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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1-ETTN-G (13))
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Contreras, Miguel A
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General Biotechnology, LLC
United States
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Yuan, Ye; Yang, Ying; Tian, Yuchen et al. (2016) Efficient long-term cryopreservation of pluripotent stem cells at -80?°C. Sci Rep 6:34476