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Caco-2 Thawing Cells From Frozen

Use this page for thawing CaCo2 cells from frozen in -80° freezer. From Wine Lab SOP and cell culture guidebook.

Purpose

When beginning a new cell line to grow for experiments, they need to be brought “back to life” from frozen. They need to be thawed and stored in a sterile environment to grow and seed the cell flask.

Safety

• Complete work in the BSC.
• Spray down all materials brought into the BSC with 70% Ethanol.
• Try not to touch the sides of the flask opening with pipette tips.
• Do not touch the rims of the flasks or tubes to work with.

Materials

• Frozen cell cryovial
• Complete culture DMEM growth medium
• Culture vessel (Flask)
• Centrifuge tube
• 37°C Water Bath
• Styrofoam tub

Procedure

1. Prepare the complete growth media using the Caco-2 media recipe and place an alliquot in the 37° water bath to pre-heat.
2. Take the cryovial with the frozen cells and place in the styrofoam tub filled with ice to transport to the warm bath.
3. Place the cryovial with the frozen cells into the 37° water bath for less than 1 minute, gently swirling until there is a small amount of ice left in the vial.
4. Directly place the cryovial and balance in the centrifuge for 4 minutes to form a pellet.
5. Remove and spray down with 70% ethanol before removing the supernatant and resuspending with 1mL of advanced DMEM to wash. If you see the pellet, do as written. If not, then try again to see pellet.
6. Centrifuge again for ~4 minutes to form pellet again. Then remove the supernatant, put complete DMEM into the cryovial, resuspend, and place all of the liquid into a centrifuge ependorf tube so the total volume of the tube is 5mL (so place ~3-4mL in the tube before putting cells in).
7. Mix by gently pipetting and releasing the 5mL of cells + DMEM.
8. Fill the red-capped cell flask with ~10mL of complete DMEM. Pipette and add the suspended cells and growth media into the flask.
9. Label and swirl on the table in gentle figure 8 motion at least 10 times.
10. Leave in the incubator.

Quality Control

• Check the clarity of the supernatant and the visibility of the pellet.
• Make sure to complete the methods quickly and accurately.

Revision History