26 replies to this topic

[Minnie Mouse]

Freezing cells protocol by Abcam


http://www.abcam.com...g...e&rid=11742

[ecgian]

Thank you for this protocol. It is very helpful.

I have a few questions about freezing cells.
1) How important is it for the freezing medium (DMEM complete + 10% DMSO) to be ice-cold v. RT?
2) While I'm aliquotting the cell mixtures into the cryovials, the cells are sitting in the freezing medium at RT (either in the aliquots or in the centrifuge tubes). Is this terrible for the cells? It probably is ~10 minutes??
3) Is it terrible if the cryovials aren't chilled?

Thank you. I hope I am not destroying my cells and stocks of them.

[davidpatternsh]

Hey there,, i am not about cell culture ..can you describe this topic ...Due to this i could leave my post on this topic....

Alta White Teeth  

   Nitro Force

[raaann]

ecgian, on 18 March 2010 - 09:09 AM, said:

Thank you for this protocol. It is very helpful.

I have a few questions about freezing cells.
1) How important is it for the freezing medium (DMEM complete + 10% DMSO) to be ice-cold v. RT?
2) While I'm aliquotting the cell mixtures into the cryovials, the cells are sitting in the freezing medium at RT (either in the aliquots or in the centrifuge tubes). Is this terrible for the cells? It probably is ~10 minutes??
3) Is it terrible if the cryovials aren't chilled?

Thank you. I hope I am not destroying my cells and stocks of them.

Hello,

Im sure there is no problem if the cells are in the freezing medium at RT for abt 5-6 minutes while you are aliquoting
them to cryovials. i dont chill my cryotubes too..
Just aliquot and tranfer them all quickly to -20..
Ive never had any problems in rethawing them when ive done it like this. .  

[MJD]

You should not have any problems if your freezing media is at RT until you get your freezebacks aliqouted.  I've done this at least a dozen times and I have never had any problem with the cells that come back out of the nitrogen tank.

[chowyc2]

http://addexbio.com/...tdetail?pid=115

[PamioCrazia]

I am having quite problem when I freeze my cells (prostate cancer cells) and re-use them.
1) is it normal for about 1/4 of the cells to die during freezing and thawing?
The protocol really helped. thanks for it.

[Neanderthal]

i am trying to freeze down a number of vials of HepG2 cells at once. I added the freezing medium ( I am trying to add 1.5ml medium drop by drop) in to the cells and try to re-suspending the cell pellet. By the time I finished re-suspending the last one, I could see that the cells in the first vial have settled down. They again I had to re-suspend it before putting it in cryo vial.

Why is this settling down of cells happen> Any thoughts? Also, how important in adding the freezing medium drop by drop to re-suspend the cells?

Thanks in advance.

[science noob]

PamioCrazia, on 26 September 2011 - 05:44 PM, said:

I am having quite problem when I freeze my cells (prostate cancer cells) and re-use them.
1) is it normal for about 1/4 of the cells to die during freezing and thawing?
The protocol really helped. thanks for it.

Yup, you'll loose some cells after the freeze thawing process.  This is unavoidable since the cells have to go through so many temperature changes (RT, -200, 37, RT).  We can do a few things to minimise cell loss - e.g. not leaving cells in DMSO for too long at RT, introduce thawing media into cells + DMSO drop-wise etc.  The amount of cell loss depends on how robust your cell type is -  immortalised cell lines tolerate harsh conditions better, some are more sensitive.

Neanderthal, on 27 November 2011 - 03:13 AM, said:

i am trying to freeze down a number of vials of HepG2 cells at once. I added the freezing medium ( I am trying to add 1.5ml medium drop by drop) in to the cells and try to re-suspending the cell pellet. By the time I finished re-suspending the last one, I could see that the cells in the first vial have settled down. They again I had to re-suspend it before putting it in cryo vial.

Why is this settling down of cells happen> Any thoughts? Also, how important in adding the freezing medium drop by drop to re-suspend the cells?

Thanks in advance.

What I normally do is I prepare the freezing medium + cells in bulk.  So say if I have 10 million cells, I would resuspend all the cells together in freezing medium.  E.g. Add 10 ml to the spun down cell pellet, resuspend, then aliquot them into 10 cryovials.  The more volume/cells you have in your tube, the more times you need to resuspend the cells (use the pipette to resuspend it, or give the tube a shake).  This is the most efficient process I know of, instead of doing them one by one.

