Maintaining the integrity of research and obtaining reliable, repeatable data depends on the stability of a research model. Given the cost and complexity of managing model breeding, cryopreservation is often viewed as a highly efficient insurance policy for a colony.
Cryopreservation brings significant welfare, logistical and cost benefits to modern research and, consequently, should be “woven into the fabric” of each and every research project.
It is a primary role of researchers to continually consider how to avoid uncertainty and generate reproducible data by minimising variability throughout the span of their studies. One key area of potential variability is highlighted by remembering that an animal model is a potential variable in research … rather than just a static research tool.
As modern research in many disease areas continues to leverage increasingly specialised transgenic lines, such variability becomes a significant risk — not only to research outcomes, but also to budgets and timelines.
These inherently unique models often demand a large upfront investment of time and resources. Once established, sustaining a genetically modified colony requires ongoing monitoring for genotype as well as genetic drift caused by natural point mutations, which may eventually lead to a change in gene function and phenotype.
Day-to-day upkeep of an established cohort occupies valuable facility space and requires the ongoing efforts from animal care technicians to maintain a supply of healthy, research-ready animals. With time, these costs add up, quickly impacting the overall research budget, especially if models are not immediately needed or only require intermittent use in the future.
Colony maintenance must also account for the possibility of natural disasters, microbial contamination, breeding cessation or even facility failures — unforeseen issues that can threaten difficult-to-replace strains and cause catastrophic losses.
Similar to the way we have become familiar with backing up valuable electronic records, cryopreservation provides a method for maintaining a strain at a specific genetic state in time and enables a colony to be “refreshed” back to that genetic moment.
Not only does this “back-up” serve as an insurance policy in case of unexpected problems, it can reduce the space and resources required for animal care and allow for a more targeted focus on actively used colonies.
Beyond protecting from loss and providing on-demand recovery for a colony, cryopreservation has other recognisable benefits with logistical processes.
In some countries, such as Japan and Australia, sending live models is a cumbersome, paperwork-intensive process that can take weeks before approval.
Using cryopreserved material to transport a line can bypass this wait and get a colony back up and running in much less time.
Freezing a line also makes economical and ethical sense by reducing the overall number of animals needed. Maintaining live colonies requires ensuring the breeding, maturing and health status of animals, with associated technician, facility and health monitoring costs.
Freezing and rederiving a line as and when needed not only eliminates potential waste from continued breeding when a study is on hold, but also frees up positions and space in the facility. Licensing bodies also look favourably on this “welfare mind set,” incorporating three R (replace, reduce and refine) practices to demonstrate the responsible use of models.
Incorporating best practices in cryopreservation
Depending on the line, there are certain time points when cryopreservation is ideal. First, cryopreservation should take place when the proper background is established for the line and before the genetic background has a chance to shift.
Another time point that makes sense is when the first transgenic animals (known as founders) are produced to save the mutation itself. Last but not least, freezing a multiple transgenic line really must be considered, as it can take a lot of effort to reach a particular complex genetic status.
Cryopreservation captures a snapshot from which animals can be rederived back to this status, should it be needed in the future.
Two standard techniques are available: sperm and embryo cryopreservation. The former is the most cost-effective but only provides one copy of the genome. Typically, with a minimum of two males, approximately two dozen frozen straws provide enough material to freeze a line.
Embryo cryopreservation typically requires 20 females and 10 breeder males. Although the costs of embryo cryopreservation are higher compared with sperm cryopreservation, this process ensures an exact copy of the genotype. Depending on whether the model is bred as a heterozygote or homozygote, anywhere from 150–300 embryos are captured.
Ensuring a quality process
Regardless of the cryopreservation choice, quality control (QC) is an important consideration. Providers must validate and demonstrate, both before and after cryopreservation, that the material is properly preserved and can successfully reproduce live animals.
It is equally important to ask a provider how they perform QC and confirm the efficiency of their process. Many providers will also offer dual storage of frozen materials, separating samples to ensure that at least one set remains viable should a facility fail.
Just as a considerable amount of effort goes into developing and maintaining a line, responsible researchers should commit to developing an accompanying high-quality cryopreservation process as an integral part of each and every research project.
Cryopreservation offers a cost-effective insurance policy that not only eliminates the constant resources needed to maintain a live colony, but also provides an opportunity to quickly restore a line and ensure ongoing research continuity.
