Tips and FAQs about freezing and thawing cells - Abcam (Feb/01/2009 )

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??

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!

science noob on Sun Nov 27 12:35:20 2011 said:

PamioCrazia on Tue Sep 27 01:44:47 2011 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 Sun Nov 27 11:13:08 2011 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?

Robert Allaway on Thu Jan 5 17:24:18 2012 said:

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?

What freezing medium is better for ADSCs (Adipose derived stem cells)?Should freezing medium be contained antibiotic or not?
helps are appreciated in advance

I have a basic question. whenever we prepare cell seed, we inoculate FTM for checking the sterility of cell seed.

Within 24 h. FTM changes to yellow with a thin line of pink ring at the top............i don't think it is contamination........... I inoculated FTM with serum alone, freezing medium and DMSO separately....but FTM resembles control.(no color change)......
please clarify my doubts..........


Thanks
Brahmavidhya K.V.

Brahmavidhya Arun on Wed Mar 14 08:33:54 2012 said:

Hi. I need help to culture cell line that I brought from USA to Brazil. When I thaw the cell, the cells are very good, but into one/two days all cells die. I have put growth factors, more FSB, but nothing worked. What is supposed I do? This cell is IL-7 dependent.

Hi,

Can anyone please tell me if I can use Pen/strep in the freezing medium?