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Protocols: ES Cell Culture - Gene Targeting - Protocols

Freezing and Thawing Cultured Cells





Freezing Cells:



	1.	Trypsinize cells and harvest in the normal way.



	2.	Count a 200 ul aliquot and determine the total cell 

            number.  From this,  calculate the volume of media required 

            to give a final freezing density of 3.0 x 107 cells/ml.



	3.	Collect the cells by centrifugation @ 1,000 rpm for 7 minutes.



	4.	Aspirate off the supernatant and resuspend the pellet  

            in 1/2 the volume calculated in Step 2 above.  Use 

            media appropriate for the cells being frozen (i.e., M15 

            for ES cells or 7% FCS, 1% GPS for STO's).



	5.	Dilute the cell suspension 1:1 with 2X Freezing Media 

           (60% DMEM, 20% FCS, 20% DMSO; freshly prepared).  Add the 

           media dropwise, mixing well after each addition.



	6.	Aseptically aliquot the suspension into sterile freezing  

            vials, label each vial with the date and cell type/clone 

            number, and place the vials into a styrofoam container.



	7.	Freeze the cells overnight @ -70o C, then transfer to the 

            -135o C freezer.



Thawing Out Cells:



	1.	Remove vial of frozen cells from the -135o C freezer and 

            transfer to 37o C water bath to thaw (thawing generally 

            takes only 1-2 minutes).



	2.	Transfer the cell suspension to a sterile 15 ml tube.  

            Add appropriate media dropwise, shaking the tube well 

            after each addition.  "Top up" the tube with additional media.



	3.	Collect the cells by centrifugation @ 1,000 rpm for 7 minutes.



	4.	Aspirate off the supernatant and resuspend the cell pellet in 

            12 ml of media.  Plate out the cells on a 10 cm plate 

            (use a gelled plate if plating STO's; use a 10 cm feeder plate 

            if plating ES cells).