and arterial tissue models and a panel of assays for evaluation of the histopathology and physioloan livers in a subsequent Phase II SBIR proposal. PUBLICH HEALTH RELEVANCE: Livers destined for transplantation are currently stored using static cold storage in a chemically defined solution at 4¿C for periods of up to 6 hours. However, the application of static cold storage has proven to be insufficient to fulfill the organ demand for patients with irreversible liver failure, because marginal livers can not be preserved optimally, for long enough, by static cold storage to permit organ evaluation to occur. These limitations should be overcome by use of a lower, sub-zero hypothermic temperature (-10¿C) to further reduce metabolism in combination with insect-derived antifreeze proteins to inhibit ice nucleation resulting in increased availability of organs for transplant.