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.....

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....

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