Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations

Abstract

The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000 degrees C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l 1,2-propanediol and 0.5 mol/l trehalose and achieve survival rates of 90.0%(36/40). Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.

Figure 1

Quartz capillary (QC) vitrification device and oocyte loading method. (a) The QC device; (b) schematic illustration of the QC loaded with oocytes; and (c) photomicrograph of oocytes loaded into QC and loaded oocytes (arrowheads). VS = vitrification solution.

Figure 2

Oocyte survival rate after vitrification in slush nitrogen in the two different vitrification solutions, 0.5 mol/l trehalose + 1.5 mol/l propane-1,2-diol (PrOH) in HEPES-buffered physiological salt solution containing 20% fetal bovine serum (FHM20) or 0.5 mol/l trehalose + 2.0 mol/l PrOH in FHM20, immediately and 2 h after warming.