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

描述（由申请人提供）：本提案的总体目标是设计和开发一种冷冻保存介质和一种易于使用的冷却装置，允许在-80 ℃下长期储存和安全运输动物模型种质（胚胎和精子）和胚胎干细胞（ESC）。将动物模型物种的有价值基因型作为活动物系储存和维持是完全不切实际的[Critser，2000]。在世界各地的许多实验室和动物资源中心，将细胞系作为冷冻保存的种质和ESC进行库化，并将这些细胞恢复成活的、可繁殖的动物是常规的。然而，由于使用液氮（液氮），液氮杜瓦瓶和复杂的冷却装置，这些冻存细胞的储存和运输是这些研究所的日常运作的一个很大的负担。因此，降低这些程序的成本每年将节省数十万美元。在本提案中，我们计划开发一种含有冷冻保护剂（CPA）的水溶液，该溶液在高达-80 ° C的温度下具有化学稳定性，因此冷冻保存的样品可以分别使用传统的“-80 ° C冷冻机”和干冰进行储存和运输。还将生产一种廉价、易于使用的自成核冷却系统，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-80 ℃下稳定的冷冻保存介质;（II）通过冷冻保存啮齿动物种质和ESC来经验性地测试新的冷冻保存介质的功效。对于第二阶段：（III）设计和开发一种新型的、易于使用的冷却装置，该装置将为第一阶段开发的培养基提供限定的恒定冷却速率和自动接种;（IV）确定使用所开发的装置和培养基在-80 ° C下冷冻保存啮齿动物种质和ESC的功效。最后，将在适当的工程设计控制下生产申报器械和培养基，并通过生物物理试验进行确认。 公共卫生相关性：动物模型种质和胚胎干细胞的冷冻保存对于生物医学研究至关重要。该提案的总体目标是开发一种冷冻保存介质和一种易于使用的设备，使这些细胞类型能够在-80 ℃的冰箱中冷却和储存很长一段时间，并在干冰上安全运输。这些方法将完全避免使用液氮或复杂的冷却装置，并显著降低冷冻保存细胞的储存和运输的相关成本。

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.