(a) What is the rationale of doing a slow freezing (controlled rate freezing)?
(b) Why a quick thawing of frozen vials at 37 degree C?
I read some research articles , but confused about crystallization and vitrification.
Please help me if you can explain the reason in a simple way !!
Cheers
Nimal
a)At present, glycerol or DMSO is used as a protective agent for cryopreservation of cells, which can improve the membrane permeability of water. The process of is that slow freeze intracellular water exudate by glycerol or DMSO to reduce the intracellular ice crystals. Thereby reducing cell damage due to ice crystal formation.
b)When recovery of cells, it should be quickly melted. So as to ensure extracellular crystallization can dissolve in a very short period of time, and avoid the water into the cells to form intracellular recrystallization damage to cells.
Hope can give you some assistance.
Good luck.
a)At present, glycerol or DMSO is used as a protective agent for cryopreservation of cells, which can improve the membrane permeability of water. The process of is that slow freeze intracellular water exudate by glycerol or DMSO to reduce the intracellular ice crystals. Thereby reducing cell damage due to ice crystal formation.
b)When recovery of cells, it should be quickly melted. So as to ensure extracellular crystallization can dissolve in a very short period of time, and avoid the water into the cells to form intracellular recrystallization damage to cells.
Hope can give you some assistance.
Good luck.
my understanding is that long thin ice crystals penetrate cell membranes and kill cells but the antifreeze effect of dmso frozen slowly is to generate more rounded crystals which do less damage. I think that the dmso is lethal to cells at high concentration and high temperature so when thawing cells we thaw in 37c water bath just until the ice just disappears then add culture medium while the mix is still cold so that as the cells warm they are now in diluted dmso which is less harmful. Often the medium is added in small amounts with mixing to avoid the osmotic shock of 10% dmso inside the cell and none outside so adding and mixing minimises the salt /dmso gradient across the cell membrane as the dmso leaks out
Microscope of cells show slow shrinking with quick thawl yields most viable outcome. Still with a loss of a good freeze thawl of 50% in dmso.serum freezer medium. It is not easy for them to go through.
Ice is lighter than water because the structure is a rigid matrix, in which air gets trapped. The rigidity of these crystals is what causes damage. Look at any tissue with swiss cheese ice artefacts and you will get a feel for that.
However, melting ice dissolves the crystal lattice and the fluidity of the water molecules removes the distortion, only of course, if the tissue has not already been permanently damaged or fixed in place.
I really recommend the BBC video on ice crystal formation. It gives you a good feel for how these processes proceed at the molecular level.
https://www.youtube.com/watch?v=3Qasw7lb2UM
Think about a plant wilting after a quick freeze overnight. The cell walls of a plant can become damaged and literally burst. Ice is less dense than water (reason ice floats)... the molecules spread out and form a lattice as they freeze and take up more space than the liquid phase. Mammalian cells do not have cell walls, but a quick freeze without the additives (DMSO, etc.) can wreak havoc on the cell membrane, which is needed for the cell to remain alive, along with distorting tertiary and quaternary bonding of proteins, etc.
Oh, and quick thaw because you don't want things growing, bacteria reproducing, living off the remains of the cells that were damaged from the freeze. There will always be some cells lost.
I think you asked this question in relation with tissue cell cultivation, freezing and re-freezing the cell suspension:
a) to protect the cells (not to be demaged by ice crystalization, not to burst by ice) you need to dehydrate them, and this dehydration is doing by slow freezing; the first ice crystals will be around the cell in medium, and in the process of slow growth of crystals, the cell will be slouly dehydrated (the water is going from the cell- to medium)
b) the "quick" is not so quick,
first, you have to use ice (water) and put the vials with frozen cell susension into melting ice (ice slush), the cell will be slowly thawing and slowly sucking the water inside, so they will reassembly vitals without cell demage. If you thaw them quickly, cells will be not in isotonic solution!!!! and therefor start to go to necrosis or burst (by hypotonic solution)
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