Cryoconservation. Everything you wanted to know about it but were afraid to ask.

What is cryoconservation? What is it needed for? How does it work?

Ice
Do you know why fresh meat is tastier than the frozen one? And why you can’t freeze a person and store them up till better days?
The answer to both questions is the same. Any cell and any tissue contains a large amount of water. At a temperature higher than zero degrees, water in an organism exists in the form of a liquid or in a special state resembling liquid crystals when molecules are densely packed around various macromolecules or ions forming something like a fur coat. The latter is called structured water and in accordance with contemporary ideas, the major part of water in any organism remains in this state. In any case, in both states water molecules are lined quite densely and do not form the so-called “long-range order” – a crystal structure or a large supramolecular pattern. The density of such water is close to 1.
When the temperature drops lower than a certain value (which is not necessarily 0°C), water, just like the majority of other liquids, starts to crystallize forming ice. Simultaneously, a crystal pattern appears being much bigger than the size of the molecules, hence a long-range order is formed. In contrast with the majority of other liquids, the density of ice is lower than the density of liquid water because the resulting crystal pattern is fairly lacelike.
However, despite the beauty and the laciness of ice structures (try to recall snowflakes or ice patterns on glass), they are absolutely detrimental to a cell. If we place a bottle filled with water to the top in a freezer, it will burst. Similarly, when intracellular water is crystallized, it tears the intracellular structures apart and kills a cell.
Which is why meat after defrosting is by far not the same product that it was before.

Protection against ice
Still, it is not the whole truth. Why is it then that a frog can freeze into ice, only to come back to life next spring?
There are special substances called “cryoprotectants” that hamper the formation of ice crystals. Their mechanism of action is quite simple: these are either large molecules making up a thick solution and functioning as “barriers” against the growth of ice crystals, or fairly small molecules replacing water inside a cell and thus preventing crystal formation: no water equals no problem. The former are the so-called “non-penetrating cryoprotectants”, the latter – “the penetrating cryoprotectants”. The former include sucrose, other sugars and ficoll, the latter – ethyleneglycol, dimethyl sulfoxide and glycerin (that can be placed in either group).
The frog makes use of glucose and glycerin. For human cells, these are usually not enough but one can find an optimal combination of cryoprotectants so that to sufficiently dehydrate a cell by means of penetrating cryoprotectants and to sufficiently protect it on the outside with the help of non-penetrating cryoprotectants. The problem forcing us to search for their optimal combination is the necessity to make the dehydration and the freezing reversible. Excessive dehydration of a cell may also prove detrimental: the intracellular structures stuck together in the absence of water may never recover. Besides, there are many toxic substances among cryoprotectants.

Cryoconservation in Biology and Medical Science
However, for some types of cells, an optimal cryoconservation method was found, namely, such a sequence of solutions that a cell, an embryo or a piece of tissue can go through and be frozen afterwards. At the same time, these solutions (1) protect cells from ice crystal formation fairly well (if cooling dynamics is proper) and (2) are not so toxic as to poison an embryo (or a tissue) during the manipulation.
A “sequence” of solutions means that a tissue or an embryo is at first placed in one cryoprotectant solution (for a strictly specified amount of time) and then into another, the more concentrated one, et cetera. This gradual increase in cryoprotectant concentration up until the final concentration (necessary for freezing) helps to avoid the osmotic shock phenomenon – a rapid irreversible damage to intracellular structures brought about by too quick a change in the concentration of ions and cryoprotectants.

There are two essentially different approaches to the freezing of biological objects.

Slow freezing
This is the first approach to ever appear. It showed good results in its time but is mostly abandoned today. During slow freezing, an object (that was held in a cryoprotectant solution for a specific amount of time) is placed into a capillary tube with a freezing solution and is gradually cooled down to the temperature of crystallization of this solution, and then somewhat lower. The solution reaches the so-called “overcooled state” when it is ready to crystallize in response to any external impulse. Then, by cooling the capillary tube in certain spots far from the object, we trigger an avalanche-like crystallization of the solution.
Try to freeze mineral water. You will learn that as more and more ice is formed, the remaining liquid becomes saltier and saltier. The reason behind this is that ice crystals mostly contain clean water whereas salts remain in the part of the solution that is still not frozen.
Similarly, as more and more ice is crystallized in a capillary tube with a cryoprotectant solution, the remaining part of the solution that is still not crystallized becomes more and more concentrated. Thus the concentration of salts and cryoprotectants around the object (an embryo or a piece of tissue) will increase.
As we have already seen, cryoprotectants prevent the formation of excessively large crystals. Hence ice crystals that eventually form around and inside the object will be so small that they will not be able to “spoil” either cells or intracellular structures.
The primary objective in this case will be to safely preserve the object and to quickly defreeze it when necessary so that the rise of temperature should not give small ice crystals enough time to recrystallize into large crystals that can damage cells.

Vitrification
This approach gives an opportunity to avoid crystal formation altogether.
The formation of a large crystal lattice, though quick, still takes time. If a liquid could be instanteneously cooled down to a very low temperature, it would freeze in a liquid, non-crystallized state forming something like glass. This is what this process is called – conversion into glass, or vitrification.
Vitrification of clean water is possible only in very exotic conditions (with the cooldown rate of 1 million degrees per second, or under a very high pressure). However, the addition of an optimal combination of cryoprotectants enables vitrification of the solution as well as of biological objects in it under normal pressure and with an attainable cooldown rate.
During vitrification, embryos (or tissues) are also successively held in several cryoprotectant solutions increasing in concentration, whereafter they are immediately placed into liquid nitrogen. At the same time, due to the combination of cryoprotectants and the partial dehydration of an embryo (a tissue), water in a cell does not have enough time to crystallize, and an embryo remains “as is”. The main aim is still the maintenance of stable temperature during storage (so that to avoid water recrystallization) and the most rapid possible defreezing.
When defreezing, one should again get rid of cryoprotectants as quickly as possible (yet gradually so that to avoid osmotic shock) and transfer an embryo into a normal cultural medium.

Modern cryoconservation
At present, we have fully functional freezing methods for sperm cells, egg cells and embryos.
Usually, it is slow freezing that is employed for sperm, with an average efficacy of 60-70%. Apart from cases of severe pathospermia, such efficacy for sperm is more than enough. There are also a few laboratories developing methods of sperm vitrification.
For egg cells and embryos (starting from the pronucleus stage to the blastocyst stage) we mostly use vitrification. Good vitrification media and strict abidance by the protocol enable the efficacy of almost 100% for embryos and 70-100% for egg cells. The efficacy of egg cell freezing – thawing largely depends on their initial quality. The same holds true for embryos, but in contrast to egg cells, unpromising embryos can be easily identified making it possible to refrain from their freezing altogether.
Still unresolved is the problem of the freezing of whole tissues. The difficulty consists in a much larger size of pieces that have a much longer equilibration time in cryoprotectants (which increases the toxic effect) and freeze much slower (which increases the risk of crystal formation). In addition, unlike embryos and gametal cells, thawed tissues must be transplanted immediately (which is associated with a number of complications and risks).
Still, there are methods of ovarian and testicular tissue freezing that although regarded as experimental seem to have a fairly high efficacy. These approaches are still studied at many laboratories around the world but are already employed in a number of leading clinics when the need arises.