Cryopreservation FAQ

Security Cryopreservation provides a reserve population that will guard against the loss of your strain due to accident, disease, or disaster.
Cost If your strain is not being actively used in experiments, but you might need to use the mice at a later date, it is much cheaper to maintain the strain in the freezer instead of as a live colony.
Space Mice in the freezer free up space in the mouse room.
Ease of distribution It is much easier to distribute frozen material than it is to distribute live animals. Animal shipments are subject to environmental hazards in transit; this is not the case of frozen material, which travels in a container that can maintain liquid nitrogen temperature for weeks. Further, frozen material is almost never subjected to the quarantine restrictions that face live animals, since the act of recovery also serves to re-derive pathogen-free mice.

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1.2 Which of my mouse strains should I cryopreserve?

It is a good idea to freeze all your strains. Nonetheless, resources will probably limit the number of strains that you can preserve. In order to prioritize your strains, consider the following points:

How easy would it be to replace these animals? The harder it would be to replace the animals, the stronger the argument for cryopreservation.
How active is the use of these animals in your current research? If you are simply maintaining these animals for some future use, they are strong candidates for cryopreservation.
How valuable are these mice? If these animals are an important and unique research model, they are strong candidates for cryopreservation.

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1.3 Should I freeze embryos or sperm?

The choice depends on three factors:

Genetics Strains that must be maintained by brother-sister matings must be cryopreserved as embryos. Strains that can be maintained by mating males with commonly available wildtype inbred strains can be cryopreserved as sperm.
Practicality There are practical considerations that will determine the suitability of a particular strain for a particular method of cryopreservation. For example, embryos of some strains do not recover particularly well from the freezers, or sperm of some strains does not fertilize well in vitro.
Price Consideration of price should include both the cost of cryopreservation, and the price of recovery. Cryopreservation of embryos is more expensive, due to the costs of the mice to produce the embryos. However, the cost of recovery of cryopreserved embryos is lower than that of frozen sperm, because in the latter case mice must be purchased to provide a source of oocytes that are then fertilized in vitro by the recovered sperm.

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2 Sperm Cryopreservation

2.1 How is sperm cryopreservation done?

Sperm is collected from 2 - 4 males, pooled and frozen. A sample is then thawed and used to produce embryos by in vitro fertilization. These embryos are then transferred to foster mothers. If pups are recovered in this quality control (QC) step, the strain is considered to be successfully cryopreserved.

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2.2 How do you collect sperm? Will I get my mouse back?

The usual practice is to sacrifice the male and remove the epididymides for sperm collection. In theory, one can collect ejaculated sperm from the uterus of a female bred with the male, or one could do a unilateral epididymectomy, thus preserving the male. In practice, this is done in only the rarest of circumstances.

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2.3 What age males are used for sperm cryopreservation?

Males aged 10 - 20 weeks are best, but older animals can be used.

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2.4 Should I simply freeze sperm without recovery (the QC step)?

This is advantageous because it is considerably less expensive then doing cryopreservation with the quality control step. However, the disadvantage is that there are no guarantees that live mice can be recovered from this material.

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2.5 If I freeze sperm without recovery, how many males should I use?

There is no real answer for this. More is better, because fertility of individual mice will vary, so by freezing a single male there is a risk that this one won't be fertile. On the other hand, more is more expensive.

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3. Embryo Cryopreservation

3.1 How is embryo cryopreservation done?

Embryos are produced either by in vitro fertilization or by natural mating with superovulated females. These embryos are then frozen. An aliquot of frozen embryos is then thawed and transferred to foster mothers. If pups are recovered in this QC step, the strain is considered to be successfully cryopreserved once an appropriate number of embryos are frozen.

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3.2 How many embryos are frozen?

On average, about 25% of cryopreserved embryos will develop into live pups. However, the recovery of pups is very dependent on the genetic background of the strain. The target is to freeze enough embryos to recover 25 - 50 pups carrying the gene(s) of interest. For most strains, this means that 100-200 embryos are frozen; however, the actual number frozen is determined on the recovery of pups in the QC step.

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4. Storage and Recovery

4.1 Do I have to pay for storage?

No.

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4.2 How secure are the frozen samples?

Material from each strain is split between at least 2 different storage freezers, and an aliquot of also stored off-site.

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4.3 How long will frozen material remain viable?

For all practical purposes, forever. As long as it is kept at liquid nitrogen temperatures, the material will remain viable with the only theoretical loss being due to damage caused by cosmic radiation. Half-life is estimated to be measured in millenia.

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4.4 How long does it take to recover mice?

Recovered animals are available 6 weeks after thawing the frozen material. This represents the 3-week gestation time of the mouse plus the 3 weeks between birth and weaning.

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