Schlapp Geraldine, Meikle María Noel, Goyeneche Lucía, Dos Santos Neto Pedro, Crispo Martina
Laboratory Animal Biotechnology Unit, Institut Pasteur de Montevideo;
Laboratory Animal Biotechnology Unit, Institut Pasteur de Montevideo.
J Vis Exp. 2025 Jun 6(220). doi: 10.3791/68176.
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.
本研究描述了一种高效且经济的玻璃化方法，用于小鼠和绵羊胚胎冷冻保存，即蒙得维的亚刮铲法（MVD）。该方案使用自制刮铲作为载体装置，它由凝胶加样器吸头制备而成。胚胎在两种浓度递增的自制冷冻保护剂溶液中快速平衡，装入刮铲，然后直接浸入液氮中。大约30 - 40个植入前阶段的小鼠胚胎可以装载在刮铲上，随后用0.5 mL细管密封。使用刮铲而非常用于储存玻璃化胚胎的冷冻管，可优化液氮杜瓦瓶中的空间。此外，解冻过程仅需一种浓度递减的自制蔗糖溶液来使胚胎再水化并恢复其形态。我们实验室成功使用蒙得维的亚刮铲法处理小鼠和绵羊植入前胚胎已有10多年。在小鼠胚胎中，蒙得维的亚刮铲法的平均回收率（回收/玻璃化胚胎）为84%，存活率（存活/回收胚胎）为97%。小鼠胚胎的玻璃化冷冻效果优于慢速冷冻，尽管在生殖结果（妊娠、分娩和出生率）方面未观察到差异。在绵羊胚胎中，与商业设备相比，蒙得维的亚刮铲法取得了相似的存活和胚胎发育率。尽管商业设备的妊娠率更高，但各设备间的羔羊存活率无显著差异。蒙得维的亚刮铲法玻璃化冷冻技术是一种简单的冷冻保存技术，可用于储存多余胚胎、备份转基因小鼠品系和绵羊模型，并便于在全球范围内的研究人员之间进行交换。
