Cell Freezing Protocol

To continue the cell culture theme of the previous two posts^1,2, I thought I would share my protocol for freezing down cell culture stocks.

Lets assume that I have HeLa cells.

Defrosting Cells

If you receive a cryovial of cells shipped in dry ice, either store the vial of cells in liquid nitrogen or defrost the contents. To defrost, immerse the entire vial in a 37 degree waterbath. When you see that the contents of the vial has defrosted to a partial liquid state but still has a frozen block inside, spray and wipe down the vial with 70% ethanol, open the vial in a laminar flow hood and empty the contents into a sterile tube containing warm (heated to 37 degrees) cell culture media. Spin down to pellet the cells. Discard the supernatant and resuspend the cells in 5ml of fresh cell culture media. Transfer the contents into a T25 flask for culturing and expansion.

 

Cell Expansion

With a T25 flask of cells, grow it to full confluence and transfer (by trypsinising or whatever protocol you use to detach/passage your cells) all the cells to a T75 flask. From here, grow the T75 flask to full confluence and split the cells into two T175 flasks. Again, grow the cells to confluence and expand them into a further two T175 flasks. When the cells in all 4 flasks are at full confluence and ready for passaging, use your standard protocol for cell detachment, but take an aliquot to count on a haemocytometer. Centrifuge the tube to pellet the cells as you carry out the cell counting. Calculate how many cells you have in your cell pellet. You will need ~1x10⁷ cells/ml for the Master cell stocks, thus for 2-3 vials, you will need at least 3.5x10⁷ cells (the extra cells you will need for further culturing and expansion).

Freezing

Prepare your freezing media. I would recommend using freezing media made up of 90% FBS + 10% DMSO for the Master and Submaster cell stocks; and 10% DMSO + cell culture media containing 10% FBS for the Working cell stocks. Make sure your DMSO is cell culture grade and sterile. Your FBS should be sterile. If you are using a new bottle of FBS, it should be sterile but if there is any uncertainty just filter it with a 0.2um pore filter.

Have your sterile cryovials ready and labeled. After your cells have pelleted, in a laminar flow hood, pour off the supernatant and resuspend your cell pellet in 5mls of freezing media. Aliquot 1ml per cryovial, cap your vials, and place them into a freezing container (e.g. Mr. Frosty or a DIY chamber). Store the freezing container of cells in -80 degrees for 24 hours.

With the cells that are left over, dilute them in serum-free media and spin down. Pour off the supernatant to remove the freezing media and resuspend the pellet. Transfer everything into one T175 flask. Repeat the expansion process to freeze down your Submaster cell stocks (@ 5x10⁶ cells/ml; ~8 vials) and then again for your Working cellstocks (@ 1x10⁶ cells/ml; as many vials as you need).

After freezing the cells in the freezing container at -80 degrees for 24 hours, transfer the cells to liquid nitrogen for long-term storage. If you need to transport the freezing container from one location to another, use dry ice to keep the contents frozen.

Note

There may be times when a collaborator will give you a T25 flask of cells with media filling up the entire flask. If this is the case, spray and wipe down the flask and put it in the incubator for a couple of hours so that the cells can settle and acclimatize (cells tend to shrivel when cooled).

In a laminar flow hood, open up the flask and pipette out the excess media and filter it into a sterile tube or bottle for later use. Leave enough media in the flask for the cells to continue to grow.