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Freezing Procedure 

  1. Grow cells until 80 % confluent, cells should be healthy and split no more 2-3 days before the freeze down.

  2. Trypsonize Cells.  Remove media from plate. Wash with 2 ml of Trypsin.  Tilt plate in both directions making sure the entire plate is covered.  Aspirate trypsin.  Add another 2ml of Trypsin.  Tilt again to ensure full coverage of plate.  Incubate cells at 37°C in CO2 until they release from plate.  Usually 5-10 minutes.

  3. Lable Tubes using the standard format given below.

  4. Examine plates with the Fisher Micromaster Microsope to ensure that all cells are unattached from the plastic.  Add 2ml of complete media.  Pipitte media over tilted plate to wash down cells where they can then be collected into a 15 ml falcon tube.  Centrifuge cells for 1 min @ 1500 RPM.  

  5. Remove Media and Trypsin from tube, without disturbing cell pellet (tilt tube and aspirate media with Pasteur where tip is at least 1 inch away from cell pellet).  Add desired amount of Freeze Down Media (Usually 3-5 mls/15 cm plate) and pipette vigorously with P-1000 (generally 10 x) to break up all cells.  

  6. Aliquot 1 ml of resuspended cells to each cryo tube.  Labeling info is also listed below.

  7. Put these aliquots in the “Tissue Culture Freeze Down Chamber”.  The cooler should be at room temperature when the cryo tubes are added.  Use autoclave tape to seal the edges of cooler.  Place in -80°C.

  8. The Freeze Down Chamber should remain at -80° C  for 24 hours.  The tubes will then be transferred to the appropriate box in Liquid Nitrogen.  

  9. IMPORTANT:  Record the box, and the position numbers in the cell tracking spreadsheet and in the LIMS. 


**NOTE:  Whenever the lid is removed from the liquid nitrogen tank, the levels must be checked when the lid is replaced.  

Freeze Down Media                                              

50% FBS (Fetal Bovine Serum), 25 ml

40% Complete Media, 20 ml

10% DMSO, 5 ml


Labeling Information

*all labeling should be done with a fine tip Sharpie marker

*Label cryo tube with the date that the cell line is being frozen

*Cell line C#

*Name of cell line

*If the cell line being frozen is a stable, V# is necessary.


Defrosting Procedure 

  1. Utilize spreadsheet of Cell Line Locations to locate the cell line.

  2. Remove the entry from the spreadsheet that corresponds with the tube that is being defrosted.  Keep track of the number of tubes that are left from each cell line so that more freeze downs can be made, if necessary.

  3. Thaw tube quickly at 37°C.

  4. Pipette contents to 15 ml tube with 1 ml of complete media.

  5. Spin at 1500 RPM for 1 minute.

  6. Remove excess media, while not disturbing the cell pellet.  

  7. Resuspend the cell pellet in complete media using P-1000.  Pipette vigorously to ensure to dispersion of cells.  

  8. Plate cells in complete media.

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