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Surviving the Big Chill: Freezing and Thawing Mammalian Cells in the Lab

A picture of an ice cream cone that has been dropped and started to melt to represent freezing and thawing cells

Cryopreservation is crucial to the long-term maintenance of cells in the lab, so it’s important that you’re clued up on the process of freezing and thawing cells.

Not even the most diligent and dedicated scientist is capable of maintaining a cell line day in and day out for years.

In addition, cells can undergo genetic changes, senesce, or, ooops (!) become contaminated as the length of time in culture increases. Freezing can protect cells from these processes.

Freezing and thawing cells can be done easily with just a handful of reagents, and storing cells in liquid N2 will ensure a reliable, career-long source of cells.

Here, we’ll take you through the ins and outs of freezing and thawing cells.

Freezing and Thawing Cells

How to Freeze Cells

Special materials needed:

  • cultured cells
  • cell growth media
  • solutions for detaching cells from the plate if using adherent cells (e.g. balanced salt solution, trypsin/EDTA)
  • tissue culture grade dimethylsulfoxide (DMSO)
  • fetal bovine serum (FBS)
  • cryovials
  • cell freezing chamber
  • –80°C freezer
  • liquid N2 tank for long-term storage.

1.   Passage Cells

Healthy, actively growing cells should be used for cryopreservation. I always try to freeze large numbers of cells that have not been passaged in culture too many times.

I passage the cells 1–2 days prior to freezing to ensure that they are in a log rhythmic phase of growth at the time of freezing.

Some labs recommend changing the growth medium 24 hours prior to freezing if the cells have not been passaged at that time.

2.   Prepare Cells

Remove cells from the dishes following the usual method for passaging adherent or suspension cells. Pool all cells and centrifuge.

3. Resuspend Cells in Freezing Medium and Aliquot to Cryovials

Freezing cells can be lethal. To avoid the damage that can be caused by, for example, ice crystal formation, osmotic stress, or membrane damage, a cryoprotectant is used to lower the cells’ freezing point.

DMSO, as a 10% stock solution, is the most commonly used cryoprotectant.

Caution: Wear gloves when working with DMSO as it easily penetrates the skin.

The most common freezing medium is 90% FBS/10% DMSO. For less finicky cells and for tissue culture on a budget, 10% DMSO in cell growth medium can also be used.

After centrifugation, resuspend the cell pellet in 1 mL of freezing medium per cryovial.

Make sure you have cryovials designed for liquid N2 storage. I typically plan on 1 100-mm dish of cells per cryovial. Vials should be labeled using a lab marker that will withstand alcohol and liquid N2.

Be careful to include passage/lot numbers when labeling cryovials.

4.   Freeze Cells

To allow water to move out of the cells before freezing, freeze cells slowly. This is accomplished using a cell freezing chamber. Pricey freezing chambers pulse in liquid N2 periodically to control the freezing rate.

Less expensive options include chambers that use room-temperature isopropanol. Vials are placed in the chamber, isopropanol is added, and the chambers are placed at –80°C for at least 4 hours.

Those of us on a tight budget (like me!) use homemade chambers. I keep the Styrofoam™ racks that come with 15-mL conicals, place a cryovial in each hole, cover with a second rack, tape the racks together, and place at –80°C.

5.   Remember To Move Your Cells to the Liquid N2 Tank

Probably one of the hardest things in this protocol is remembering to move your frozen cryovials to the liquid N2 tank! I usually allow my cells to freeze overnight and then quickly place the cryovials into the liquid N2 tank.

How to Thaw Cells

Special materials needed:

  • frozen cells
  • cell growth media
  • 37°C water bath.

1.   Remove Cells From the Tank and Thaw

For the greatest cell viability, it is important to freeze the cells slowly. The opposite is true for thawing—thaw quickly! Remove cryovials from the liquid N2 tank and immediately place them in a 37°C water bath.

Keep the cryovials in the water bath until just the tiniest ice crystal is left in the cryovial.

