Embryo cryopreservation is an indispensable technique in reproductive programs and in animal facilities where genetically modified mice are used extensively. Here we report the use of a vitrification spatula (VS) that can be readily homemade and has a large holding capacity to vitrify preimplantation mammalian embryos in a micro-drop employing ultra-rapid cooling in liquid nitrogen (LN₂). Vitrified one-cell embryos and morulae have high survival rates after thawing, and the fertility of the derived progeny is comparable to that of the control unvitrified group. The large holding capacity (up to 50 embryos per VS) does not only allow rapid expansion of storage capacity for additional mouse strains but also opens up the possibility to streamline transgenic mice generation procedures in transgenic facilities.

Cryopreservation of mammalian preim-plantation-stage embryos serves a wide range of purposes, including routine assisted reproduction programs for humans and livestock, and animal management in institutional animal facilities. The first case of successful mouse embryo cryopreservation by slow controlled-rate freezing was reported in 1972 (1), but a more effective cryopreservation method—vitrification, or ultra-rapid freezing—was developed in 1985 (2). The success of vitrification is based on procedures that minimize the formation of intra-cellular ice crystals when an embryo and the surrounding vitrification solution are “glassified.” The reduced exposure time of the embryos to osmotic stress and toxic cryoprotectant leads to a high revival rate of the stored embryos.

Different devices were used as containers for preimplantation embryo vitrification. Plastic straws (3) are widely used for vitrification. Open pulled straws (4) and the double straw system (5) were produced to improve sample cooling rate and to isolate samples from potential infectious agents accumulated in the storage vessels. Electron microscope grids (6), Cryoloop (7), Cryotop (8), nylon mesh (9) and metal mesh (10) were recently adopted as an open device to maximize the cooling rate. However, these devices are only available commercially. They do not have a large holding capacity nor can they achieve a high embryo revival rate after vitrification is performed as a routine procedure in mouse facility. We hereby report an inexpensive and easy-to-assemble device—a vitrification spatula (VS)—as an alternative. This device, which allows easy handling, does not only allow an ultra-rapid cooling of samples, but also stores vitrified samples in a closed system. Most important, the VS has the highest effective embryo holding capacity ever reported.

To assemble a VS, the tip of an autoclaved gel-loading tip (Cat. no. 1022-000; USA Scientific, Ocala, FL, USA) was crushed with a pair of fine forceps (N4, Regine, Switzerland) that had been gently heated with a Bunsen burner (Model no. F4003; R & L Enterprises, Bramley, Leeds, UK) to generate a petal-like plate of 1 mm² (see Supplementary Materials for the monitoring of the heating status of the forceps and the softening of the spatula). The distal edge was heat-sealed to avoid liquid infiltration. The other end of the gel-loading tip was removed to shorten the spatula stalk. The cut end was mounted onto the underside of a cryogenic vial cap (Cat. no. 430488; Corning, Corning, NY, USA) by heat (Figure 1; see Supplementary Materials for a detailed protocol of VS assembly, vitrification and thawing of embryos). On average, a VS can be assembled in 1–2 min. We did not observe any detachment of the stalk from the cap, even when an assembled spatula, with or without liquid N₂ (LN₂) cooling, was allowed to free-fall to the ground from 1 m.

To evaluate the efficacy of vitrifying preimplantation embryos on a VS, one-cell embryos, morulae, and blastocystes were first harvested from super-ovulated and mated C57BL/J6/CBAF1 females and were vitrified as follows. Since it is the most commonly used in transgenic mouse generation by DNA microinjection (11), this mouse strain was tested to enable a good comparison with other studies. To eliminate the fluctuating contribution of low-quality one-cell embryos to the survival assay, only one-cell embryos with two prominent pronuclei and polar bodies confirmed under inverted DIC microscopes were selected for experimentation. After ~20 embryos were incubated in previtrification and vitrification solutions [Ethylene glycol (Cat. no. 102466; Sigma-Aldrich, St. Louis, MO, USA), DMSO (Cat. no. D2650; Sigma-Aldrich), Ficoll PM70 (Cat. no. F2878; Sigma-Aldrich), and M2 medium (Cat. no. MR-010P-5F; Specialty Media, Phillipsburg, NJ, USA)] (each for 30 s), the embryos were loaded onto the surface of a VS with approximately 0.5 µL vitrification solution and cooled down by dipping the droplet into LN₂ by holding the attached cryovial caps. The VS was then inserted into a precooled cryogenic vial and stored in a LN₂ cell storage vessel for 1–3 months before their viability was assessed. The embryos were then thawed and released from the VS by dipping the tip containing the droplet in 2 mL of 0.5 M sucrose solution. After the embryos had fallen from the spatula, they were transferred to a droplet of 20 µL 0.5 M sucrose solution and then 20 µL 0.25 M sucrose solution (each for 2 min) to remove the vitrification medium. The embryos were then washed with one drop of M2 medium before they were transferred to M16 medium (Cat. no. MR-015P-D; Specialty Media) for in vitro survival tests and subsequently implanted into the foster mother for in vivo survival tests.