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).
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