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Surviving the Big Chill: Freezing and Thawing Mammalian Cells

Cryopreservation is crucial to long-term maintenance of cells. Not even the most diligent and dedicated scientist is capable of maintaining a cell line day in and day out for years. Additionally, cells can change properties, senesce, or, ooops (!) become contaminated as the length of time in culture increases. Freezing and resuscitating your cells can be done easily with just a handful of reagents. Final storage of cells in liquid N2 will ensure a reliable, career-long source of cells.

Freezing 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, and 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 media 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; therefore a cryoprotectant is used to lower their freezing point. DMSO is the most common cryoprotectant used and is used as a 10% stock solution.

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 1mL of freezing medium/cryovial. Make sure you have cryovials designed for liquid N2 storage. I typically plan on 1 100mmdish of cells/cryovial. Vials should be labeled using a lab marker that will withstand alcohol and liquid N2. I am 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 some type of 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.

Thawing cells

Special materials needed:  frozen cells, cell growth media and a 37°C waterbath.

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 waterbath. Keep the cryovials in the waterbath 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 media (~10mL/1mL aliquot 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. In some labs, the cells are centrifuged and the cell pellet is resuspended in fresh cell growth media prior to placing cells in a culture dish. This is the way I first learned to thaw cells. However some data suggest that cells are extremely fragile after thawing and centrifugation may increase cell death. Therefore it is recommended to plate the cells and change the media at a later time. For adherent cells you can change the media as soon as the cells have attached to the dish. I switched to this method and I generally take my cells out just before leaving the lab for the night and change the media first thing the next morning.

3)   Check your cells.  About 24 hours after thawing cells I look them over carefully under the microscope to make sure they are healthy and behaving normally.

Knowing how to correctly freeze and thaw your cells will save you the embarrassment of asking a colleague for yet another aliquot of their cells or the expense of purchasing new cells.

If all else fails, remember to freeze slowly and thaw quickly!

 

3 Comments

  1. 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

  2. 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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