A Novel, Inexpensive Cryopreservation System for Stable -80 degree C Storage of A

描述(申请人提供)：本提案的总体目标是设计和开发一种低温保存介质和一种易于使用的冷却设备，使动物模型种质(胚胎和精子)和胚胎干细胞(ESCs)能够在-80℃下长期储存和安全运输。将有价值的动物模式物种的基因类型作为活体动物品系进行存储和维护将是完全不切实际的[Critser，2000]。在世界各地的许多实验室和动物资源中心，储存超低温保存的种质和ESCs，并将这些细胞恢复为活的、可繁殖的动物是例行公事。然而，由于使用了液氮、液氮杜瓦和复杂的冷却设备，这些低温保存细胞的储存和运输给这些研究所的日常运营带来了很大的负担。因此，降低这些程序的成本每年将节省数十万美元。在这项提议中，我们计划开发一种含有低温保护剂(CPA)的水溶液，该溶液在高达-80oC的温度下热力学稳定，因此低温保存的样品可以分别使用传统的-80oC冰柜和干冰储存和运输。还将生产一种廉价、易用、自成核的冷却系统，以进一步降低相关成本并改善这些程序的结果。为了实现这些目标，我们提出了以下具体目标：对于第一阶段：(I)设计和开发一种在-80oC下热力学稳定的低温保存介质；(Ii)通过对啮齿动物种质和ESCs的低温保存来经验地测试新的低温保存介质的有效性。对于第二阶段：(Iii)设计和开发一种新型的、易于使用的冷却设备，该设备将为第一阶段开发的介质提供定义的恒定冷却速度和自动播种；(Iv)使用所开发的设备和介质确定在-80℃下冷冻保存啮齿动物种质和ESCs的效果。最后，所提出的装置和介质将在适当的工程设计控制下生产，并通过生物物理测试进行验证。 公共卫生相关性：动物模型种质和胚胎干细胞的超低温保存对生物医学研究至关重要。这项提议的总体目标是开发一种低温保存介质和一种易于使用的设备，使这些类型的电池能够被冷却并在零下80摄氏度的冰柜中长期储存，并在干冰中安全运输。这些方法将完全避免使用液氮或复杂的冷却设备，并显著降低低温保存细胞的相关存储和运输成本。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to design and develop a type of cryopreservation media and an easy-to-use cooling device that allow the long-term storage and safe shipment of animal model germplasm (embryos and spermatozoa) and embryonic stem cells (ESCs) at -80oC. Storage and maintenance of valuable genotypes of animal model species as live animal lines would be wholly impractical [Critser,2000]. Banking lines as cryopreserved germplasm and ESCs, and restoring of these cells into live, reproductive viable animals is routine in many laboratories and animal resource centers across the world. However, due to the use of liquid nitrogen (LN2), LN2 dewars and complicated cooling devices, storage and shipment of these cryopreserved cells is a large burden to the daily operation of these institutes. Therefore, lowering the cost of these procedures will save hundreds of thousands of dollars annually. In this proposal, we plan to develop an aqueous solution with cryoprotectants (CPAs) that is thermodynamically stable at temperatures as high as -80oC, so that the cryopreserved samples can be stored and shipped using conventional "-80oC freezers" and dry ice, respectively. An inexpensive, easy-to-use, self-nucleating cooling system will also be produced to further lower the associated costs and improve the outcomes of these procedures. To achieve these aims, we propose the following Specific Aims: For Phase I: (I) Design and develop a cryopreservation media that is thermodynamically stable at -80oC; (II) Empirically test the efficacy of the new cryopreservation media by cryopreserving rodent germplasm and ESCs. For Phase II: (III) Design and develop a novel, easy-to-use cooling device that will provide defined constant cooling rates and automatic seeding for the media developed in Phase I; (IV) Determine the efficacy of cryopreserving rodent germplasm and ESCs at - 80oC using the developed device and media. At the end, the proposed device and media will be produced under appropriate engineering design control and validated through biophysical tests. PUBLIC HEALTH RELEVANCE: Cryopreservation of animal model germplasm and embryonic stem cells is of critical importance for biomedical research. The overall goal of this proposal is to develop a cryopreservation media and an easy-to-use device that allow these cell types to be cooled and stored in a -80oC freezer for a long time and safely shipped on dry ice. These approaches will completely avoid the use of liquid nitrogen or complicated cooling devices, and significantly reduce the associated costs for the storage and shipment of the cryopreserved cells.