Freezing and thawing of cells - (Sep/09/2004 )

If you have an adherant cell line or any cell line for that matter which has poor viability when thawed sometimes it helps to plate it in a small space such as a six-well plate. This means that even if you have poor recovery the chances that some cells will survive and grow out are increased. Also if your cell line is adherant you can plate onto a collagen or gelatin coated dish ... this helps sometimes too.

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I've been working with NIH 3T3 fibroblast cells. generally speaking i agree with anil about using glycerol in freezing media. i hav'nt had a problem with freezing them for longer than a year. but perhaps this should also be viewed in terms of which cell line is being used?

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hi shilipi,
since you are not able to get any viable cells after thawing,
i would think about following issues,
1, freezing conditions(physical) are not supporting their survival
2, DMSO present in the medium might be toxic for your cells
3, u can try to supply more nutrients to cells before freezing or after thawing.

i dont doubt spinning because if there are any viable cells after thawing they should get setteled by spinning, i prefer 1000 rpm speed,
if there are no viable cells after spinning, concentrate on 1 and 2 isses. becasue cells already died because of one of the above reasons.
incase cells start dying after 12 hours then try to concentrate on supplying additional nutrients 20% of FCS for culturing.
according to my knowledge (1 or< 1%)DMSO enhances the growth for some extent. give a try with and withour DMSO.
inconclusion
for the first issue u can use isoproponol croyo box.
for the second issue u can use 5%DMSO in freezing medium instead of 10%
for the third issue use 10% DMSO and 90% FCS as freezing medium.

i hope this will help
cheers
sravan.

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Hello,

In my experience, most viability issues that arise after thawing can be remedied by freezing down an absolute ton of cells in each vial. When I freeze cells (which is a pain and I don't like to do very often) I freeze many vials that are very heavy. I take T150 flasks (usually at least two per cell line I am freezing) and wait until they are in log-phase. The flask is usually 80% confluent. You want to make sure they are ramping up growth wise and not too confluent. Then when they come out of the freezer they will still be in that mode. I change the media 24h before I plan to freeze the cells.

I freeze about 5 cryo vials per 150 flask. If it is Friday and there are not as many cells as I would like, I use less vials. The important part is lots of cells. I freeze in regular complete medium with 10%FBS and 7.5% DMSO. No antibiotics. I keep the cryo tubes on ice while I am working and keep everything as cool as possible. Then aliquot the cells out into the tubes (on ice) and put into -80C. You want them to cool at the rate of 1 degree per minute. I usually leave them overnight or a couple days if I forget about them.

The reason you cool the cells slowly is that between 0 and -20C ice crystals begin to form increasing the solute concentration of the medium (which is why I use complete medium to freeze in, it is osmotically balanced). As this happens, water moves out of the cells, dehydrating the cells and shrinking them. If you cool to rapidly, ice crystals can form inside the cells since the water has not had time to move out. Then the cells will die when you thaw them. Although if the cooling is too slow the cells will suffer from the dehydration and die when you thaw them. Mammalian cells have a very narrow window for freezing, which is why cryopreservation agents are used. These don't really effect ice crystal formation, they just protect from the dehydration effects.

Once the cells are moved to -130C or below, time essentially stops for them since that is the point where water is like glass. No biological processes occur. Storing cells at -80C still allows cellular damage and changes to occur.

Thawing: I thaw at 37C, but don't let the cells get that warm, when there is still a bit of ice left, move to a tube of media. I pellet to get rid of the DMSO, but I only pellet at 200xg. Then plate, change media in 24h, split 24h after that.

A note on cryo tubes: It is dangerous to store tubes with no gasket in liquid nitrogen. They are only designed for the part of the freezer where there is only gas. If liquid nitrogen gets inside these tubes, they can explode. I currently have tubes like this in the liquid nitrogen, so inorder to save valuable cells, I immediately loosen the caps on these tubes when I take them out. Not very far, just enough for them to hiss a bit. But don't tighten the cap back down, that can force crap into your vial.


Sorry for the super long post. Hope this info helps,
Beverly

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are u dumping the tube of cells straight in2 37 or u put in for 20-30seconds at a time?

the other way which ive found it better way take out from N2 and leave on bench to self thaw.

doesnt take that long to thaw say 3mins

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Hi

Cells are sometimes very sensitives.
Freezing : 50% normal growing medium / 40% HI-FBS / 10% DMSO. Heat to 37°C, ressuspend cell pellet in it, put vial in isoprop-containing cryorack, put at -80°C for 24h (the T gradient should be 1°C / min and not more...
Thawing : Keep vial in your hand or water bath for 5' (not more). When "icecube" is 1/2 of volume, add hot medium, and plate the cells (try a smaller flask to reach confluency faster. It might be your problem).
Increase (2X) FBS concentration, remove all antibiotics, and change the medium the next day (to remove dead cells). If it doesn't work, your stock might be the problem. Trash and buy new...
Maybe check for mycoplasmas...
Hope it helps...

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Hi
I am having trouble getting thawed cells to grow, and I read your post in the BioForum, and decided to email you to ask a specific question. You mentioned that one of the ways to increase the chance of cells surviving thawing is to try a smaller flask in order to reach confluency faster. I normally use a T75 flask to grow a thawed 1mL vial of cells (CHO, HEK, etc). Would you know is it OK to thaw in a T25 flask? The surface area will be significantly smaller, but I was worried that it might be too crowded for the contents of the usual 1mL of frozen cells. So my protocol is thaw and put cells into warm media, then I change the media the next day to get rid of DMSO. Works well, but not recently. Thanks.

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Hello,

I thaw my cells into a smaller surface area flask when there are less cells than usual. When I thaw, I put the 1mL of cell suspension into complete media and pellet the cells (a wash essentially) at low speed (200xg) then remove the supernatant and resuspend in fresh complete media. At that point you can tell how many cells there will be based on the size of the cell pellet. If the pellet is small or I know that the cells have spent a while in -80 or someone not as trustworthy had frozen them down, I will plate into a small flask, because I know there will either be a lot less cells than usual or many more will die than usual. They just seem to do better with the decreased volume. Less time to condition the media, etc. The cells need buddies that are pretty close spatially when you thaw them.

Hope that helps,
Beverly

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I learned that if cells are preserved in DMSO, the temperature and osmolarity are critical. Here is is what was recommended to me:

Thaw cells by placing tube in a 37C water bath-thaw until cells just reach a liquid state. Then with cold media, start doubling the volume slowly: 1 ml of cells to 1 ml of cold media, 2 mls cold media, 4 mls cold media, etc...until you get the desired dilution for a flask. Gradually increasing the temperature and osmolarity works well for the most sensitive cells-hybridomas are supposed to respond well to this method.

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Sorry, I am an big blue apple.It is not wise to offer assistance at my level.but I still want to say that not so sad ,maybe luck deside every thing
try more, luck will be prent

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