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

hi shilpi

Though my experience of handling the cells is less than 2years. I would like to suggest you some things. Even though if you leave DMSO in the media after thawing the cells the dmso in low cocentration is not toxic 2 cells more over once you dilute the small volume of cell suspension in 5-10 ml of media, it further dilutes the dmso. It looks to me like the protocol you are following may be causing the cells to die, I think you recognise the fact that centrifuging at high speed can lead to cell necrosis. One method is to lower the speed of centrifuge maybe 900-1000 rpm for 4-5 min should be adiquate.
Alternatively you should also consider the method you are using for preserving the cells. If you freeze the cells directly in liquid nitrogen this can also lead to cold shock induced cell lysis or damage the membrane, one way of preventing this is gradually lowering the temperature such as storing 4 degrees for few hours then transfering to -20 degree for overnight and then storing in -70 degrees for an couple of days then transfering to liquid nitrogen tanks, by following this method u r not only making the cells to adjust to the lower temp you are also ensuring that they donot undergo cold induced necrosis leading to cell membrane damage and lysis.

I hope this information is of use to you.

If you need any further information you can mail me at orexin2003@yahoo.com

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I dont agree with preethi or anil as one cannot preserve the cells in glycerol for more than 6-8 months.

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I agree with Anil.
Biomed

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I am experiencing similar problems with my LCL b cell line. See topic title:

LCL B cell culture - Death within 24 hours of thawing.

I might try some of the suggestions here.

Andrew

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This is how to freeze and thaw cells:

Always check the cell viability before freezing. They should be highly viable: about 95%. keep your freezing media ( 10% DMSO in FBS) cold. Centrifuge cells for 1000 RMP for 5 minutes. Prepare labeled cryogenic vials. Cell concentration should be about for e.g. collected from a 50 ml flask, about 50 million cells. You can have 10 vials and about 0.5 milion cells per vial. After you centrifuge, get rid of media completely, gently tap the pellet to make it loose. Add 5 mls of cold freezing media resuspend with a pipet and transfer 0.5ml to each vial. Close the cap tight and place cryogenic tubes in special cryogenic container that has alcohol at the bottom and cool down gradually, they usually hold up to 20 vials. Close the top and transfer the container to -70 freezer. Wait 24 hours and no longer than 15 days before transferring them out of the container into the liquid nitrogen boxes.

To thaw the cells, be very quick, take the viall out still with some liquid nitrogen, walk to the water bath. Take the vial, make sure the cap is very tight, sometimes it becomes loose. thaw the vial holding the opening upward so the water from the water bath does not contaminate cells or if there is some detegent in the water it doesn't become in contact with the inside of the vial . When frozen cell media is almost half thawed (about few minutes), take it under the hood and add 0.5 warm media to the cells and immediately transfer to a flask with about 10 to 15ml warm media in it. DMSO will be diluted and you can check the viability.

Good luck and let us know if it had worked.

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Hi all,

When I went through top to bottom of this page, it remind me something I could tell in general

As the wise words (Auguste Rodin) say
"Nothing is a waste of time if you use the experience wisely"

We have -85 Deep Freezer for cell storage in our lab. I got normal revival of cells even after an year, when I preserved them in glycerol. Those were cell lines, not primary cells. The case may be different with primary cells. Yet to get experience with it.

Anil

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The protocol described by soundi is the same as i use and it works well.
Highly suggested...

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Hi,

I work with cells not more than half year, but I did met with the similar problem with you. And it's strange that I did all the operation right: thaw quickly, no shaking, and no bubbles when adding medium. But still, I can't get the cell alive, just they don't settle down or very very slow. Plus, I never spin my cell, because I always dilute them into T-75 flask at which concentration DMSO will not be toxic. So the only changes I made I guess is to make sure medium really warm, not just warm them up in 37 degree for 15mins, but 30mins to make sure.

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Hi, there
I think the cell line is more tough. I do the similar way to freeze and thaw cells as you suggest above. It works fine. But when I come to fresh spleen cells, I have only 10 percent cells viable after freeze and thaw. My question is that if I am not going to culture the cells after I thaw them, instead I want to do FACS staining and cell sorting, should I dilute them in cold media or warm media after I thaw them in the water bath? I always leave cells in 37 degree water bath for just one min, but they are still crystal. I don't know whether I can leave them in the water bath for a little bit longer. Thank you!

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Hi,

Spleen cells could survive freeze and thawing as well. I would freeze the same number of cells in two separate containers and thaw them in warm or room temperature media to see the difference atfer 24 hours in 37C0 incubator. I usually take the tube out of the liquid nitrogen then warm it up in the water bath for may be one minute and while still is half frozen i add 37degree media to dilute the DMSO. If spleen cells have been treated with amonium chloride for lysing red blood cells they will become very sensitive to freez/thawing procedure.

Good luck,
Soudi

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