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Cell freezing & thawing

Mammalian cell culture

Cryopreservation of mammalian cells in liquid nitrogen. Always freeze backups before transfecting / engineering / passaging extensively.

Freezing medium

• Complete growth medium (e.g., DMEM-10) — 90%
• DMSO (cell-culture grade) — 10%

Or use Bambanker / CryoStor CS10 — ready-to-use, more forgiving for sensitive lines (iPSCs, primary cells).

Freezing procedure

1. Trypsinise cells from one healthy ~80% confluent 10 cm dish (see HEK293T culture).
2. Collect into a 15 mL conical. Pellet 200 × g, 5 min. Aspirate.
3. Resuspend in 1 mL freezing medium per vial you want to make. Aim for 1–5 × 10⁶ cells/mL.
4. Aliquot 1 mL per cryovial. Label: cell line, passage number, your initials, date.
5. Place in a Mr. Frosty (or similar slow-cooling container with isopropanol) at −80 °C overnight. This gives ~−1 °C/min cooling.
6. Next day, transfer vials to liquid N₂ for long-term storage. Log location in your freezer database.

Thawing procedure

1. Pre-warm a 10 cm dish with 9 mL DMEM-10 at 37 °C in the incubator.
2. Take the vial from LN₂ on dry ice. Thaw rapidly in a 37 °C bead bath, swirling — should take ~60–90 s. Do not let it sit warm in the bath — DMSO is toxic at room temp.
3. Wipe vial with 70% ethanol. In the hood, transfer cells dropwise into 9 mL pre-warmed DMEM-10 in a 15 mL conical.
4. Pellet 200 × g, 5 min. Aspirate (DMSO removed).
5. Resuspend in 10 mL fresh DMEM-10. Transfer to the pre-warmed dish.
6. Return to 37 °C, 5% CO₂.
7. Change media after 4–24 h to remove dead cells and residual DMSO.

Tips

• Always freeze when cells are healthy and in log phase — never freeze sick or contaminated cultures.
• Track passage number on the vial AND in your records. Cap at ~30 passages from the original stock for HEK293T.
• For very valuable lines (engineered, custom CRISPR, etc.), make ≥ 5 vials in a single freeze, store across two LN₂ tanks.
• Test thawed cells for mycoplasma after the first passage post-thaw.