Efficient vitrification of mouse embryos using the Kitasato Vitrification System as a novel vitrification device

Abstract

Background: Currently, the cryopreservation of embryos and oocytes is essential for assisted reproductive technology (ART) laboratories worldwide. This study aimed to evaluate the efficacy of the Kitasato Vitrification System (KVS) as a vitrification device for the cryopreservation of mouse embryos to determine whether this novel device can be adapted to the field of ART.

Methods: In Experiment 1, blastocysts were vitrified using the KVS. Vitrified blastocysts were warmed and subsequently cultured for 72 h. In Experiment 2, 2-cell-stage embryos were vitrified using the KVS, and vitrified embryos were warmed and subsequently cultured for 96 h. In Experiment 3, we evaluated the in vivo developmental potential of vitrified 2-cell-stage embryos using the KVS, and in Experiment 4, we evaluated the cooling and warming rates for these devices using a numerical simulation.

Results: In Experiment 1, there were no significant differences between the survival rates of the KVS and a control device. However, re-expanded (100%) and hatching (91.8%) rates were significantly higher for blastocysts vitrified using the KVS. In Experiment 2, there were no significant differences between the survival rates, or rates of development to the blastocyst stage, of vitrified and fresh embryos. In Experiment 3, after embryo transfer, 41% of the embryos developed into live offspring. In Experiment 4, the cooling and warming rates of the KVS were 683,000 and 612,000 °C/min, respectively, exceeding those of the control device.

Conclusions: Our study clearly demonstrates that the KVS is a novel vitrification device for the cryopreservation of mouse embryos at the blastocyst and 2-cell stage.

Keywords: Cryopreservation; Embryos; KVS; Ultra-rapid cooling; Vitrification.

Fig. 1

The Kitasato Vitrification System (KVS) for embryo vitrification. A: (a) The KVS consists of a gripper made of acrylonitrile butadiene styrene resin and a support. (b) The protective straw-cap of the device. B: The support is composed of polyethylene terephthalate (PET) film and the vitrification solution absorber, which consists of a porous membrane and is placed on the PET film (both ends of the vitrification solution absorber adhered to the PET film). Scale bar = 0.5 cm

Fig. 2

Embryo vitrification procedures using the Kitasato Vitrification System. A: The vitrification solution absorber of the device is set on a stereomicroscope. B: After an embryo with a small volume (≤0.4 μL) of vitrification solution is placed dropwise on the vitrification absorber (a) that embryo can easily be observed under the stereomicroscope (b). C: Subsequently, the vitrification solution surrounding the embryo is absorbed by the absorber within less than 5–6 s, and the embryo, with a small remaining volume of vitrification solution, can easily be observed

Fig. 3

The vitrification dynamics of the Kitasato Vitrification System (KVS) and the Control device. a-b: The embryo temperature in the vitrification procedure was calculated using a thermal transfer-based simulation method. The temperature in the center of the embryo during the vitrification procedure was plotted using the simulation result for the KVS and the control device. Temperature changes are shown every 0.02 s from liquid nitrogen (LN₂) immersion for 1 s (a) and every 0.002 s from immersion for 0.05 s (b). c-f: Illustration of the thermal distribution of a wide cross-section of embryos on the KVS support (c and e) or the control device sheet (d and f) in the vitrification procedure. Thermal distributions immediately after immersion in LN₂ are shown in (c and d). The thermal distributions at 0.05 s after immersion are shown in (e and f)

Fig. 4

Embryo temperature in the warming procedure was calculated using a thermal transfer-based simulation method. a-b: The temperature in the center of the embryo during the warming procedure was plotted using the simulation result for the Kitasato Vitrification System and the control device. The temperature changes are shown every 0.02 s from thawing solution immersion for 1 s (a) and every 0.002 s from immersion for 0.05 s (b)