Embryo Cryopreservation
Introduction
Mouse embryos stored in liquid nitrogen (-196°C) are metabolically inactive, therefore, they can be stored for a conceivably infinite period of time and then used to regenerate a colony of live animals. There are several advantages to banking embryos in LN2:
- Provides “insurance” in case of animal loss due to breeding failure, pathogens, or natural disasters
- Reduce cage costs when mice are no longer needed for experimental purposes
- Reduce demand for limited animal housing space
- Simplifies shipment of animals and avoids importation quarantines
- We will provide the hormones for super-ovulation.
- We will harvest embryos from a maximum of 15 super-ovulated donors per session.
- Embryos are washed, sorted, and frozen at a slow rate to -35°C.
- The frozen embryos are stored in 2 separate liquid nitrogen containers.
There are several protocols available for freezing fertilized mouse embryos. The TRP freezes 8-cell pre-implantation stage embryos (E2.5) using 1.4M glycerol as the cryoprotectant. Cryoprotective agents improve the survival rate of frozen cells by decreasing the temperature at which ice crystals form.
We collect the E2.5 embryos from the oviduct side of the utero-tubal junction. Embryos are selected for cryopreservation by assessment of normal developmental progression and an intact zona pellucida. They are washed through a trypsin-EDTA solution to remove any contaminating pathogens sensitive to trypsin that may have adhered to the zona. The embryos are placed in the freezing media and allowed to equilibrate for 30 minutes prior to starting the freezing process. Up to 30 embryos are loaded per straw (enough for 2-3 transfers upon thaw). The straws are placed in a Bio-Cool Controlled-Rate Freezer at a starting temperature of -7°C, and frozen at a rate of -0.3°C/min. down to -33°C, then -0.1°C/min. down to -35°C. When the slow freeze is complete, the straws are quickly dunked into liquid nitrogen.
The straws are loaded with an excess of 1M sucrose separated from the embryos by an air bubble. This is useful upon thaw, as straws need only be shaken to drop the embryos into 1M sucrose for removal of excess cryoprotectant. This method was developed so that no special media need be made upon thaw. There is no impact on survival due to method of thaw, whether by step-wise cryoprotectant removal or the 1-step method described above.
- You need to provide up-to 15 transgenic male breeders. Males must be of breeding age (not too young, too old, or inexperienced).
- You may also provide up-to 15 transgenic female embryo donors if it is necessary to maintain homozygosity. Otherwise, we will use WT female embryo donors.
- Mice will be transferred to the TRP via protocol transfer in AOps.
- Many factors can influence the number and viability of embryos collected including the age & weight of the embryo donors, age and experience of the stud males, transgenic alterations, and genetic background strain.
- For best results, use experienced, proven male breeders.
- We recommend that you store at least 150 homozygous embryos.
- It may be necessary to perform more than one cryopreservation session in order to freeze an adequate number of embryos.
- Cryopreservations are charged on a per session basis and includes LN2 storage. A maximum of 15 donor females per session. A test straw will be thawed immediately after freezing in order to provide information on the survival and developmental potential of your stored embryos. An 80% survival rate is expected upon thaw. If we experience a failure as determined by < 50% survival, the session will be repeated at no charge.
- We cannot offer any guarantees as to how many embryos will be harvested and frozen in any given session as many factors can influence the number and viability of embryos collected.
- Click here for current rates.