Hi,

We have frozen and thawed many cell types.  7.5% DMSO in the cells' usual medium + serum is often optimal.  Mix the DMSO in before adding to the cells, because neat DMSO kills cells on contact.  To thaw cells, the most critical step is diluting out the DMSO.  Here is our core protocol, which gives 90-98% viability for most cell types, so long as they were healthy and dividing well at the time of harvesting for freezing.

To recover stocks: vial is thawed quickly, e.g. in warm waterbath. Cells are resuspended from bottom of vial by gentle pipetting, transferred to a 20-ml tube and diluted by gradually adding 20 ml medium with serum. Important: dilute very slowly, dropwise. E.g. add first 1 ml with mixing (by swirling) over about 30 sec, second 1 ml over 10 sec, then remaining 18 ml over 30 sec. (Note that freezing medium is highly hypertonic: over 4 x isotonic.) The DMSO needs time to diffuse out of the cells, or they burst. Centrifuge, resuspend in complete medium and plate out at desired density (generally higher than normal).

If the cells have been kept in a Liquid Nitrogen tank the cells will be fine. Remember to thaw fast and freeze slowly. I would have a tube of at least 14mls of media, warmed to 37degrees C, ready for the cells. I take a vial from the LN2 and warm quickly in a 37 degree C water bath by agitating the cryo vial. To thaw should only take 1min or less. once thawed pour into the tube with the 14mls of media and centrifuge @ 1000 RPMs for 10 minutes. remove the supernatant. Add 1 to 2mls of media and add to 1-2 T75 flasks that has 14mls of media in it. This is for 1x106 cells/ T75 flask if you have less put into a T25 with 8mls total of media. If they have been stored in a -80 do the same as above, the only difference may be that it takes a bit longer to grow at first due to percent viability.

DEAR BETHANY SKOVAN

as you mentioned they are stored in a liquid nitrogen tank, but after 24 hours I find the cells floating in medium, it looks like they are alive but not attached, will you please guide me?

What media are you using? What kind of flasks are you using and where did you get these cells from?

Medium = RMPI , 10% FBS , 1% antibiotic PH=~7.3 - 7.4

Flask = T25 with 5 mls of medium

here I have AGS cell line

they were frozen for more than 6 months, too.

I've got them from pharmacology and biotechnology research center of Tabriz university of medicine science, Tabriz, Iran, ( 5 subcultured vials, P=10) which they originally came from Tehran pastor institute research center, Tehran, Iran.

my supervisor said they are all right, just wash the cells and replace the medium with fresh one, I did it, but the results is same, no attached cells, low growth rate.

then I'v added some more FBS  to improve the growth rate, now they are floating in RPMI, 15% FBS, 1% antibiotic, I need to check them for possible changes one more time.   

6 months of being frozen in LN2 shouldn't affect the cell adherence or viability. I have frozen stock of MCF7/s that are 25yrs old and they thaw fine.

Do you know how many cells were in the vial you thawed? I always that in to a tube with media and spin it down at 1000 RPMs for 10 minutes before plating. This take off the DMSO and gives you a better start for the cells.

They could be Mycoplasma+ which can cause changes in the cell line.

I am assuming they are in a 5%CO2 Incubator at 37C. Are the flasks vented or not? If they are not vented you will need to loosen the cap to let the CO2 in. Also, make sure the flasks are for adherent cells and not suspension cells.

You stated that you had 5 vials, I am assuming you have used one and have 4 left. I would thaw another vial and put the cells into a tube with at least 14mls of media and spin them down at 1000 RPMs, remove the media add 5msl of media and plate in your T25 flask. I would check them in 4-6hrs to see if they have adhered to the flask.

Dear Ditte Lundvig

2 days ago I had began to thaw the cells, after 24 hours they were not attached, so I tried to replace the medium, now, after 24 hours (second day ) they began to attach but their growth rate looks like to be very low, but some of them are now attached and growing up. I wondered why almost of them never attached and so they were washed out when I was replacing the medium? the concentration of cells is very low, now....

thank you in advance for your helps

dear Bethany Skovan

yes, they are in 5% CO2 incubator at 37.5 degrees of Celsius, and the flasks are vented (they have filter on their caps), but I have no idea about the count of cells, I'll check it out tomorrow,

I have 4 more vials of AGS cells to thaw theme, I don't worry about them, I worry if the medium has a problem for my MCF7 cells because I don't have any other stock of these cells, or if my protocol was wrong,

now, after 48 hours my AGS cells began to attach and grow up, but only 5 or 10% of them.

do I need to reproduce new medium and check theme with new AGS vial?

