Cryopreservation: The Basics


Cryostorage of cells, in its simplest form, involves several steps. Because cells are mostly water, and water when solidified can form ice crystals that can damage the cell, the first step is to dehydrate or remove some or most of the water from the cell. The next step involves addition of a cryoprotectant, usually an alcohol, that will enter the cell and bind to the remaining water, so that it cannot organize into ice crystals when cooled to solid form. Next, the cells are cooled until everything is solidified, usually below -80°C, and then stored in liquid nitrogen (-196°C). When it comes time to use the cells again, they must be thawed or warmed. This is done relatively quickly by submersion of the container with the cells into a water bath. The cells rapidly warm to near room temperature at which point they are rehydrated and the cryoprotectant removed through a series of wash steps.


Slow-Cooling:

The first theoretical basis for cryopreservation of cells was proposed by Mazur, 1965 and later applied to mouse oocytes (Mazur et al., 1984). For example, human embryos are exposed to a simple salt solution containing a permeable cryoprotectant (1,2-propanediol) and usually, a low concentration of non-permeable cryoprotectant (sucrose). After a brief exposure time to allow uptake of cryoprotectant, the cells are cooled rapidly to a temperature slightly below the melting point of the solution (usually around -7°C). At this point the container with the cells is “seeded” so that ice forms in the extracellular solution. Basically, to obtain seeding, metal forceps are cooled in liquid nitrogen and then touched to the outside of the container with the cells. This cools the media in the container around the forceps and causes ice to form. This localized spot of ice then grows like frost on a window. Upon further cooling (slow cooling to below -30°C), the osmolarity (concentration of salt in the solution) of the extracellular solution increases as water freezes out as ice. With the increasing salt concentration, the cells dehydrate further. Dehydration continues during slow-cooling until the cells are plunged into liquid nitrogen, usually at a temperature below -30°C. At this point (the cells are still in liquid form) the intracellular cryoprotectant concentration is high enough that the remaining intracellular water will vitrify, preventing intracellular ice formation. During thawing, the dehydrated cells are rehydrated along with cryoprotectant removal.




Rapid-Cooling (Vitrification)

All of the rapid-cooling forms of vitrification procedures for human oocytes and embryos described in the recent literature are, in principle, the same. They all involve exposure of oocytes or embryos to high concentrations of cryoprotectant(s) for brief periods of time at or near room temperature followed by loading onto or into a tiny container (cryo-loop, cryo-top, cryo-leaf, cryo-tip, etc.) that may or may not be sealed, then submerged directly into liquid nitrogen and stored. The high osmolarity of the vitrification solution rapidly dehydrates the cell, some of the cryoprotectant enters the cell and binds the remaining water, and submersion into liquid nitrogen quickly solidifies or vitrifies the cell so that any remaining intracellular water not bound by cryoprotectants, does not have time to form ice crystals. The embryo is effectively vitrified without intracellular ice, similar to slow-cooling. From these descriptions, both techniques (slow-cooling and rapid-cooling/vitrification) although seemingly very different, have the same outcome of vitrifying the cell.

Compared with slow-cooling, rapid cooling vitrification has allowed for improved survival and pregnancy rates. Reasons for this are numerous, despite the fact that both procedures vitrify the cell. The methods are different enough that, despite posing a greater risk from the potential toxicity of the highly concentrated cryoprotectants used and the relatively high exposure temperature, rapid-cooling has met with greater success in most instances. To combat the toxic effects of elevated cryoprotectant levels (mainly DMSO), exposure to the final vitrification solution is usually limited to around 45-90 seconds or less before plunging in liquid nitrogen. Also, to promote faster solidification, minute amounts of vitrification media are used, usually under 2ul. For example, when using the cryo-top device, the cell(s) are placed on the tip and excess media is removed leaving the cell(s) covered in a very thin film of media before plunging directly into liquid nitrogen. This allows for an extremely rapid cooling rate of over 20,000°C/min. However, this technique is, at times and for some people, difficult to perfect.




Large-Volume Vitrification (ICE Vitrification):

There is another method to obtain a vitrified cell. It is very similar to the rapid-cooling method described above. However, this method uses different cryoprotectants and no DMSO. Dr. James Stachecki developed this method of vitrification numerous years ago. The main differences are that a normal 0.25cc straw can be used and rapid cooling rates were not necessary. There is plenty of time in 3 vitrification solutions for dehydration and uptake of cryoprotectants to occur before plunging and storage into liquid nitrogen. The principles are the same, as described above. The benefits are that a more relaxed protocol and larger container eliminate the difficulties associated with using tiny containers and micro volumes of solution, as in the rapid-cooling method described above. Since most everyone used 0.25cc straws during slow-cooling, many labs are familiar with these inexpensive and easy-to-use storage devices. This method offers a third option for vitrifying oocytes and embryos. More information on "ICE vitrification" can be found elsewhere on this website.