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

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hi,i am using hepg2 cells for last 5 months and iuse this protocol for thawing, hope this will be useful.

in a 15ml falcon add 8ml of complete medium and put around 1ml of C.m in the frozen cells, spin for 4 min at 1000g, throw supernatant, add 10ml c.m and spin for 4min at 1000g, trow supernatant and resuspend in 5ml C.m . then spread in flask.

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Dear All,

This is what I think is important in freezing cells after 30 years of freezing cells down :-

Use DMSO as the cryopreservative at a final concentration of 10%.

If the cells are primary's or sensitive to DMSO, use 20% FCS instead of 10%.

Always make sure that the concentration of DMSO in the resultant cell culture flask is < 0.1%. At a concentration higher than 0.1%, DMSO is CYTOTOXIC.

Use a controlled rate freezer if you have one....this reduces the temperature 1 degree/minute.

If you do not have a controlled rate freezer, use a polystyrene box.....cells at 4 degrees for 10 mins, -20 degrees for 2 hours and then overnight at -80 degree. Next day transfer to liquid nitrogen.

For cells like HL-60's DO NOT USE DMSO as this will DIFFERENTIATE THE HL-60's. Use glycerol at 20%.

CELLS FROZEN IN GLYCEROL WILL BE LESS VIABLE. THEY WILL ALSO NOT LAST AS LONG STORED IN LIQUID NITROGEN.

Thaw the cells in your hand, this is easily quick enough and will reduce the chances of contamination that will occur if you use a waterbath.

Use Cryovials that have an internal seal.

Label them with Cell type, Passage number if known, when they were frozen and the name of the person who froze them.

Always keep your records upto date, and have people log what vials they have taken from a central repository.

Always have a tissue culture grade DMSO that you have locked away JUST FOR YOUR USE....and used for tissue culture only.

Always Mycoplasma test your cells PRIOR to freezing and storage. Do it the way ATCC do it....Hoescht staining/Culture method identification and NOT PCR.


It is really easy....honest.

Rhombus

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my 2 cents...
I have found that, with some stable lines I've used, they recover better if frozen down with 10% sterile glycerol instead of DMSO. This really depends on the cells, though.
Also, I like to condition my "receiving" media (ie: the media that the cells will go into after thawing) by putting it into the incubator for an hour or so before putting the thawed cells in. So for a T-75, I put 10-15 ml of media in the flask and pop it into the incubator: I have better results when the media's CO2 level is ready to go.
Cheers,
mito1

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Hi Penny,

Did you get a reply for this question - Placing all the ingredients in the incubator for ~1hour or so - will this lead to FBS inactivation? I too do this quite often. Is this question answered specifically? In this thread I did not find an answer to this.

Thanks.

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hi shilpi
there is nothing serious wrong in your procedure, add warm water (37C) when you take out cell from liq. N2., and while freezing the cells don't store the cells at -20C for more than 1hrs.

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Hi Scifi,
I am sorry I did not check out this thread for a while.
But I think, that it is best to place plain media without FBS in incubator for 30 minutes at the max.
That balances pH (due to the CO2)...take out flask, add right amount of FBs( generally 10%) and then thaw the cells and add suspend them in this media.
I have personally tried with and with out FBS and this works the best.
Penny

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Hi,

I took my keratinocyte cell line out of liquid nitrogen yesterday. I looked at the flask today and non of the cells have adhered! They are all just floating about. Should they have adhered by now? does this mean they are dead? This is the first time I have took cells out of liquid nitrogen so I don't know what to expect.

Thanks

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Hi LittleMiss,

I had a similar problem with the thawing of my vascular smooth muscle cells! However, my vascular smooth muscle cells were freezing, in liquid nitrogen, with DMSO 5%. Therefore in my case, the death of my cells was due to too slow thawing; in fact, DMSO is cytotoxic for cells at room temperature.
Thus, the presence of DMSO and slowness of thawing are two parameters to consider!

Good luck

Boumbo_doc

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hi everyone, i am new to cell culture, i wonder are there anybody doing malaria culture? Cause i need to advices in my malaria culture. it seems that i am unable to retreive the malaria from LiqN2, as a results my malaria culture dint grow at all. Anyone can help me?

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Hi,
If I am reading this right, and I may not be (as the coffee has worn off ), it seems that 37 degrees for 1 hour should NOT inactivate the serum: after all, the cells grow in this media, sometimes for a few days. To actually heat-inactivate serum, you need temperatures around 56 - 60 degrees.
mito 1

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