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

Service Description

  • 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.

Our Cryopreservation Protocol

There are several protocols available for freezing fertilized mouse embryos. The PRTS 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.

What You Need to Do

  • You need to have/produce up to 15 transgenic male breeders. Males must be of breeding age (not too young, too old, or inexperienced). For best results, males should be “practiced” (given a chance to breed), and then given one week to recover in order to get their sperm count back up.
  • Order 15 wild type C57BL/6 females at 3-weeks-of-age to be super-ovulated. The strain and age is crucial to success. C57BL/6 mice respond best to superovulation when hormoned at 12-14 grams body weight. Some strains do not respond to superovulating hormone therapy.
  • The hormone injections and matings take place in your colony by your lab to prevent transmission of disease into the K-wing transgenic mouse colony. We will provide the hormones for you.
  • Deliver the time-mated embryo donors to K-024D by 9:00 AM on day E2.5.

Superovulation Protocol

There are 2 separate hormones that must be administered by I.P. injection 48 hours apart. PMSG (pregnant mare’s serum gonadotropin) is given at noon on day one. PMS is used to mimic the oocyte maturation effect of the endogenous follicle-stimulating hormone (FSH). 48 hours later, hCG (human chorionic gonadotropin) is administered at noon in order to mimic the ovulation induction effect of luteinizing hormone (LH). Immediately after receiving the injection of hCG, each female should be placed with a transgenic male (in a ratio of 1:1). Males are housed individually, and the female should always be introduced into the male’s cage. All of the females should be checked for plugs the following morning and removed from the male's cage. It’s helpful to keep a record of each male's plugging history. As long as the females are all the same genotype, you can pool them back together, up to 5 mice per cage.

Hormones should be kept frozen until ready to use!

Mid-day

  • 5.0 IU (0.1 ml) Pregnant Mare’s Serum Gonadotropin (PMSG), injected I.P.
  • Thaw tube of PMS and add 1ml PBS or 0.9% saline
  • Inject 0.1ml per mouse, I.P.
  • Do not re-freeze

48 hours later

  • 5.0-7.5 IU (0.1 ml) human Chorionic Gonadotropin (hCG)
  • Thaw tube of hCG and add 1ml of PBS of 0.9% saline
  • Inject 0.1ml per mouse, I.P.
  • Immediately after injection, each female is placed 1:1 with transgenic males.

E0.5 = the morning after hCG (day of copulation plug ),
E2.5 = the morning of 8-cell embryo collection.

For example, if embryos are to be collected on Thursday :
PMSG is given Saturday
hCG is given Monday
E0.5 is Tuesday
E2.5 is Thursday

Some Things to Consider

  • It is extremely important to be aware that many factors can influence the number and viability of embryos collected: age & weight of embryo donor, light cycle, hormone dosage, transgenic alterations, and genetic background strain.
  • Ideally, female donor mice should be hormoned between 3-4 weeks-of-age and weigh about 14 grams (for C57BL/6).
  • It is vital to use experienced, proven male breeders.
  • We recommend that you store at least 150 homozygous embryos. If heterozygous embryos are frozen, we recommend the storage of approximately twice that number.
  • It may be necessary to perform more than one cryopreservation session in order to freeze an adequate number of embryos.

Prices and Guarantees

  • Cryopreservations are charged on a per session basis, with 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.
  • The number of embryos cryopreserved depends on the number of embryo donors supplied by the client, their age and response to superovulation, and the fertility of the stud males. Therefore, we cannot offer any guarantees as to how many embryos will be frozen in any given session.
  • Click here for current rates.

Reserve a Cryopreservation Session

To order an embryo cryopreservation session, go to our TRP Forms page. Fill out a “Request for Embryo Cryopreservation” form and e-mail it to Bob Hunter bhunter@uw.edu