2.   Transfer, Spin (?), and Plate

Immediately transfer the cells to a large volume of pre-warmed cell growth medium (~10 mL/1-mL aliquot of cells). For the next step, you have a decision to make.

There are two schools of thought on whether the cryoprotectant should be immediately removed from the cells:

  1. In some labs, the cells are centrifuged and the cell pellet is resuspended in fresh cell growth medium prior to placing cells in a culture dish. This is the way I first learned to thaw cells.
  2. However, some data suggest that cells are extremely fragile after thawing and that centrifugation may increase cell death. Therefore, it is recommended that the cells are plated and that the growth medium is changed at a later time.

For adherent cells, you can change the medium as soon as the cells have attached to the dish.

I switched to this method and now I generally take my cells out just before leaving the lab for the night and change the medium first thing the next morning.

3.  Check Your Cells

About 24 hours after thawing, I look my cells over carefully under the microscope to make sure they are healthy and behaving normally.

Summary

Freezing and thawing cells effectively will save you the embarrassment of asking a colleague for yet another aliquot of their cells or the expense of purchasing new cells.

And if all else fails, remember to freeze slowly and thaw quickly! For more on cryopreservation, check out our article on preserving microorganisms. Let us know about your top tips for freezing and thawing cells!

Originally published April 3, 2013. Reviewed and updated May 2021.

A picture of an ice cream cone that has been dropped and started to melt to represent freezing and thawing cells

6 Comments

  1. sarath chandra on September 25, 2018 at 7:04 am

    I’m working on CHO suspension cells, in our institution there is no place in -196 to store freezed cells. So
    1.Can we store freezed CHO cells at -80 ?
    2. What is the appropriate number of cells to freeze?



  2. Sarath on September 25, 2018 at 7:01 am

    How long can we store CHO suspension cells at -80?



    • Dr Amanda Welch on January 28, 2019 at 9:47 pm

      Depends on whether you have a cryoprotectant. But generally they can last for a very long time.



  3. Garima Joshi on June 5, 2017 at 6:32 am

    Any info on how many cells should be plated on a flask of a particular size?



    • Dr Rebecca Tirabassi on June 5, 2017 at 9:06 pm

      Hi Garima,

      It really depends on the type of cells you are using. They can be very different sizes.
      Sometimes you can get an idea by looking at the ATCC website and look at the culture conditions.

      When freeze thawing, I put as many cells in to the flask as were in the flask when I harvested.
      If you think you cells will not survive freezing well, you can put them in to a smaller flask.
      Rebecca



  4. Tijs Claessens on October 6, 2016 at 3:25 pm

    I was taught by some guy who made transgenic mice to slowly add DMSO seperately to your freezing media. This allows the cells to get used to the DMSO, since it is toxic for cells, especially at higher temperatures. I now use 2 parts of medium to freeze my cells in. I use normal medium (DMEM, 10% FCS, Gentamycin) to resuspend my cells in and 2x freeze medium (DMEM, 30% FCS, 20% DMSO, Gentamycin). After centrifugating my cells, I resuspend my cells in half the end volume in ice-cold normal medium and then I gradually add the ice-cold 2x freeze medium, swirling the cells between each addition. I keep the cells on ice to keep them cool until they go into the -80C (in whatever container that is available).

    For the thawing part, I thaw the cells in a 37C water bath and put them on ice once the ice in the tube is almost gone. Then is put the cells into an ice-cold 14ml Falcon tube on ice and gradually add ice-cold DMEM, 10% FCS, Gentamycin, again swirling between every addition. (I usually use ¬6ml to add, but you can add more if you want). After spinning my cells down, I resuspend my cells in room temperature DMEM, 10% FCS, Gentamycin and put them into the incubator.

    It may seem overly complicated (for some cell lines it certainly is), but if you have to wait every timeuntil your cells are healthy to do experiments with, it may just be worth the effort. Good luck!



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