From what I gather, you're freezing your HepG2 cells one by one, adding freezing medium to each cryovial?

[Rute]

Sometimes the number of cells lost when bringing the cell back its not related to the freezing method but due to the thawing method. If your cells are not that resistant you should thaw them very quickly, my suggestion is thaw them by adding warm media and ressuspend rather than waiting for the vial to thaw and then use it.
Another thing that can influence is the amoutn of cells frozen. You should freeze up to 1/3 of the destination culture flask.
and change the media the day after you thaw the cells. These are some tips that reduce the amount of cells lost durin this whole process.
But everyone has their way of working.

[Mohamed Abd EL Salam]

i need help


i performed a biotransformation reaction using suspension culture.
i dissolved the substrates in methanol (10mg/1ml methanol).
and then i added each 1 ml methanol into 200 ml medium, so is methanol at that concentration toxic to the cells??

[Robert Allaway]

Hi all --

I froze down some cells yesterday (A549) and accidentally left them at -20 overnight (~16h). I have transferred them to -80 now, but I am wondering, has anyone accidentally done this before? Were the cells fine? I'll probably plate out one of the vials to see if they are still viable, but I want to know ASAP whether they will be OK so I can know whether or not to plate more cells out for freezebacks. Thanks for any help!

[Neanderthal]

science noob, on 27 November 2011 - 04:35 AM, said:

PamioCrazia, on 26 September 2011 - 05:44 PM, said:

I am having quite problem when I freeze my cells (prostate cancer cells) and re-use them.
1) is it normal for about 1/4 of the cells to die during freezing and thawing?
The protocol really helped. thanks for it.

Yup, you'll loose some cells after the freeze thawing process.  This is unavoidable since the cells have to go through so many temperature changes (RT, -200, 37, RT).  We can do a few things to minimise cell loss - e.g. not leaving cells in DMSO for too long at RT, introduce thawing media into cells + DMSO drop-wise etc.  The amount of cell loss depends on how robust your cell type is -  immortalised cell lines tolerate harsh conditions better, some are more sensitive.

Neanderthal, on 27 November 2011 - 03:13 AM, said:

i am trying to freeze down a number of vials of HepG2 cells at once. I added the freezing medium ( I am trying to add 1.5ml medium drop by drop) in to the cells and try to re-suspending the cell pellet. By the time I finished re-suspending the last one, I could see that the cells in the first vial have settled down. They again I had to re-suspend it before putting it in cryo vial.

Why is this settling down of cells happen> Any thoughts? Also, how important in adding the freezing medium drop by drop to re-suspend the cells?

Thanks in advance.

What I normally do is I prepare the freezing medium + cells in bulk.  So say if I have 10 million cells, I would resuspend all the cells together in freezing medium.  E.g. Add 10 ml to the spun down cell pellet, resuspend, then aliquot them into 10 cryovials.  The more volume/cells you have in your tube, the more times you need to resuspend the cells (use the pipette to resuspend it, or give the tube a shake).  This is the most efficient process I know of, instead of doing them one by one.

From what I gather, you're freezing your HepG2 cells one by one, adding freezing medium to each cryovial?

Ya, that is right, I am doing it one by one..
Your's is a good idea. Thanks a lot. May be I should try this out!

[Neanderthal]

Robert Allaway, on 05 January 2012 - 09:24 AM, said:

Hi all --

I froze down some cells yesterday (A549) and accidentally left them at -20 overnight (~16h). I have transferred them to -80 now, but I am wondering, has anyone accidentally done this before? Were the cells fine? I'll probably plate out one of the vials to see if they are still viable, but I want to know ASAP whether they will be OK so I can know whether or not to plate more cells out for freezebacks. Thanks for any help!

I guess the cells will be fine. But I am curious to know what you saw after plating??

[daywalkerbyday]

I am pretty much at my wits end which is why I have come here to see if I can get any solution. I have been working with THP-1 cells for about 3 years now. Whenever I freeze them down (after putting them into the -80 freezer overnight/few days and then into liquid nitrogen) they will never come back. Before Christmas, stocks that I kept in the -80 freezer would come back, but this has since stopped working. I am using the same freeze media for RAWs, SW480s, U373s and I haven't had a problem. Has anyone any thoughts?