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Cryopreservation refers to a process of preserving living cells, tissues, organs or other biomaterials in an ultra low temperature environment. Under cryogenic condition, chemical and biological activities within the living cells are dramatically reduced, and thus biomaterials can be kept structurally intact for an extended period of time. However, successful cryopreservation requires optimization of multiple factors. Any inappropriate handling can lead to cryoinjury, which ultimately leads to low viability or even unsuccessful recovery.

Cryopreservation, or low temperature preservation, was first studied in the early twentieth century. Starting from 1930, a Catholic priest and biophysicst named Basile Luyet started to study what he believed to be “latent life”[1], a form of suspended animation. He said:

“If the coefficient of decay in storage is of the same order as that for several enzymatic reactions and if the principle holds at very low temperatures, the decay which takes place in 3 weeks in material stored at +2 °C would take 15 years at -78 °C (in a bath cooled by dry ice), and some 53,000 years at -196 °C (in liquid nitrogen).” [2]

Basile Luyet was considered to be the “father” of cryobiology by many [3]. Although what he said sounds reasonable, the freezing process is very often lethal to living things. That’s because many biological structures and processes are temperature dependent. One theory, the solution-effect theory, explains that when we try to freeze some tissues (which can contain 80% of water) the liquid will have an increasing concentration of solutes, as the water becomes ice. This can kill cells. Even though low temperature can suppress biochemical activity and help to “suspend” life, the cooling process is a dangerous journey and the cells might have been destroyed before they reached the safe temperature.

In 1949, Polge, Smith, and Parkes published their important paper [4] which showed that the inclusion of 10%-20% of glycerol enabled the spermatozoa of the cock to survive prolonged freezing at -80°C. Later in the 1950s, in a series of classical papers[5] [6], James Lovelock gave strong evidence that it was the salt concentration, rather than ice, that causes cryoinjury to cells. Glycerol can protect cells in the cooling process because it balances the rise in salt concentration. The discovery of glycerol leads to the adoption of other cryoprotectants. Some might work better than others in different cell types. They include dimethyl sulfoxide, ethanediol, propanediol and others.

Yet it seems there are more factors than salt concentration that affect cryopreservation. The cooling rate and warming rate also matter. In 1963, Mazur published a paper, explaining that the rate of temperature change was important because it controlled the transport of water in and out of the membrane[7]. Warming rate is crucial for a similar reason: if the sample is warmed too slowly, ice can form again and the high concentration of salt would damage the cells.

As the technology of freezers improved, and the preservation process got simplified, its application gradually became wider and wider. In the 1960s, it started to be used in different fields, such as cattle breading to genetics, physical anthropology, biomedicine and public health [8].

As ultra low temperature devices became more accessible and reliable, anthropologists even set out to collect bloods from tribes in places like Africa, Central America, which later facilitated the research and understanding of deadly diseases such HIV/AIDS and Ebola [9].

In the past few decades, many biobanks and repositories have been established in different areas around the globe. They were created to collect and accumulate precious biological samples, which can provide hints on what causes different diseases, who might be at risk, and how to cure them. Samples quality matters, and therefore, highly developed and sophisticated cryopreservation technology is much needed.

However, despite of the fact that people recognize the importance of cryopreservation, its current process is still similar to that of 1960s, which is plagued by uncertainty and unreliability. Cryopreservation is widely used, but not mastered. There are still many, if not most of researchers, who try to freeze their samples without really understanding what happens to them in low temperature, and pray that they can be revived decades later.

In Fibulas, it is our goal to understand cryopreservation fundamentally, and develop new technologies to reduce the uncertainty and unreliability. We strive to improve the recoverability of samples, and help life scientists to discover more from their precious collections.

Preserving and reviving cells should be based on science, not luck.