Cryopreservation of samples.

The use of Liquid Nitrogen to store animal cells dates to the late 1940’s. Initial studies involved experimenting with various excipients known as cryoprotectants, to enable preservation of poultry semen. These experiments led to procedures for cryopreservation of bull semen; a commercial breakthrough allowing breeders access to semen from preferred bulls for selective breeding programs, something previously impossible due to the lifespan of unfrozen sperm.

It is hard to imagine that those early innovators would have envisaged the huge potential that cryopreservation now provides in helping preserve endangered species around the world. A recent article in “Reproduction & Fertility” publication outlines how these techniques could help overcome the largest predicted loss of biodiversity in 65 million years, with 41% of amphibians, 26% of mammals and 14% of bird species assessed by the International Union for the Conservation of Nature (IUCN) being under threat of extinction.

Here is where cryopreservation and biobanking can aid species survival. Habitat restoration alone is not enough as some species have dropped to levels where population clusters are fragmented, leading to unsustainable populations with low genetic diversity. Breeding programmes that increase gene diversity by introducing new genetic information to a different region, however, transporting animals between locations for breeding purposes has its challenges including logistics, animal welfare, as well as the potential for disease transmission. Cryopreservation (freezing cells with cryoprotectants enabling long-term viable cell and tissue storage), followed by cryobanking (indefinite storage of viable cells and tissue in liquid nitrogen at −196°C or ultra-low freezers) and assisted or advanced assisted reproductive technology (ART/aART) can save the genotypes that are being lost today without the issues involved in transportation of live animals.

When looking at species preservation using induced pluripotent stem (iPS) cells, which can be differentiated into oocytes or spermatozoa, we can use the rhinoceros as an example (Fig. 1). After a biopsy of a recently deceased animal, cells can be cryopreserved until the moment they need to be differentiated in order to create oocytes or spermatozoa. Those differentiated cells will first need to be reprogrammed to obtain pluripotency. Afterwards, growth factors can re-differentiate the cells into the desired cell population (oocytes or spermatozoa). In vitro fertilisation will result in viable embryos, which can be transplanted into a surrogate mother leading to offspring.

Figure 1 - Species Preservation using iPS Cells (1)

The article (1) outlines the many variations in freezing techniques and cryoprotectants used, the same can be said for the cryogenic storage systems. In cryogenic terms the general rule is ‘the colder the better’ and preserving material below the Tg of your product is crucial for their future viability.

However, there are other areas to consider such as cross-contamination and container leakage so choosing the correct vessel to store valuable and sensitive samples in is paramount. Not only is the safety of the material important but also considerations of the safety of the users, our article on “Considerations for your storage facility” (2) details some of these areas.

Many facilities also operate shared storage containers, meaning access to your valuable samples should also be a consideration. Security in some facilities has been an issue in the past, therefore ensuring only authorised access has become an increasingly important factor. The new “Revolution” range of automation ready cryostorage systems provides this feature with PLC controllers acting as local inventory management system. Samples can be identified within the controller and access to segments within the freezer lockable through a password protection system, thus offering piece of mind that your samples (up to 94,000 vials) are stored safely.

To discover more, speak to our product specialists

The extinction assessment by the IUCN is a sobering and thought provoking report, but now after more than 80 years of development and innovation, cryopreservation has reached a level where it now has an invaluable role to play in the conservation of endangered species.

References:

1: L Boulton, A. Mooney et al 2022, Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking. Reproduction and Fertility 3, 3 (https://raf.bioscientifica.com/view/journals/raf/3/3/RAF-22-0005.xml)

2: I. Blackham, G. Strongitharm 2021, Consideration for your storage facility. (https://cryopreservation.co.uk/2021/02/11/considerations-for-your-storage-facility/)