Dear Arvind, interesting question. First of all, it just doesn't work without additional procedures. Under normal circumstances the water crystals that form during freezing would destroy all organelles and membranes (not the increased volume, which is around 10% and easily sustainable for a flexible membrane). So, we have to give a freeze protective agent, in most cases 10% DMSO, so that only very, very tiny water crystals form. It's important to add the DMSO stepwise not to create an osmotic shock. We also need to lower the temperature stepwise, around one degree Celsius per minute (until -80 degrees), again to prevent crystals from forming. If one would leave the cells now at -80 degrees after two to four weeks (depending on the cell type) only very few cells would still be viable, since at this temperature still some biochemical processes work also very, very slowly. Thus, we put the cells into liquid nitrogen or another freezing device to lower the temperature to around -160 degrees. Now that said, if the temperature changes regularly in your liquid nitrogen tank or cells are taken out from time to time (maybe to look for a tube) and put them back in, the viability rate drops significantly since crystals will form. Obviously, when the cells are thawed again, it should be done relatively slow (not as slow as freezing) and the DMSO concentration should also be lowered stepwise. Hope my answer helps you something. You will find good protocols for freezing and thawing cells in any good lab guide. Daniel

Dear Arvind, interesting question. First of all, it just doesn't work without additional procedures. Under normal circumstances the water crystals that form during freezing would destroy all organelles and membranes (not the increased volume, which is around 10% and easily sustainable for a flexible membrane). So, we have to give a freeze protective agent, in most cases 10% DMSO, so that only very, very tiny water crystals form. It's important to add the DMSO stepwise not to create an osmotic shock. We also need to lower the temperature stepwise, around one degree Celsius per minute (until -80 degrees), again to prevent crystals from forming. If one would leave the cells now at -80 degrees after two to four weeks (depending on the cell type) only very few cells would still be viable, since at this temperature still some biochemical processes work also very, very slowly. Thus, we put the cells into liquid nitrogen or another freezing device to lower the temperature to around -160 degrees. Now that said, if the temperature changes regularly in your liquid nitrogen tank or cells are taken out from time to time (maybe to look for a tube) and put them back in, the viability rate drops significantly since crystals will form. Obviously, when the cells are thawed again, it should be done relatively slow (not as slow as freezing) and the DMSO concentration should also be lowered stepwise. Hope my answer helps you something. You will find good protocols for freezing and thawing cells in any good lab guide. Daniel

Dear Arvind,

The reason behind slow freezing is not just to minimize harmful ice crystal formation but also (and equally importantly) to allow water to leave cells. Hence, most of the water will exit the cells. Secondly, we always add a hydrophilic cryoprotectant (such as 10% DMSO as mentioned Daniel) which helps in sequestering water. Thirdly, I do not completely agree with Daniel's statement that thawing should be done relatively slow. In fact, if i am not wrong, thawing should be done rapidly.

As far as your second question regarding molecular mechanism is concerned, I guess molecules are not active in liquid nitrogen and hence there are no issues of molecular mechanisms. Molecules just stop working and there is nothing in liquid nitrogen that could permanently inactivate them.

Thanks,

Ishan

Interesting! I agree with Ishan, thawing should be done rapidly. I do it holding the tube in my hands.

Manuel de Miguel

I also used to thaw cells from the liquid nitrogen rapidly, by holding the tube in the 37 degrees waterbath. After thawing I take the cells up in medium with serum and use this for washing a couple of times to get rid of the DMSO before plating the cells. I used to freeze lots of human primary cells straight after cell isolation (so very early passage number). I got best results when using 10% DMSO and 90% serum as a freezing medium.

Thank all for your valuable feedback. I am still trying to understand the thermodynamics which comes into play in liquid nitrogen.....

Maybe you guys are right about the fast thawing. Anyhow, I just always had the feeling that I had higher viability thawing the cells at rt and not at 37 degrees or in hands. Once thawed, I agree that the DMSO should be washed out with warmed medium several times, to get rid of the DMSO.

I suppose I took the middle road by allowing the cell suspension to thaw briefly (enough for the 1.5 cc block of frozen suspension to move in the cryovial) either in my hand or by holding in a 37C water bath. I then pour the block of frozen suspension directly into 10 ml media + 10% FBS. This enables the DMSO to be quickly diluted as the cells thaw. The cells are then pelleted and resuspended in the appropriate media for plating.

you cant store cells untill unless you add cryoprotecant.

that serve as a protector prevent them from dying

Water can exist in liquid, crystal or amorphous state. Addition of DMSO or glycerol prevents crystallization of water thus preventing cell damage. Cell damage during crystallization of water occurs because volume of crystalized water is larger than the volume of the same mass of liquid water. I you fill a bottle up to the top, close it tight and put it in a freezer than next morning you will find this bottle broken. The same occurs with cell when water crystalizes inside of cell.

One little addition. DMSO or glycerol forms hydrogen bonds with protons of water thus preventing formation of regular structures (crystal) . These compounds can migrate through cellular membrane because they not charged.

I always heard that there are two types of water solidification. The slow one makes cristals that can kill your cells. The really fast one should solidy the water in a way that it resembles glass, almost a vitrification. This kind of freezing don't result in much volume increase, as the molecules don't have enough time to rearrange (as they do when the freeze normally). However, I think we don't have ways to freeze cells using the second method yet (maybe liquid nitrogen straight?). But maybe someday, we can do that. This way I don't think there will be the need to add any cryopreservant.

