An Overview of Cryopreservation


Cryopreservation is the procedure that makes it possible to stabilize the cells at sub-freezing temperatures. Many advances in this technology have led to the emergence of methods that allow for long-term, low-temperature maintenance of sperm, ova, and embryos.

Cryopreservation is an integral part of assisted reproductive therapy, and is vital for the preservation of fertility for cancer patients, particularly before radiotherapy or chemotherapy. Cryogenic storage appears to be the only proven solution that offers men a chance of having children in the future: cancer therapy and its adjuvants often lead to reproductive cell damage, resulting in subfertility or sterility due to gonad removal or permanent damage to germ cells. Even nonmalignant diseases such as diabetes and autoimmune disorders may lead to testicular damage. Cryopreservation is advisable in this regard.

For patients diagnosed with azoospermia (i.e. absence of sperm), cryostorage is also used to avoid having to repeat these procedures. Furthermore, cryopreservation is often performed in patients who want to freeze the semen sample preemptively to avoid inconveniences due to failed ejaculation. Finally, sperm cryopreservation is recommended to preserve fertility in men who have a history of exposure to potentially toxic agents that may interfere with sperm production.

There are two main conventional freezing techniques used in sperm cryopreservation: slow freezing and rapid freezing. The slow freezing technique consists of progressive sperm cooling over a period of two to four hours in multiple steps using a programmable freezer.

The manual method is performed by simultaneously decreasing the temperature of the semen while adding a cryoprotectant in a stepwise manner followed by submerging the samples into liquid nitrogen. The sample is cooled from room temperature to 5°C at a rate of 0.5–1°C per minute. Then the sample is frozen from 5°C to −80°C at a rate anywhere from 1–10°C per minute. Upon reaching -80˚C, the specimen is plunged into liquid nitrogen at −196°C.

Rapid freezing technique requires contact between the samples and nitrogen vapours for eight to ten minutes followed by immersion in liquid nitrogen. The sample is mixed in with an equal volume of cold cryoprotectant; the mixture is loaded into straws and left to incubate at 4°C for 10 minutes. The straws are then placed at a distance of 15 to 20 cm above the liquid nitrogen level for 15 min. After this stage, the straws are submerged.

Cryopreservation of sperm requires the use of cryoprotectants: chemicals that are highly permeable and have low molecular weight. These are used to protect spermatozoa from damage from ice crystallization. Glycerol is the most widely used cryoprotectant for human sperm.

The thawing procedure is an equally important step: the cell must be allowed to recover its normal biochemical functions while avoiding abrupt thermal changes. The cryopreservation protocols use a thawing temperature of 37°C even if higher thawing temperatures allow for more rapid heating. The risks associated with higher temperatures can lead to cell damage. Once the semen is thawed, it is separated from the cryopreservation medium by washing in culture medium and centrifuging.