Spatula Montevideo Device for the Vitrification of Mammalian Embryos

Summary

Here, we present the Spatula Montevideo protocol for vitrifying mouse and sheep pre-implantation embryos, consisting of a homemade spatula and utilizing homemade solutions. Embryos are rapidly equilibrated in two cryoprotectant solutions of increasing concentration, loaded onto the spatula, immersed in liquid nitrogen, and finally sealed with a 0.5 mL straw.

Abstract

This study describes an efficient and cost-effective vitrification method for mouse and sheep embryo cryopreservation known as the Spatula Montevideo (MVD). This protocol utilizes a homemade spatula as the carrier device, which is prepared with a gel-loader tip. Embryos are rapidly equilibrated in two homemade cryoprotectant solutions of increasing concentration, loaded into the spatula, and then directly immersed in liquid nitrogen. Approximately 30-40 pre-implantation stage mouse embryos can be loaded on the spatula, which is subsequently sealed with a 0.5 mL straw. The use of spatulas instead of cryotubes, which are frequently used to store vitrified embryos, optimizes space in the liquid nitrogen dewars. Additionally, the warming process requires only a homemade sucrose solution of decreasing concentration to rehydrate the embryos and restore their morphology. Our laboratory has successfully used the Spatula Montevideo for mouse and sheep pre-implantation embryos for over 10 years. The Spatula Montevideo has demonstrated an average recovery rate (recovered/vitrified embryos) of 84% and a survival rate (viable/recovered embryos) of 97% in mouse embryos. Vitrification of murine embryos has yielded better results than slow freezing, although no differences in reproductive outcomes (pregnancy, delivery, and birth rates) have been observed. In sheep embryos, similar survival and embryo development rates have been achieved with Spatula Montevideo compared to a commercial device. Although the pregnancy rate was higher for the commercial device, the lamb survival rate was not significantly different among the devices. The Spatula Montevideo vitrification method is a straightforward cryopreservation technique that can be used to store surplus embryos, back up genetically modified mouse lines and sheep models, and facilitate their exchange among researchers worldwide.

Introduction

The efficiency of genetically modified (GM) animal generation has significantly increased due to the development of endonuclease-based methods, particularly CRISPR/Cas9. Many laboratories worldwide produce various GM mouse and rat lines that should be cryopreserved to prevent genetic drift and maintain the integrity of the original phenotype¹. Additionally, archiving eliminates the need for constant breeding, which often results in excessive animal numbers. Some laboratories send GM mouse lines as cryopreserved embryos or sperm to international repositories, facilitating the exchange of these models while avoiding the transport of live animals. This ensures accessibility to lines without the repetitive generation of identical GM animal models². For these reasons, embryo and sperm cryopreservation are paramount in any GM animal facility. Generally, embryo cryopreservation is preferred for storing GM lines because embryos contain the complete diploid genome with modifications and can be easily recovered via embryo transfer.

Vitrification is a non-equilibrium process characterized by an imbalance between water entering the cell and water forming extracellular crystals around it. This method prevents the formation of intracellular ice crystals, distinguishing it from the slow freezing method (SLF)³ (see Figure 1). The high concentration of cryoprotectants and direct contact with liquid nitrogen (LN₂) enable ultra-rapid cooling (~40 °C/s), achieving a vitreous state of the samples within seconds. Moreover, vitrification is faster, easier, and less expensive than slow freezing⁴. It requires minimal LN₂ and typically employs simple equipment that can be commercially purchased or homemade⁵ (see Figure 1).

Additionally, the warming process is generally fast and replicable. It should be performed quickly to avoid cryoprotectant toxicity and to maintain the vitreous state³. When freezing embryos, many laboratories consider the convenience of the carrier device, its reliability, robustness, holding capacity, and costs.