About the molecular mechanism: protein enovelation is a process in which the polypeptide chain try to achieve a state of lowest energy possible (thus more stable). Generally, all proteins do that espontaneously (except the ones that need chaperons, or other ways of folding). I guess that when the temperature falls, they either maintain themselves that way (since this is already the state that requires less energy), or can change to another way, that would be automatically reverted when the temperature rises (since it is entropy dependant). Of course, that would be defects, some proteins may never return to the original state. In this case, if the protein is too important and can't be replaced in time, the cell die. And you always have cells that die during thawing. But in those that the proteins fold correctelly, or the protein can be replaced in time, the cell survives.

Dear Arnind,

I frequently Used the -80C procedure to Freeze the cell lines and Primary cell cultures also from PDAC (Pancreatic Ductal Adenocarcinoma). I left more than 2 years the cells in -80C refrigerator and I had not the problem. So far I would like suggest you to reduce the amount of DMSO (only 5%) of total volume. Using this procedure is it possible to observe the attached cells after 24h. Unfortunately, I have not experience with the procedure as well you used. However I agree with Luiza about the vitrification using the water. The ice could be dangerous for the surface of the cell.

Good luck. Niccola

Also, regardless of all the treatments discussed above, much of that 80% of water is weakly bound to/mixed with other molecules, thus notnecessarily available for large crystal formation. So the cells may have some limited tolerance to freezing damage of their own. Cells with vacuoles might be an exception.

Mammalian cells (in 10 % DMSO or 15% glycerol) can be stored in liquid nitrogen (-169 deg C) vapor or liquid for decades with negligible loss of viability. I have thawed 50 year old cultures and retrieved them. When I repeated experiments on them, the same results were obtained. All cell lines should be stored in liquid nitrogen with aliquots (vials) being removed to re-establish the line approximately every 3-6 months, and new stocks grown and frozen from the thawed cells (to maintain the frozen stock). This prevents genetic drift that otherwise lead the same line in different labs to diverge from each other or to make your experiments unrepeatable years later. This is one of thew first principles of running a cell culture lab.

I think your question is genuine and should be answered carefully but it is not the case. I think how particular organs or tissues remains live when we stored it for long time under different condition or what is the minimum energy requirement of a cell to be in quiescent phase without being dead....i dont think any have answered it.

Besides the freezing procedures: A lot of cells doesn't survive this procedure. You freeze ways more cells, than finally get back in culture, my guess would be 90-95% doesn't survive the treatment for various reasons. Problems during the freezing (too fast freezing), cells doesn't tolerate DMSO too well, wrong thawing can cause this. And this freezing and reviving is only possible to single cells, whole organs suffer too much damage.

I would like to add a important things to the other answers listed above to your questions;

1. DMSO is widely preferred for animal cell cryopreservation and glycerol is used for bacterial cell cryopreservation.

2. It is important to preserve cells that are growing in the log phase (75-80% confluence in mammalian cells) otherwise revival percentage will be very less.

3. While thawing,

(a) bacterial cells need to be thawed slowly. Because rapid thawing will lead to aggregation of the cells and they wont get resuspended properly in the culture.

(b)animal cells need to be thawed very rapidly (At 37C) and immediately after taking out of liquid N2 tank or - 80C freezer. Because DMSO is toxic to cells and slow thawing will allow the DMSO to enter the cells and cell kill them.

4. While freezing, bacterial cells can be frozen rapidly but animal cells should be frozen slowly. This is because, bacteria are slight resistant to freezing due to their cell wall and presence of some compatible solutes, but rapid cooling of animal cell will lead to intracellular ice crystal formation and kill the cells.

You can read the following articles also for more info:

http://staging2.orthofix.com/ftp/assets/Product/Product_Files/Trinity_Evolution/Mazur.pdf

http://catalog2.corning.com/Lifesciences/media/pdf/Cell_freezing_protocol.pdf

All the best!

@Ganeshkumar: You can use DMSO also very well for freezing bacteria. It has a number of advantages above glycerol.

@Christian: DMSO can also be used to freeze bacteria. But at room temp DMSO has high toxicity and has bactericidal activity against some bacteria. So it is better to use Glycerol for freezing bacteria since it has less toxic to bacteria when compared to DMSO at room temperature.

This is an old conversation, but I should add that for mammalian cells, in 10% DMSO, the optimal freezing rate is -1 deg C/minute. Your cryobank can be calibrated by placing a vial (same size as you will use for your cells) containing water or better, the same medium (with DMSO) that you will use for your cells, and a thermocouple probe in the same location in the freezer neck as you will use for freezing the cells, and then measure the temperature as a function of time. Move this position up or down, as need be, to get a cooling rate of -1 deg C/minute from room temperature down to -20 deg C. That gets the cells through the ice point which would otherwise (without the cryoproctectant (DMSO)) lead to ice crystal formation tearing the cell's membranes and killing the cells. The cells should be healthy, in mid-log phase of growth at the start of the procedure. With fast thawing, recovery rates in the 50% to 90% range have been obtained (only slightly lower than the unfrozen cells' plating efficiencies). Upon thawing, dilution of the ca. 1 ml of thawed cells into 5 to 15 ml of complete medium for growth (in T25 or T75 flasks) suffices to dilute the DMSO to non toxic levels.

Arvind Kumar Daniel Besser Ishan Deshpande

I am embarking on a research that requires collection of oral fluid samples, this samples after the first round of work should be stored for atleast 6months for the next phase of work.

Unfortunately the sample collection device I am using for this does not come with buffer or transport media. How do I extract the sample and store for a relatively long time from the collection device? Christopher Larbie