Freezing and Thawing Cultured Cells

Freezing Cells:

1.Trypsinize cells and harvest in the normal way.

2.Count a 200 μl aliquot and determine the total cell number.From this,calculate the volume of media required to give a final freezing density of 3.0×107 cells/ml.

3.Collect the cells by centrifugation at 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 2× 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 at -70℃, then transfer to the -135℃ freezer.

Thawing Out Cells:

1.Remove vial of frozen cells from the -135℃ freezer and transfer to 37℃ 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 at 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).

From the Laboratory of Dr. Allan Bradley

Baylor College of Medicine, Houston, Texas