Cell Transformation Group:Protocols/Cell Culture
FREEZING AND THAWING CELLS
- 1. Lift cells when 70 – 80% confluent and resuspend in 10% FCS/DMEM (~8 ml) (75 cm2 FLASK = 1 X 107 Cells/ml).
- 2. Spin at 250 g for 5 min. Discard S/N. Resuspend in 2 ml of DMEM (or 4 ml for large flasks). Put on ice.
- 3. Prepare freezing mix: per ml add 600 μL DMEM (+10%FCS) with 200 μL FCS and 200 μL DMSO (add DMSO [flammable goods cupboard] last as a precipitation forms) and chill on ice. This is the freezing mix. IMPORTANT - adjust the final volume of freezing mix (need 1 mL per mL of resuspended cells).
- 4. Mix at a ratio of 1:1 the freezing mix and cell mix, by adding dropwise the freezing mix to the cells. gently shaking to ensure mixing.
- 5. Add 1 ml of mix to each cryovial on ice. transfer to “Mr Frosty” and freeze at -70 °C for 24 hrs. Make sure to mark off Mr Frosty usage by crossing out number on lid. Refill with isopropanol after 5 usages.
- 6. Transfer to liquid nitrogen and add to cell lines log book.
Speed is essential for the first few steps as DMSO is harmful to cells and the less time they are in contact with it the better.
- 1. Remove cells from liquid nitrogen. Thaw quickly at 37 °C.
- 2. Pipette into warm complete DMEM (10 ml).
- 3. Spin at 250 g for 10 mins.
- 4. Resuspend cell pellet in 5 ml of DMEM and transfer to medium flask (75 cm2) containing 20 ml of DMEM.
- 5. Incubate in 37 °C CO2 incubator. Delete from cell lines log book.
If Thawing Lots of Samples (>5)
- 1. Fill chilly bin with dry ice from -70 °C freezer
- 2. Remove cells from liq N2 and place on dry ice
- 3. Place chilly bin (with cells) in -70 °C
- 4. Pre-label a medium flask† and a plastic universal for each cell line being brought up. Add appropriate amounts of DMEM to each
- 5. Take 4 (or appropriate number) vials out at a time and thaw at 37 °C
- 6. Pipette entire contents of thawed vial into its corresponding universal
- 7. Spin at 250 g for 10 min [removes DMSO]
- 8. Use a 10 ml pipette to resuspend the cell pellet in 5 ml DMEM and transfer suspension to a medium flask
- 9. Incubate at 37 °C. Delete from cell lines log book.
† Choose the size of the vessel you are using for the cells based on the size of the cell pellet you get out of the tube when thawed. Most cell lines will be OK in a medium flask, but some will need a small flask or even a 35 mm dish if there are few cells or they are damaged.