A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen

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Abstract
Double packaging, in which an inner straw containing the specimen is inserted into an outer, larger straw (here termed `straw-in-straw') to prevent the inner straw from coming into direct contact with liquid nitrogen provides a simple strategy for reducing or eliminating the potential contamination risk associated with storage in liquid nitrogen. This approach has in the past been used in conjunction with cryopreservation by slow cooling, but has not previously been tested for use throughout an entire rapid cooling and warming procedure. This study determined whether keeping the straw containing the embryos inside a second protecting container throughout the cryopreservation and storage protocol would compromise embryo viability. We established that a cryoprotectant containing a high polymer concentration (35% dextran or Ficoll) together with 25% ethylene glycol (as the penetrating cryoprotectant) was highly effective for day 2 and day 3 mouse embryos in both single and double straws. The survival and development of all cryopreserved embryos, as assessed both in vitro and in vivo, was not statistically different to their untreated controls. This established that a protein/serum-free cryoprotectant solution supplemented with polymers could provide complete protection of mouse embryos. It also shows, for the first time, that embryos can be cooled by direct immersion in liquid nitrogen and warmed by direct immersion into a waterbath within a double straw arrangement to reduce the likelihood of contamination.

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