Cryopreservation is the process of preserving living cells, tissues, or microorganisms at ultra-low temperatures (typically −80 °C or −196 °C in liquid nitrogen) so they remain viable for future use. A well-managed cell bank ensures experimental reproducibility over years.

Principles

Ice crystal formation is the primary cause of cell death during freezing. Intracellular ice punctures membranes and disrupts organelles. Cryoprotective agents (CPAs) protect cells by:

• Penetrating the cell (e.g., DMSO, glycerol) to displace water and lower the freezing point.
• Non-penetrating (e.g., sucrose, Ficoll) to dehydrate cells before freezing.

The cooling rate must balance two competing effects: too slow causes osmotic damage from prolonged exposure to concentrated solutes; too fast causes lethal intracellular ice. Most mammalian cells are optimally cooled at −1 °C per minute.

Cryoprotectants

• DMSO (dimethyl sulfoxide): the most widely used CPA. Typical concentration is 5–10% (v/v) in culture medium with 10–20% FBS or serum-free freezing medium. DMSO is toxic to cells at room temperature over time — work quickly and keep cells on ice.
• Glycerol: 10–20% (v/v). Less toxic than DMSO but less penetrating. Commonly used for bacteria, yeast, and some cell lines.
• Serum-free freezing media: commercial formulations (e.g., CryoStor, Recovery Cell Culture Freezing Medium) use defined CPAs without animal serum.

Freezing Protocol

1. Harvest cells in log phase at >90% viability.
2. Count and centrifuge at 200–300 × g for 5 minutes.
3. Resuspend in cold freezing medium at 1–5 × 10⁶ cells/mL.
4. Dispense into cryovials (1 mL per vial). Label with cell line, passage number, date, and operator.
5. Cool at −1 °C/min using a controlled-rate freezer, an isopropanol freezing container (Mr. Frosty), or a passive cooling device.
6. Transfer to −80 °C for 24 hours (short-term), then move to liquid nitrogen (−196 °C) for long-term storage.

Thawing Protocol

1. Remove vial from storage and immediately place in a 37 °C water bath with gentle agitation.
2. Thaw until only a small ice crystal remains (about 1–2 minutes).
3. Wipe the vial with 70% ethanol.
4. Transfer contents dropwise into 10 mL of pre-warmed culture medium.
5. Centrifuge at 200 × g for 5 minutes to remove DMSO, resuspend in fresh medium, and plate.

Bacterial and Yeast Cryopreservation

Bacteria and yeast are simpler to preserve. Mix an overnight culture 1:1 with sterile 50% glycerol in a cryovial, vortex, and freeze at −80 °C. For recovery, scrape the surface of the frozen stock with a sterile loop and streak onto an agar plate — do not thaw the entire stock.

Good Cell Banking Practice

• Create a Master Cell Bank (MCB) at the lowest possible passage number, then derive Working Cell Banks (WCBs) from the MCB.
• Test MCBs for mycoplasma, bacterial contamination, and identity (STR profiling for human cell lines).
• Store MCB vials in two physically separate locations.
• Monitor liquid nitrogen levels and maintain a temperature log.