We prefer the minimum volume spatula vitrification method for routine cryopreservation of sheep and mouse embryos, using the Spatula Montevideo (MVD) device⁶. Initially, we employed the slow freezing method⁷ but quickly transitioned to vitrification. To create a simpler and more space-efficient device for the LN₂ dewar, we modified the homemade spatula from Tsang and Chow (2009)⁸, which is plugged into a cryotube, to develop the Spatula MVD which is sealed with a 0.5 mL straw (see Table of Materials). The spatula features a gel-loader tip (see Table of Materials), where a platform of approximately 1 mm² is formed by gently melting the tip with fine forceps, creating a petal-like plate for loading the embryos. This platform can accommodate up to 40 mouse embryos in a small vitrification drop. An identification plug rod (see Table of Materials) is inserted into the open end of the gel-loader tip.

The aim of this method is to vitrify embryos in a simple, fast, and inexpensive method that, in addition, saves space in the nitrogen dewar. This system has been successfully used for many years, providing excellent embryo survival rates (~97%) and reproductive outcomes, particularly in mouse embryos. Since Spatula MVD is prepared in the laboratory, it is a low-cost method compared to commercial devices⁹. Moreover, it saves space in the nitrogen dewar in comparison to the use of cryotubes^8,10.

Figure 1: Comparison of the main two techniques used for embryo cryopreservation. This figure has been modified with permission from Crispo et al.¹. Created in BioRender.com. Please click here to view a larger version of this figure.

Protocol

Animals used to obtain mouse embryos were housed and handled according to national law 18.611 and international animal care guidelines¹¹. Experimental protocol (permit number #007-18) was opportunely approved by the Institut Pasteur de Montevideo Animal Care and Use Committee (written consent was given). Experimental protocols for sheep oocytes/embryos were approved by the Institutional Animal Care Committee Fundación IRAUy (protocol #001-2017), complying with the ARRIVE guidelines¹² and were carried out in accordance with international animal care guidelines¹¹.

