Most investigators use 10% DMSO and 90% FBS (Cell freezing medium or CFM). After suspending cells in chilled CFM, store cells on ice for 20 min and then store at -80 degree C for few weeks. Longer storage needs to be in liquid nitrogen.

10% DMSO plus 90% FBS could be the first step forward. You may also want to consider doing controlled freezing (-1C/min; you may use the so called "Mr. Frosty" for this. See the link here https://us.vwr.com/store/catalog/product.jsp?product_id=4600983. its available with other brands too. 

Most importantly, you want to ensure that you are storing the cells in vapor phase in liquid nitrogen (if you are storing in liquid nitrogen tank) and not in liquid phase. 

Hope these tips helps. Good Luck!

No need to take 90% FBS. Take exact medium which u r using for culture with 10 % DMSO. As soon as u add cryomedium to cells. Freeze cells at -80 degree freezer for atleast 24 hrs. Then transfer in liquid nitrogen and the cryotubes should be dipped properly in liquid nitrogen. If cryotubes are not dipped property cells will not revive. That is the reason ur cells die and u cannot revive ur HT - 29 cells.

I agree with mansoor on that.

On the other hand, how do you unfreeze them? Do you make sure that you remove all DMSO from the medium when you unfreeze ?

I think also that it is fully sufficient to use the normal cell culture medium with 10% antifreeze supplemented . However, I would take glycerol instead of DMSO, which has the advantage that some (not all!) cells/ cell lines can be sown directly after thawing in the culture vessel and the washing step with PBS for removing the toxic DMSO (10%!) can be omitted.

i agree with manssor. No need to take 90% FBS. Take exact medium which u r using for culture with 10 % DMSO.

after preparing and aliquotting cells in freezing media, place in alcohol bath and store at -80C overnight for a slow freeze.  There are special containers you can buy for this. Then move to liquid nitrogen next day.

• I use 30% FBS+ 60% RPMI + 10% DMSO for cryopreservation. Please incubate the cells at -20C for 1.5 h after aliquoting in cryopreservant; then incubate overnight at -80C before liquid nitrogen freezing. 
• Remember to remove the DMSO after overnight culture when you try to revive freezed cells. Best of luck 

Not enough information here. If it's taking you too long to freeze the cells once you put them in the freezing media, the DMSO may be hurting them. I personally use my regular media + 10% DMSO but work really fast to get them to the freezer. The other issue is to remove/dilute the DMSO enough when you are unfreezing them. In my experience, if you wait too long to put the cell in the fresh media, the DMSO may also hurt them during that time. How are you freezing and storing may also affect a little.

You could also try to freeze in 100% FBS, it works good for many cell lines.

For HT-29 10% DMSO+20%FCS in culture medium worked well. Use a cell freezing container in -80°C to freeze them down slowly and defrost them quickly in a 37°C water bath. I drop the defrosted aliquot in 20ml culture medium and centrifuge them, completely remove the supernatant and dissolve the cell pellet in fresh medium. Second thought: did you check for mycoplasma? 

Don't use FBS in the medium only use 10% DMSO. It should work fine.

You should use 10% DMSO plus the cell culture media you used for culturing HT-29  cells.

ATCC recommends 5% DMSO and 95% complete growth media. However if you wish to prepare high serum stocks you can use a freezeing media with 5% DMSO and 70% Heat inactivated FBS/FCS and 25% DMEM-HG or whichever culture media  you use to maintain your cell line. But u need to have some growth media in your freezeing media.. Also freeze down your stocks in 1°C cooler inthat has IPA as coolent (this is very importatnt) place the 1°C cooler containing stocks vials in - 80°C freezer overnight. 

 According to my knowledge, first you make freezing media in 9:1(for 10 ml - 9 ml fbs and 1 ml dmso) ratio and then use 50% freezing media and 50% cell suspended media. and then freeze it  slowly and make sure try to freeze earlier passage cells. and also check for the long time preservation, which condition is suitable for your cell in liquid nitrogen vapor or liquid phase?. 

be careful at the time of revival. thaw the vial in the normal temp water and re suspend the cell in new fresh media. try to wash the cell one time in media to remove all the content of DMSO. 

good luck

I'm still having problems after defrost HT 29 vials. I'm trying two criopreserving methods. One of then with McCoy 5A medium + 10% FBS + 5% DMSO and another option with McCoy 5A medium + 20% FBS + 10% DMSO. After defrost we put the cells in culture medium, centrifugue and resuspend in McCoy 5A + 20% FBS medium, but a lot of cells aren't attached on the flask surface. Could someone give me some advices?

Thank you!

Dear stefano Clerici

your defrosting procedure seems fine

You can't use 90% FBS. 10-20% FBS and 5-10% DMSO with cell culture media are good enough.