At first glance, it seems paradoxical: living cells that are frozen at -196°C can be viable and functional after thawing. How is this possible when the water inside them turns to ice - a material that is known to form sharp-edged crystals and whose expansion can burst pipes?
The answer lies in cryobiology - the science that studies the effects of low temperatures on living organisms and cells. Since the groundbreaking discovery that glycerol as a cryoprotectant can protect sperm from freezing (Polge, Smith and Parkes, 1949), cryobiology has developed into a discipline in its own right, the findings of which form the basis of all successful cryopreservation.
In this article, we offer a clear introduction to the basics of cryobiology - the foundation on which the entire cryotechnology of Consarctic® is built.
Water makes up 60-90% of the volume of a living cell. If this water freezes uncontrollably, ice crystals form - and these are deadly for the cell.
If ice crystals form inside the cell, they penetrate the sensitive cell structures: the cell membrane, the mitochondria, the endoplasmic reticulum and the cell nucleus. This leads to irreversible damage and cell death.
If ice crystals form outside the cell, they draw water from the environment. The resulting increase in the extracellular salt concentration (osmolarity) draws water out of the cell - the cell dehydrates. With moderate dehydration the cell can survive, with excessive dehydration it collapses.
In the 1960s, cryobiologist Peter Mazur formulated the influential two-factor hypothesis, which still forms the conceptual foundation of cryopreservation today:
If a cell is frozen too slowly, it has too much time to release water to the growing extracellular ice crystals. The resulting extreme dehydration and high intracellular salt concentrations damage the cell membranes and denature proteins.
If a cell is frozen too quickly, the water does not have time to leave the cell. It freezes inside the cell and forms destructive intracellular ice crystals.
Between these two extremes lies the optimal cooling rate - the range in which the cell is frozen slowly enough to avoid intracellular ice crystals, but fast enough to minimize the harmful dissolution effects. This optimal rate is different for each cell type and must be determined experimentally.
Cryoprotectants (CPAs) shift the balance in favour of cell survival by reducing the formation of ice crystals and stabilizing the cell membrane. DMSO, glycerine and trehalose are the best known representatives.
Vitrification completely avoids the problem of ice crystal formation: Ultra-fast cooling in combination with high CPA concentrations transforms the water directly into a glassy (amorphous) state without passing through the crystalline state. No crystals mean no mechanical damage.
Vitrification is now successfully used for the preservation of oocytes and embryos in IVF and is an active field of research for organ preservation.
The theoretical findings of cryobiology would be worthless without the right technology. A controlled rate freezer such as the BIOFREEZE® from Consarctic® translates cryobiological theory into reproducible practice:
Every piece of equipment and every process in a cryobank is based on the principles of cryobiology. Understanding these principles allows you to make more informed decisions about freezing protocols, storage methods and equipment selection.
Would you like to find out more about the scientific basis of your cryopreservation? Our application scientists are available to answer technical questions and optimize protocols.