I never want to lose my MFC7 cells. Please guide me.

thank you for your helps

Aref Sobhkhizy,

    If you are having some growth, even slow, I would give them a little more time to see if they just came out of thaw badly. Sometimes this happens. You could add 1mM more of L-glutamine to the media to help with stress and give the cells more energy. If your media is sterile you shouldn't have to make new. How old is the media? If the media is more than a month old you may not have any L-glutamine left. L-glutamine breaks down with in a month of adding FBS and antibiotics.

Dear Bethany Skovan

thank you for your offers and guiding me to over come the problem.

medium is fresh, I made it 3 days earlier when I thawed the cells. as you mentioned it looks like to be there some problems with thawing steps. now, they are growing up, but slowly.

and I guess my second mistake was replacing the medium after 24 hours before giving them enough time to attach. I know that in normal situation several hours are enough for attaching, but here, I would give them some more time.

I decided to thaw one more AGS vial with your protocol and test the cells again before thawing MCF7s and T47Ds with this medium.

What are you freezing them in? (Or what were they frozen in?)

I've used the technique of spinning the cells down before plating, to remove any DMSO rather than having to do a media change the next day. Then resuspending them in their suggested media. (T47D being RPMI/10% FBS)

Also if you have multiple vials, they can be combined together and spun down and added to a single plate. I had some success with that when trying to thaw a cell line that had difficulty proliferating.

I'm just a summer student but I hope that helps.

Dear Aref

you mentioned abt duration but not the condition (liq nitrogen or -80C). If it is from liq N2 no need to afraid of duration. if you are storing them at -80C, cell survival comes down slowly after 2-3 months. So it depend on the procedure you adapted.

regarding thawing we generally follow routine thawing by rubbing between the palms. also some time keeping the vials in water bath at 37C. These are the two best procedures as for as my knowledge. one more suggestion is, after thawing the vial add some fresh media to the vial before you centrifuge the cells. this will reduce the stress on the cells. all the best..........     

dear Naveen Kumar

they were in LN tank, thank you for your offers, as I find out, my protocol for thawing was wrong, that was why cells were not attaching very well, now they are growing up. thank you

I am sure you have taken care well when the cells were frozen, the start point if that was done well, off course the shelf life in the freezer or in LN is there.

Important or keys point is the cell line growth rate after freezing? My experience has been that reduce the stress to zero what I mean is NO spinning, NO hand thawing, NO thawing for that matter, very quickly bring the vial to the hood add 1-0.5 ml of desired media into the vial suck back the cells and media gently and plate in the Smallest petri dish possible( extremely important) the cells need to cling to each other for support and signalling to return to life, second day look under microscope for growth, chnaage half the media not all, do like wise for 2-3 days and one can have the cells back to life with minimal growth rate known..

Hope this helps, it is simple thing sin life.

All the best, keep growing in all aspects.

Mary

How are you cells doing?

They are fine, they're growing, I have checked them 1 hour earlier, they're fine, thank you for your helps

dear colleagues

thank you all for your suggestions and helps

Thaw your cells as quick as possible (37°C water bath. take care not to contaminate cells) and put them instantly in preconditioned growing medium (~20 min at 37°C and 5%CO2, humidity (in your cell culture incubator). Change medium after cells adhered to remove residual DMSO /in case you have frozen your cells with DMSO ( Alternatively, you may centrifuge the thawed cells in 10 ml growing medium remove medium, resuspend pellet in 10 ml medium … ) good luck

I always warm up my medium, dispense something around 10 ml of the medium and keep warm it in the incubator while I am thawing the cryovial in my hand. once the icy cell suspension gets detached from the cryovial wall I directly drop it into the 15 ml tube with 10 ml warm medium. the rest of icy suspension will thaw within the fresh warm medium, immediately diluting the DMSO. Then I spin it at 220-300 RPM for 5 min, discard the supernatant and resuspend the cell pellet in fresh warm medium, perform a cell count and seed them accordingly.   

