The overall goal of this procedure is to cryopreserve mouse embryos in liquid nitrogen and to thaw them for recovery of live mice. This is accomplished by first immersing mouse embryos in equilibration medium, transferring them into vitrification medium in a cryo tube, and then placing them in a liquid nitrogen tank. Later embryos can be thawed in a high osmotic medium and incubated in culture medium until they are transferred into recipient females.
Ultimately, results show survival of more than 90%of the embryos after vitrify thawing, and birth rates between 30 to 70%after embryo transfer. The main advantage of this technique is that the permeable cryoprotectant glycol is less toxic to embryos than conventional cryoprotectant. Demonstrating the procedure will be a technician from my laboratory Prior to beginning this protocol.
Prepare two cell mouse embryos in culture obtained by natural mating or conventional IVF techniques to a cryo tube for eventual storage, add 50 microliters of embryo freezing solution EF S 40. Then prepare a 35 millimeter or 60 millimeter plastic Petri dish with 50 microliters of room temperature. EFS 20.
Use a glass capillary to transfer up to 30 embryos to the dish. Be careful to avoid transferring the culture medium. Also to ensure the embryos dehydration, place them in the bottom of the drop.
After about 60 to 90 seconds, use a capillary tube to draw up dehydrated embryos. With a shrunken morphology. Move these embryos into EFS solution in the cryo tube, transferring as little EFS 20 as possible.
Once the embryos are transferred, allow them to settle in the EFS 40 for one minute before freezing them in liquid nitrogen. Before thawing the embryos place in a culture dish at least two drops of 10 microliter high osmolarity embryo culture medium, such as M 16 medium. Then cover the drops completely with silicon or mineral oil, and place the dish in a carbon dioxide incubator until use.
Next, warm the TS one solution to 37 degrees Celsius while wearing a face mask and cryo gloves. Retrieve the embryos from the liquid nitrogen tank. Now quickly open the cryo tube and discard any liquid nitrogen to the tank or any appropriate container.
Then wait 30 seconds. Now add 850 microliters of warm TS one solution to the cryo tube. Gently suck up and deject the solution about 10 times so it is evenly dissolved.
Then transfer the entire volume to a plastic 60 millimeter Petri dish. Or watch glass wait three minutes for the embryos to equilibrate to room temperature where they should stay for the remainder of the procedure. Do not use a warming device.
Quickly examine the embryos in the dish under a stereo microscope to know how they look at the beginning of this incubation. After about two minutes of incubation, sink the embryos by gently shaking the dish until the medium is spread over the surface of the dish. In the last minute of the incubation, eject three separate 50 microliter drops of TS two solution in a dish.
When the three minutes are over, use a stereo microscope to confirm that the embryos have shrunken slightly. If the embryos are still swollen, continue the incubation for one to three more minutes. Now, pick up the embryos with a glass capillary and transfer them to the first drop of TS two.
Wait three minutes and transfer embryos to the second drop. Followed by the third drop. Finally, retrieve the prepared embryo medium dish stored in the incubator, and use a glass capillary to transfer the embryos to the first drop of the medium.
If examined under a stereoscope, the embryos should slowly recover their normal morphology. Now, return the dish to the incubator for about 10 minutes. Then to wash out the sucrose that has been carried over from TS two.
Move the embryos to the next drop of culture medium. Continue culturing the embryos in the CO2 incubator until they're transferred to the uc of recipient females. After vitrify and recovering between 300 and 480 embryos of three different mouse strains, survivability was very high.
After thawing, the fraction of embryos showing normal morphology was between 93 and 99%and of those embryos at least 84%developed into blast cysts. Don't forget that working with leak nitrogen can be extremely hazardous and precautions to avoid direct contact and minimize short distance exposure to leak. Nitrogen should always be taken by performing this procedure.