1. Vitrification (Figure 2)

1. Spatula MVD preparation
   1. Shorten the gel-loader tip (see Table of Materials) by cutting off the first 10 mm and the tip cone.
      NOTE: This creates a shorter, straight tip, facilitating the final sealing with the 0.5 mL straw (see Table of Materials).
   2. Hold the end of the gel-loader tip with watchmaker's #5 forceps (see Table of Materials) approximately 1.0 cm from the tip. Gently place the tip into the flame of a Bunsen burner (see Table of Materials) and hold for 8-10 s to form a petal-like platform of approximately 1 mm².
   3. Remove from the flame and hold tightly with the forceps for 5 s.
   4. Using a stereomicroscope (see Table of Materials), check that the petal-like platform (~ 1 mm²) is formed and the distal edge is sealed.
      NOTE: The platform must be closed at the distal end to prevent LN₂ infiltration during vitrification. Ensure the platform is clean and free of plastic threads. If it is too small or has threads, trim the tip and repeat the procedure.
   5. Repeat steps 1.1.1-1.1.4 to prepare the necessary number of spatulas (1 spatula = up to 40 mouse embryos).
   6. Sterilize the spatulas using ultraviolet (UV) rays or ethylene oxide and store them in a sterile tube or box.
   7. On the day of vitrification, retrieve the spatulas, handling them from the opposite end of the platform. Insert an identification rod (see Table of Materials) into each spatula and place them on the stereomicroscope base. Label the rod with a permanent marker or liquid nitrogen-resistant sticker label.
      NOTE: Always avoid touching the platform when handling the spatulas. The total length of the complete device (spatula + 0.5 mL straw + identification rod) should not exceed more than 1 cm of the length of the goblet.
2. Preparation of vitrification solutions
   1. Prepare the FS solution: Dissolve 1.5 g of density gradient medium (see Table of Materials) and 0.85 g of sucrose (see Table of Materials) in 5 mL of phosphate-buffered saline (PBS; see Table of Materials). Heat in a boiling water bath until completely dissolved, then sterilize using a 0.22 µm filter (see Table of Materials).
      NOTE: This solution can be stored at 4 °C for up to 6 months.
   2. Prepare the pre-vitrification solution (PV): On the day of vitrification, mix 10% ethylene glycol (see Table of Materials), 10% dimethyl sulfoxide (see Table of Materials), and 80% M2 medium (see Table of Materials). A total volume of 100 µL is needed for 5 spatulas.
   3. Prepare the vitrification solution (V): On the day of vitrification, combine 15% ethylene glycol, 15% dimethyl sulfoxide, 10% M2 medium, and 60% FS solution. A total volume of 100 µL is required for 5 spatulas.
3. Embryo dehydration and vitrification process
   1. Prepare LN₂: Pour LN₂ into a suitable container (e.g., a 10 cm x 30 cm x 20 cm insulated polystyrene container) and place as many 0.5 mL straws as spatulas to be used.
      NOTE: To facilitate visualization of the spatula platform during manipulation under LN₂, it is suggested that a colored bottom-insulated polystyrene container be used to create a contrast effect.
      CAUTION: Wear appropriate goggles and gloves (see Table of Materials) when handling LN₂.
   2. Prepare solutions: On a 100 mm plastic Petri dish (see Table of Materials), place the following drops: 100 µL of M2, 20 µL of PV solution, and 20 µL of V solution for each spatula.
   3. Vitrify embryos: Place the embryos to be vitrified in the M2 drop. Handle 30-40 embryos at a time, incubating them in the PV solution drop for 30 s, and then in the V solution drop for another 30 s at room temperature (RT).
   4. Use a pulled glass capillary (see Table of Materials) with an aspirator tube assembly (see Table of Materials). Pre-load the capillary with at least 1 µL of the respective solution before placing the embryos.
      NOTE: Embryos will be at different heights in the drops, so adjust the stereomicroscope micrometer to locate them. Retrieve embryos from each drop when 10 s remains. All embryo manipulations must be conducted under the stereomicroscope.
   5. Load embryos: Aspirate the embryos using minimal V solution (e.g., ~ 0.1 µL) and place them quickly onto the spatula platform.
   6. Immersion in LN₂: After loading the embryos, immediately immerse the spatula platform in LN₂ for 5 s (holding the spatula by hand, by its rod). Then, while maintaining the spatula in LN₂ vapors, seal it with a 0.5 mL straw using long stainless-steel tweezers (see Table of Materials).
      NOTE: Remove LN₂ from the straw before sealing to prevent expansion during warming. Ensure the spatula and straw are aligned properly to avoid contact between the embryo drop and straw wall. For convenience, sealed spatulas can remain in the LN₂ container until all spatulas are loaded with embryos, refilling with LN₂ as necessary.
   7. Repeat procedure: Repeat steps 1.3.3 to 1.3.7 for each group of embryos, using a fresh pair of PV and V drops.
   8. Storage: Transfer the spatulas to goblets (see Table of Materials) and then to a LN₂ storage dewar (see Table of Materials).

Figure 2: Scheme of the vitrification process using Spatula MVD. See the text for more details. Created in BioRender. Meikle, M. (2025) https://BioRender.com/x96y902. Please click here to view a larger version of this figure.