For some more finicky cells, you can add a little more media than usual (15-20ml of pre-warmed DMEM to a 100mm dish). If we freeze 1ml of frozen cells, we add 0.5ml of cells to two 100mm dishes with 20ml pre-warmed DMEM immediately after thawing. Many tricky cells can die if they are spun down before plating. We do not spin our cells to avoid this. As soon as they are adherent, you can change the media.

Hi, there..

I have read almost all answers but still i am not sure about spinning after thawing cells in media. As per standard protocol for revival of MCF7 , spinning is mentioned to remove DMSO . But it seems spinning also harm the cells and they lose their attachment property. Because, I am facing similar problem with MCF 7 cells. After taking it out from Liquid nitrogen, I thawed it at 37 C , added DMEM media and centrifuged for 5-10 min at 5000 rpm. Collected the pellet and resuspended in DMEM+(10%)FBS media. The complete cell suspension was transferred in T25 flasks. However, after 2-4 days I found cells were still floating , when I washed them I didn't retrieve much of them.

Hope any expert in this forum could add something more.

I have experience with 293, Cos and BHK cells. Just directly thaw frozen cells at 37 degree C for 10-15minutes. Wipe the vial outside with 70% ethanol and plate into T25 flask (contents of one vial into one T25 flask) in DMEM with 10% serum and PS antibiotic. I would pipet back and forth the cells several times in the vial for breaking the clumps and uniformly suspending the cells before transferring to T25 flask containing the serum media. Of course everything is done in a cell culture cabinet near a flame under aseptic conditions. I would change the media after 24 hours and grow to confluence before further passage. Only about 10% of the frozen cells attach. Hope this helps your problem

I forgot to tell you I have used frozen cells that have been frozen in liquid nitrogen for several years (5-7 years). They survive well if frozen in 10% DMSO.

Hi,

We have frozen and thawed many cell types.  7.5% DMSO in the cells' usual medium + serum is often optimal.  Mix the DMSO in before adding to the cells, because neat DMSO kills cells on contact.  To thaw cells, the most critical step is diluting out the DMSO.  Here is our core protocol, which gives 90-98% viability for most cell types, so long as they were healthy and dividing well at the time of harvesting for freezing.

To recover stocks: vial is thawed quickly, e.g. in warm waterbath. Cells are resuspended from bottom of vial by gentle pipetting, transferred to a 20-ml tube and diluted by gradually adding 20 ml medium with serum. Important: dilute very slowly, dropwise. E.g. add first 1 ml with mixing (by swirling) over about 30 sec, second 1 ml over 10 sec, then remaining 18 ml over 30 sec. (Note that freezing medium is highly hypertonic: over 4 x isotonic.) The DMSO needs time to diffuse out of the cells, or they burst. Centrifuge, resuspend in complete medium and plate out at desired density (generally higher than normal).

I am not sure whether someone has already said this, but something I found useful when thawing older vials  (years old) is to change the medium as soon as you have some cells attached. I usually freeze cells in 10% dmso in fbs. When I thaw, I do it quickly in a water bath, then add medium and spin down the cells, resuspend in medium and plate everything in a T25 flask. Change the medium after 6/7 hours. This is useful if you have a lot of dead cells, the healthy cells that attach will be initially more sensitive and if you leave them in flask with a lot of dead cells that can produce an acidic environment, they can detach and die as well.

dear Valeria Padovano

do you mean I need to change the medium immediately after some hours? but still there are some detached alive cells ... by this way I will get better cells by means of eliminating stressed cells but I will lose many of my cells, too ...

thank you....

Dear D.C. Bennet and Dear S. Sridhara 

thank your for your great helps

some times earlier I've asked a question in researchgate  which led to confusing ... this is the URL of question ...

https://www.researchgate.net/post/should_I_centrifuge_the_cells_after_thawing_them_in_order_to_remove_the_DMSO_from_the_medium?_tpcectx=profile_highlights

 in this question I've asked researcher to guide me to make a decision which shall I centrifuge the cells right after thawing or not?  

once again i'm confused ... Dr.Bennet said I need to centrifuge but Dr.Sridhara said I need to give them some time to attach and after that I have to change the medium ... 

would you please help me to find the best protocol or both are good and it depends to cell types?

