Embryo Cryopreservation

Embryo cryopreservation involves the generation of pre-implantation embryos using males provided by the investigator and the controlled rate cooling of collected embryos to -30°C followed by rapid freezing of embryos in liquid nitrogen to -196°C.

While embryo cryopreservation is the gold standard for preserving mouse germplasm, not all mouse strains are suitable for embryo cryopreservation. However, we have been successful at cryopreserving embryos from several different mouse strains.

Embryos for cryopreservation can be obtained in two basic ways:

1. via in vitro fertilization (IVF) using isolated sperm from a single male and eggs collected from hormonally-treated females, a method referred to as speed cryogenics
2. via natural mating of hormonally-treated females with 6-10 fertile male

The speed cryogenics method will be used for most TTML embryo cryopreservations projects as hundreds of embryos can be generated in a single day by IVF when oocytes collected from hormonally-treated wild-type females are incubated with freshly isolated sperm. Either homozygous or hetero/hemi-zygous males can be used. If homozygous males are used, the genotype of all the preserved embryos will be hetero/hemi-zygous. Both hetero/hemi-zygous and wild-type embryos will be generated using hetero/hemi-zygous sperm.

There is inherent inefficiency in this method and it is highly dependent on the fertilization capacity of the sperm which varies substantially among lines and between males within a line. Although we have optimized the process, the queue can be lengthy.

To cryopreserve homozygous embryos, it is essential to used both homozygous females and homozygous males to generate the embryos. Due to our current staffing and animal room health status, the only way at this time to freeze homozygous embryos through the TTML requires that your lab maintains the single housed males and hormonally treats/mates the females. The TTML provide the hormones and information on preparation, dosage and timing. Working together, we have successfully frozen homozygous embryos for several investigators to date.

Project Initiation

To initiate an embryo cryopreservation project, completed an Embryo Cryopreservation Request form and submitted to TTML@northwestern.edu.

• Be sure to include background strain information on the request form
• We will review the request to determine if it is feasible and the best method

The cryopreservation process:

Generation of embryos for cryopreservation via IVF:

• The male should be between 8 weeks and 8 months of age and singly housed for two weeks for optimal sperm production.
• TTML staff will contact you ~2 weeks prior to beginning your project to confirm the schedule date and have you separate a male.
• Wild-type females are purchased and used as a source of oocytes for IVF.
• IVF procedure
  • Young wild-type females are hormonally treated to generate oocytes.
  • On the day of IVF, sperm is collected from each male and immediately incubated with freshly isolated oocytes.
  • After 5 hours, embryos are washed through fresh media and incubated overnight.
• Cryopreservation procedure
  • The next day, healthy 2-cell stage embryos are collected, loaded into straws and frozen at a rate of -0.5°C/minute to -30°C, then rapidly frozen in liquid nitrogen.
• Quality control
  • Test straws from each freeze are thawed and cultured in vitro to the blastocyst stage to assess viability and developmental capacity.
    • If the test thaw is inconclusive, a straw will be thawed and transferred into recipient female to confirm embryo viability
    • Depending on these results and the number of embryos frozen, we will contact you to either schedule another session or confirm that the project is complete.

Generation of homozygous embryos via natural mating:

• Your lab will be responsible for maintaining 6-10 singly housed homozygous males for the duration of the project.
• Your lab will also be responsible for hormonally treating, mating and plug checking homozygous females.
• The TTML will provide the hormones and information on the preparation, dosage and timing of hormones.
• Mated females are delivered to our lab and E0.5 embryos are isolated and cultured overnight.
• Healthy 2-cell stage embryos are loaded into straws and cryopreserved.
• Test thaws are performed for each freeze.
• The process will be repeated until enough embryos are successfully frozen.
• We will inform you when the project is complete.

Expected results:

• A minimum of 150-200 embryos is frozen for each line for strains with normal reproductive performance.
• For IVF projects, if fewer than 150 embryos are obtained in the first two IVF sessions, a third and final session will be performed, and the project will be completed regardless of the number of embryos frozen.
• A test straw of cryopreserved embryos from each freeze is thawed to determine the success of the procedure. Thawed embryos are assessed for viability and the ability to continue to develop in vitro.

Long-term storage and access to cryopreserved embryos:

• Cryopreserved embryos from each line are stored in two separate liquid nitrogen dewars in two different buildings.
• Currently, there is no additional cost for storage.
• Embryos can be cryorecovered at any time simply by contacting our lab.
• Embryos can also be shipped world-wide from our lab.

Costs and Services