2. Warming (Figure 3)

1. Solution preparation
   1. Prepare a 1 M sucrose solution dissolving 0.85 g of sucrose in 2.5 mL of M2 medium. Sterilize the solution with a 0.22 µm filter.
      ​NOTE: This solution can be prepared and stored at 4 °C for up to 1 week.
   2. Prepare 0.5 M and 0.25 M sucrose solutions by diluting the 1 M sucrose solution with the appropriate volume of M2 medium.
   3. Maintain the sucrose solutions at 37 °C on a hot plate (see Tables of Materials) until the warming starts.
2. Warming plate preparation
   1. On a 100 mm plastic Petri dish, place the following solution drops: a 500 µL drop of 0.5 M sucrose, a 50 µL drop of 0.5 M sucrose, and a 50 µL drop of 0.25 M sucrose for each spatula to be warmed. Place three drops of M2 medium (100 µL each) for washing.
3. Warming process
   NOTE: The warming process takes place at RT, e.g., 20-22 °C on the base of a stereomicroscope.
   1. Retrieve the sealed spatulas from the dewar. Place them in an appropriate container with LN₂. See Figure 3.
   2. Place the warming plate on the stereomicroscope. One at a time, hold the base of the spatula with stainless-steel tweezers, and with a gloved hand, pull out the identification rod and then the straw. Quickly dip the platform containing the embryos into the 0.5 M sucrose drop of 500 µL.
   3. Ensure that all embryos have fallen into the drop; if necessary, gently move the spatula to assist their release. Load the embryos into a pulled glass capillary, place them in the 50 µL drop of 0.5 M sucrose solution, and incubate for 2 min.
   4. Place the embryos in the 50 µL drop of 0.25 M sucrose solution and incubate for another 2 min.
      NOTE: Before placing the embryos in each solution, pre-load the capillary with the solution to be used.
   5. Finally, wash the embryos three times in different drops of M2 medium to remove the sucrose.
   6. Incubate the embryos under conditions appropriate for each species for at least 1 h before use.

Figure 3: Scheme of the Spatula MVD warming process. See the text for more details. Created in BioRender. Meikle, M. (2025) https://BioRender.com/k91k191. Please click here to view a larger version of this figure.

Results

We initially used the traditional slow-freezing method based on propylene glycol solutions, straws, and controlled rate freezing equipment⁷. However, after considering the vitrification method a potentially easier and more cost-effective option, we conducted a study to compare the outcomes of traditional slow freezing (SLF) vs. Spatula MVD in 8-cell in vivo derived and 2-cell in vitro produced mouse embryos¹³ (Table 1<...

Discussion

The Spatula MVD is an easy-to-assemble, low-cost, minimum-volume vitrification device with high embryo holding capacity and optimal liquid nitrogen dewar occupancy. This method has proven to be simple, fast, and robust. On average, we have achieved not only high embryo recovery and survival rates of vitrified embryos (~90%) but also good reproductive outcomes when performing embryo transfer in mice (~80% pregnancy rate and ~30% birth rate), which are comparable to other minimum volume vitrification methods

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm filter	Sartorius	16534	Minisart syringe filter 0.2 µL
0.5 mL straw	Minitube	13408/0044	French type medium paillette
Aspirator tube assembly	Sigma	A5177
Bunsen burner	Indulab	7000134
Dimethyl sulfoxide	Sigma	D2650
Ethylene glycol	Sigma	102466
Ficoll PM70	Sigma	F2878	Density gradient medium
Gel-loader tip	Eppendorf	10411193
Glass capillary	Marienfeld	2900000	Microhaematocrit capillary tube
Gloves for LN2 handling	Tempshield	CE0338	Cryogloves
Goblets	Minitube	16913/1133
Goggles for LN2 handling	3M	40661-00000-10	Anti-fog, anti-scratch coating
Hot plate	Labec	LA-SH-II-5B	Digital model
Identification rod	IMV technologies	6326	Non steril LG48 mm
Insulated styrofoam container
LN2 storage dewar	Taylor-Wharton	XT21-AI	Cryoscience
Long stainless-steel twezeers	Fine Science Tools	11000-25
M2	Sigma	M7167
PBS	Home-made		Phosphate Buffered saline. Usually 1 L of concentrated 5x buffer is prepared and then diluted to 1x. Recipe: 40 g sodium chloride, 1 g potassium chloride, 7.2 g anhydrous disodium phosphate, 1.2 g monopotassium phosphate. Add distilled water and sterilize by autoclave.
Plastic Petri dish	Falcon	351029	Not TC-treated Bacteriological Petri Dish
Stereomicroscope	Olympus	SZ2-LGB
Sucrose	Sigma	S1888
Watchmaker’s #5 forceps	Fine Science Tools	11254-20