Jacobs:Protocol Freezing and Thawing Cells

Procedure
Materials for Freezing


 * Trypsin
 * Trypan Blue Solution (0.4%)
 * Hemocytometer
 * Media for frozen cells
 * DMSO
 * Gloves, 70% ethanol, etc.

Freezing


 * 1) Check cell culture log and be sure there is room for frozen vials
 * 2) Label vials with cell type, date, passage number, etc.
 * 3) Collect cell suspension by trypsinizing (Take out media and put in 3 mL of trypsin)
 * 4) Combine all flasks into 50 ml tube
 * 5) Count cells
 * 6) 1 million cells per vial (for MC=4 vials/flasks, for UMR=6 vials per flask.)
 * 7) 1-2 million cells per vial for MLO-A5
 * 8) Make fresh freezing down media
 * 9) General (including FAK cells): 95% FBS + 5% DMSO
 * 10) MLOs: 50% α-MEM, 40% FBS, 5%DMSO
 * 11) Spin down the cell suspension and pellet the cells (1500rpm, 7-10 min)
 * 12) Aspirate media
 * 13) Resuspend cell pellet in freezing media
 * 14) Aliquot 1ml freezing media per 2ml tube
 * 15) Place tubes in round isopropanol box in -80C freezer OR
 * 16) Transfer to the -20C for 2-3 hours
 * 17) Transfer to the -80C for at least overnight
 * 18) Store permanently in liquid nitrogen within 1-2 days
 * 19) Be sure to record in the cell culture log where the cells are permanently stored

Materials for Thawing


 * Media for cells
 * Petri dishes for cells
 * Trypan Blue Solution (0.4%)
 * Hemocytometer

Thawing


 * 1) Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
 * 2) Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
 * 3) When ready to thaw, remove vial of cells from liquid nitrogen
 * 4) Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
 * 5) Place the vial in the hood and clean it with 70% ethanol
 * 6) Immediately remove the contents of the vial and place into the cold media
 * 7) Rinse the tube down with some of the cold media from the second vial
 * 8) Spin down cells at 2000 rpm for 5 min
 * 9) Aspirate off the cold media and resuspend the cells in the warm media
 * 10) Transfer the cells and the media into a petri dish
 * 11) Count cells
 * 12) Place in incubator
 * 13) Change the media once the cells have attached to the plate
 * 14) Allow cells to grow to 80-90% confluency
 * 15) Split cells (1:3) into petri dishes
 * 16) Continue to split cells and freeze down as needed

Alternative Thawing
 * From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).
 * Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.

Contact

 * History: CMBL – CRJ/JJR, last updated 8/1/07

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