Before you start
- Use healthy cells in log phase (> 90% viability).
- Label vials with cell line, passage, date and number.
Reagents and materials
Totals are for 6 vials (+10% extra). Per-vial (1 mL) amounts are in the middle column.
| Reagent | Per vial (1 mL) | Total |
|---|---|---|
| Freezing medium (90% FBS + 10% DMSO), cold | 1 mL | 1 mL |
| Cells in log phase1–5 × 10⁶ per vial | 2 × 10⁶ cells | 2 × 10⁶ cells |
Buffers and recipes
Freezing medium per 10 mL
| Heat-inactivated FBS | 9 mL |
| DMSO, cell-culture grade | 1 mL |
Alternative: complete medium with 20% FBS and 10% DMSO. Prepare fresh and keep cold.
Equipment
- Cryovials
- Controlled-rate freezing container (isopropanol or foam-based)
- −80 °C freezer
- Liquid nitrogen storage
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Harvest and count
Detach (if adherent), count and check viability.
Pellet
Centrifuge at 200–300 × g for 5 min.
Resuspend in freezing medium
Resuspend gently at 1–5 × 10⁶ cells/mL in cold freezing medium.
Aliquot
Dispense 1 mL per labelled cryovial; work quickly because DMSO is toxic at room temperature.
Freeze slowly
Place vials in a controlled-rate container at −80 °C overnight (about −1 °C/min).
Store
Transfer to liquid nitrogen (vapour phase preferred) within 1–2 days and record the location.
Critical points
- Thaw one test vial a week later to confirm recovery.
- Keep a master and a working cell bank.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Poor viability after thawing | Freeze healthier cells, slow the cooling rate, reduce time in DMSO before freezing. |
Safety
DMSO penetrates skin and carries dissolved chemicals with it; wear gloves. Use cryogenic PPE for liquid nitrogen.
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.