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Freezing and thawing of cells


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#1 shilpi13

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Posted 09 September 2004 - 10:32 PM

hi
I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?


Thanks
Shipley

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#2 labrat

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Posted 09 September 2004 - 10:42 PM

I didn't see anything wrong with your thawing procedure. Are you sure the freezing procedure was right?

#3 shilpi13

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Posted 11 September 2004 - 08:06 AM

hi,
For making the liquid nitrogen stock,we usually pellet down cells at 1500rpm for 5 min. the remove the sup.
ater that resuspend the peleet in freezing mixture 1.5ml (FCS : DMSO :: 9:1).
then place the cryovial at -20 degree for 4-5 hrs then shift it to liq. N2.
If this procedure is wrong then what all modifications do i make? PLease suggest me some way.
Thank you,
Shilpey

#4 labrat

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Posted 11 September 2004 - 09:18 AM

the general rules for freezing and thawing cells is: freeze cells slowly but thaw cells quickly. It seems to me that you have not frozen your cells in a gradual manner. There are many ways for achieving gradual decrease in temperature. Check the section for cell preservation on this site and you will find protocols of different flavors for this purpose.
http://www.protocol-...tion/index.html

#5 kant0008

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Posted 12 September 2004 - 04:53 PM

Hi,
one thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.
Good luck!

#6 anilkumarpr

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Posted 18 September 2004 - 10:48 AM

Hi shilpi,

Why do you want to pellet your cells after thawing. From your procedure you are using DMSO (1:10). DMSO will not give toxicity at low concentration
My suggestion is to add thawed cell suspn to 5-8 ml culture medium and incubate for a day then change medium. Initial pelleting could be avoided.
I never do a pelleting when I thaw cells (from -85 deg C). I never had a problem with low percentage in revial.

Good luck next time

Anil

#7 preeti

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Posted 01 October 2004 - 01:32 PM

try freezing cells in glycerol wid fcs than dmso
trust me this works

#8 anilkumarpr

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Posted 01 October 2004 - 08:07 PM

Hi

Preeti is right.
Most of the time I also use Glycerol instead of DMSO.
Glycerol with FCS in your culture medium will be excellent.

Anil

#9 tery

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Posted 07 October 2004 - 06:06 AM

What is main difference in our procedure is that we centrifuge only at 1000rpm. I think that 1500rpm might be too much for some more sensitive cells. Moreover 1000 is more than enough. The cells during and after thawing have weaker membrane and in 1500 it might not hold.

#10 JenL

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Posted 18 October 2004 - 12:56 AM

try freeze the cells at -20 for 2-3 hours, then -80 overnight before placing then in liqN2

#11 orexin2003

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Posted 24 October 2004 - 08:22 AM

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

#12 orexin2003

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Posted 24 October 2004 - 08:25 AM

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

#13 biomed

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Posted 25 October 2004 - 11:41 AM

Hi shilpi,

Why do you want to pellet your cells after thawing.  From your procedure you are using DMSO (1:10).  DMSO will not give toxicity at low concentration
My suggestion is to add thawed cell suspn to 5-8 ml culture medium and incubate for a day then change medium.  Initial pelleting could be avoided.
I never do a pelleting when I thaw cells (from -85 deg C).  I never had a problem with low percentage in revial.

Good luck next time

Anil

I agree with Anil.
Biomed

#14 Actuated_Ant

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Posted 08 November 2004 - 04:43 AM

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

#15 soudi

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Posted 01 December 2004 - 12:32 PM

hi
I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?


Thanks
Shipley

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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