The Jackson Laboratory (JAX) is one of the biggest suppliers of transgenic mice for biomedical research. JAX has a large biobank that cryopreserves and recovers thousands of different mouse strains. Biobanking mouse strains has many benefits for researchers. Biobanking can decrease the risk of important research strains being affected by disease or environmental disaster. It allows researchers to decrease the size, and therefore, the cost of maintaining active research colonies. Biobanking mouse strains also allows researchers to comply with funding requirements to make research tools publicly available.
Cold Spring Harbor Protocols recently published three different methods employed by JAX for cryopreserving and biobanking mouse embryos: slow freezing, rapid cooling and high-osmolality vitrification. Rapid cooling offers similar post-thaw recovery rates to slow freezing and doesn’t require expensive equipment. However, both rapid cooling and high-osmolality vitrification are more technically difficult than controlled-rate freezing.
Biobanking by Slow Freezing
Researchers in a number of different laboratories and biobanks have used the slow freezing method to preserve over 2 million embryos from thousands of different mouse strains. This method can be used to biobank embryos from the two-cell stage to the blastocyst stage and has post-thaw survival rates as high as 90%. The downside of this method is that it requires the use of a controlled-rate freezer. Not all biobanking laboratories have a controlled-rate freezer as it is a relatively expensive piece of equipment.
The JAX slow freezing protocol uses 1,2-propanediol as a cryoprotectant1. In this method, researchers pre-cooled a controlled-rate freezer to -7°C, loaded embryos into insemination straws filled with cryopreservation solution, then sealed the straws. Researchers then loaded the straws into the controlled-rate freezer and left the embryos to equilibrate in the cryoprotectant solution at -7°C for 5 minutes. The embryos were then cooled at a rate of 0.5°C/minute down to -30°C.
Liquid nitrogen (LN2) dewars or portable cryogenic carriers can be used to transport cooled embryos to long-term storage. This will help to avoid any transient warming events.
When using this method, researchers thawed the embryos rapidly using a room-temperature water bath.
Biobanking by Rapid Cooling
Mouse embryo cryopreservation by rapid cooling has a post-thaw recovery rate of 77% to 90% – similar to that of the slow freezing method of biobanking. Rapid cooling uses higher concentrations of cryoprotectants than slow freezing. Increasing the osmolality of the cryoprotectant solution pulls water out of cells prior to freezing. This dessication decreases the chance of ice crystals forming inside cells during cooling.
JAX used a cryoprotectant solution containing ethylene glycol, Ficoll and sucrose in their rapid cooling cryopreservation protocol2. This protocol can be used to biobank two-cell to eight-cell (morula) stage embryos. The embryos are loaded into straws containing cryoprotectant solution and allowed to equilibrate in this solution for 0.5 to 2 minutes at room temperature. The straws are then sealed and plunged into LN2 to snap freeze.
In this method, biobanking embryos were rapidly thawed in a step-wise fashion using LN2, room temperature air and a 20°C water bath.
Biobanking by High-Osmolality Vitrification
High-osmolality vitrification is a variation on the rapid cooling method that uses higher concentrations of cryoprotectants and smaller volumes to snap freeze samples almost instantaneously. The speed of this freezing method can help prevent intracellular crystal formation. This method can be used on two-cell stage embryos.
JAX uses a cryoprotectant solution containing high concentrations of ethylene glycol, Ficoll and sucrose to vitrify mouse embryos. Embryos are equilibrated at room temperature in the cryoprotectant solution, then loaded into cryotubes and plunged into LN2 to freeze3.
Vitrification protocols use much higher concentrations of cryoprotectants than slow freezing. Therefore, vitrified embryos are more likely to swell and die when exposed to thawing medium with lower osmolalities. To avoid this, embryos should be warmed at room temperature in high-osmolar medium to prevent excessive swelling.
Embryos frozen with this method can survive at -80°C for at least 5 months and at -180°C for longer periods.
You can find the full protocol for all three cryopreservation methods in Cold Spring Harbor Protocols.
Conclusion
Slow freezing is currently the most thoroughly tested and reliable method of cryopreserving and biobanking mouse embryos. However, this method does require a controlled-rate freezer. Rapid cooling and high-osmolality vitrification protocols can provide similar recovery rates to slow freezing but are technically more difficult.
All methods outlined in this article use older style cryoprotectants, which have been shown in many studies to be toxic to cells and tissues. Newer-generation cryoprotectant solutions such as multi-component osmolyte solutions could be used to adapt these protocols for other embryo types and other tissues.
References:
- Taft, R. Mouse Embryo Cryopreservation by Slow Freezing. Cold Spring Harbor Laboratory Press. 2018
- Shaw, J. Mouse Embryo Cryopreservation by Rapid Cooling. Cold Spring Harbor Laboratory Press. 2018.
- Mochida, K. Mouse Embryo Cryopreservation by High-Osmolality Vitrification. Cold Spring Harbor Laboratory Press. 2018.