I've many dead cells after thawing my MCF-7s (I've centrifuged them)

thanks in advance

best regards

Dear Ritu Raj

thank you but centrifuging cell at 5000 rpm for 10 min is really harmful for any kind of cells ... it's too strong ... I believe best centrifuging protocol is 800-1000 rpm for 2-4 min .. but still I don't know shall I centrifuge or not ...

best ...

Hi Aref,

As mentioned, we find the the main thing that can kill cells when you thaw them after cryopreservation is to dilute out the DMSO too quickly.  It can make the difference between 95% viable and 90% dead.  

I'd say it is less critical whether you centrifuge them or not.  As someone else said, most cells are ok in culture with DMSO concentrations below 0.5%.   However nor have we found that centrifugation harms the cells at this point (covering at least 20 different cell types including various normal diploid cells), so long as you do not spin too fast.  We use 5-7 min at  about 1000 rpm in a benchtop centrifuge, preferably chilled. We do spin them, so the cells can get back to normal culture conditions (no DMSO) as soon as possible.  We prefer to spin rather than to change the medium a few hours after plating.  As you said, there may still be some live cells that had not yet attached, and moreover quite a few cell types do not like to have to "condition" the medium twice in quick succession - that is another form of stress. 

One exception; if these are cells where you expect VERY low viability (like certain highly senescent or  highly differentiated cells), say only a few % alive, then they may have to be plated very densely.  Then it can help to wash away most of the dead cells so that you can see the living ones better - maybe the following day.

Hope that helps - Dot

I can only say if you have lot of dead cells after freezing either your cell type is very sensitive to liquid nitrogen freezing or you have to change the concentration of DMSO in the freezing media (either higher or lower than 10%). After re-plating, try changing the media after 6, 12 and 24 hours and see which one works best. You have to plan some standardization experiments playing with DMSO concentrations and or timing the change of media.

I had a similar problem recently. My cell type is mouse ES cell (E14).

After thawing, about 20 hours later, some live cells attached even some dead cells exist, live cells looked OK . Then I changed fresh medium for them at second day after thawing, but I found most of cells were die at day 3. what could be the problems?

Hi everyone, 

I have a similar, but a bit different, question. A colleage told me that the best way to thaw frozen cells in FBS+DMSO (from nitrogen) was in ice (that is, 0ºC) and adding slowly the medium, because the DMSO is more toxic at warm temperatures... but in all answers the temperature used is 37ºC, what would the difference be between this two conditions?

And another question: What would be better for reducing osmotic changes? Diluting DMSO in PBS or in culture medium?

Thanks in advance.

What if i do not have centrifuge facility in our lab?

am using CHO cells,,,, how to remove DMSO from vials?

I agree with the answer of Dr. Bennet and would like to add one step more to her protocol. Once the cells are attached say at 4-6 h its better to change the medium.

Secondly I would like to point that by looking at the morphology of the cells and comparing it with already reported by some source like ATCC we can be confident whether our cells are healthy or not.

In my hands, the really critical step is freezing the cells. I compared the use of a commercial container (made to decrease the temperature 1C/min) to the "poor man method" (placing them for a few hours inside a styrofoam container at -20C, then overnight at -80C and then to liquid N2) and the difference is noticeable by eye: With the commercial container the viability increases dramatically. Although both methods work, this is a purchase you should make, especially for more sensitive cells. The CoolCell, for example, doesn't even use alcohol inside, which makes it more convenient and cheaper in the long run. It is also an item that can be shared easily between labs (making sure to return it to room temperature the next day after you transfer your cells to liq. N2 instead of leaving the cells stored inside forever ;).

Soledad,

You are certainly correct that the ‘poor man’ technique you describe will give very poor viability results. However, the -20C step is the culprit, not the freezing container. If you skip this step and put your insulated box containing cell ampoules directly from room temp into the -80C freezer, then transfer the ampoules to liquid nitrogen after 24 hrs, cell viability will be excellent. We have done this for many years with many different cell types. Cells tolerate -20C freezing for any length of time very poorly, so it should be avoided. Leaving out the -20C step, even a ‘poor man’ can get excellent cell freezing viability. Plus, with fewer steps in the process, it saves time and gives less chance of error.

My best to you,

RW