Freezing and thawing cells
The rule is "freeze slowly, thaw quickly". DMSO prevents ice crystals but is itself toxic, so dilute it away immediately after thawing.
Key points
- Freeze healthy log-phase cells
- Cool slowly at about −1 °C/min
- Thaw within 1–2 min at 37 °C and remove DMSO right away
Materials
- Freezing medium (e.g. 90% FBS + 10% DMSO)
- Cryovials
- Controlled-rate freezing container (−1 °C/min)
- −80 °C freezer, liquid nitrogen tank
- 37 °C water bath
- Warm complete medium
Steps
Freezing
Thawing
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DMSO concentration
What it isConcentration of the cryoprotectant that prevents ice crystals.
Lower · lessBelow 5%: ice crystals rupture cells and viability drops.
Higher · moreAbove 15%: DMSO toxicity damages cells.
Typical choice5–10%.
Cooling rate
What it isHow fast the temperature falls. Slow cooling lets water leave the cells.
SlowerToo slow: cells shrink excessively and are damaged.
FasterStraight into −80 °C or liquid nitrogen: intracellular ice kills most cells.
Typical choiceAbout −1 °C/min (controlled-rate container).
Cells per vial
What it isNumber of cells frozen per vial.
Lower · lessToo few: too sparse after thawing, slow recovery.
Higher · moreToo many: nutrient depletion and clumping.
Typical choice1–5 × 10⁶ cells/mL (line-dependent).
Thawing speed · DMSO removal
What it isHow fast you thaw and how long cells sit in DMSO.
Slow thawSlowly at room temperature: ice recrystallises and damages cells.
Long DMSO exposureLong in DMSO: toxicity increases once thawed.
Typical choice37 °C for 1–2 min, then dilute and spin immediately.
Freezing medium
What it isThe medium that carries the DMSO.
Typical choiceWhatever the cell-line supplier recommends.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Low viability after thawing | Cooled too fast, thawed too slowly, long DMSO exposure | Controlled-rate freezing, fast thaw, spin immediately |
| Poor attachment after thawing | Too few cells, damage | Plate in a smaller vessel, change medium next day |
| Vial bursts or cap pops | Liquid nitrogen seeped in during liquid-phase storage | Store in vapour phase, wear eye protection when thawing |
⚠ Safety Liquid nitrogen causes frostbite and asphyxiation — wear a face shield and cryo-gloves in a ventilated area. Vials stored in the liquid phase can explode on thawing.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
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