Before you start
- Warm medium to 37 °C and label the flask with cell line, passage and date.
- Wear eye protection: vials stored in liquid phase can explode on warming.
Reagents and materials
Totals are for 1 vials (+10% extra). Per-vial amounts are in the middle column.
| Reagent | Per vial | Total |
|---|---|---|
| Pre-warmed complete medium (dilution) | 9 mL | 9 mL |
| Pre-warmed complete medium (plating)for a T75 or 10 cm dish | 10 mL | 10 mL |
| 70% ethanolto wipe the vial | 5 mL | 5 mL |
Equipment
- 37 °C water bath
- Biosafety cabinet
- Centrifuge
- Culture flask or dish
Procedure
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Thaw quickly
Swirl the vial in a 37 °C water bath for 1–2 min until only a small ice crystal remains. Keep the cap above water.
Disinfect
Wipe the vial with 70% ethanol and move it into the cabinet.
Dilute slowly
Transfer cells dropwise into 9 mL pre-warmed medium in a 15 mL tube.
Remove DMSO
Centrifuge at 200–300 × g for 5 min and aspirate the supernatant.
Plate
Resuspend gently in fresh complete medium and transfer to the flask.
Check next day
Check attachment and morphology after 24 h and change the medium.
Critical points
- Thaw fast, dilute slowly: slow thawing and sudden osmotic change both kill cells.
- Some sensitive cells are plated without centrifugation and the medium changed after attachment.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low recovery | Check freezing method and storage history; thaw faster; plate at higher density. |
| Contamination | Keep the cap out of the water bath; disinfect thoroughly. |
Safety
Use eye protection and gloves rated for cryogenic handling. Follow BSL rules for your cell line.
Further reading
- ATCC Animal Cell Culture Guide.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.