Cryopreservation & banking
Cooling vials at a defined rate, typically about −1 °C/min to −80 °C, either in an isopropanol- or foam-insulated container placed in a −80 °C freezer or in a programmable freezer that can also compensate for the heat released as the sample freezes.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Controlled-rate freezing
Cooling vials at a defined rate, typically about −1 °C/min to −80 °C, either in an isopropanol- or foam-insulated container placed in a −80 °C freezer or in a programmable freezer that can also compensate for the heat released as the sample freezes.
How it differs from the standard method
- Freezing medium: commonly 90% FBS + 10% DMSO, or complete medium with 5–10% DMSO.
- 1–5 × 10⁶ cells/mL for most lines, 1 mL per vial in internally threaded cryovials.
- Move vials to liquid-nitrogen vapour after 24–48 h at −80 °C.
- Programmable freezers can hold a seeding step near the freezing point to avoid supercooling.
1Stopping the clock without breaking the cells
Below about −135 °C there is not enough thermal energy for ice crystals to grow or for chemistry to proceed, so cells stored in liquid-nitrogen vapour keep for decades. The difficulty is getting them there and back.
Cool slowly
Isopropanol container in a −80 °C freezer, or a programmed controlled-rate freezer.
Store cold
Liquid-nitrogen vapour phase. At −80 °C viability falls over months to years.
Thaw fast
Swirl in a water bath until a sliver of ice remains, then dilute dropwise.
2Banks that keep a line the same
A cell bank is a set of identical vials frozen from one culture at one passage. In a two-tier system a small master cell bank is frozen once, at low passage, from an authenticated, mycoplasma-free culture; working banks are then made by thawing one master vial, expanding it a few passages and freezing many vials.
3How it goes wrong
- Freezing an overgrown or unhealthy culture: survival is poor and the dead cells inflate the count.
- Leaving cells in DMSO at room temperature while labelling.
- Plunging vials straight into liquid nitrogen: intracellular ice, nothing survives.
- Keeping the bank at −80 °C: slow, steady loss of viability.
- Thawing slowly, or adding medium all at once.
- Trusting trypan blue straight after thawing: membranes can be intact in cells that will die over the next day.
- Banking untested cells, so mycoplasma or a misidentified line is preserved along with them.
Key terms
Practise these as flashcards →An agent such as DMSO or glycerol that reduces freezing injury; permeating ones enter the cell.
Cooling at a defined rate (commonly −1 °C/min), by a programmed freezer or an insulated container.
Ice forming inside a cell when cooling outpaces water loss; almost always lethal.
Around −135 °C for water: below it the unfrozen fraction becomes a glass and ice cannot grow.
A small set of vials frozen once from an authenticated, tested culture at low passage.
A larger set of vials made from one MCB vial, used to start experiments.
Viable, attached (or growing) cells some hours after thawing, as a fraction of those frozen; more honest than immediate dye exclusion.
When to use it
- As soon as a new line arrives and has been checked, to preserve a low-passage stock.
- To store engineered lines, clones and primary cells between experiments.
- Before a culture approaches the lab's passage limit, so experiments can restart from the same point.
- To ship cells or share them between labs.
Compared with alternatives
Passaging keeps a line going in the short term; banking lets you return to the same passage instead of passaging forever.
A bank is only as good as the culture it was made from: test for mycoplasma and authenticate before freezing.
The bacterial and yeast equivalent, using glycerol stocks at −80 °C, which suits microbes far better than mammalian cells.
Counting sets the cells per vial and gives the post-thaw viability.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cooling vials at a defined rate, typically about −1 °C/min to −80 °C, either in an isopropanol- or foam-insulated container placed in a −80 °C freezer or in a programmable freezer that can also compensate for the heat released as the sample freezes.
- Freezing medium: commonly 90% FBS + 10% DMSO, or complete medium with 5–10% DMSO.
- 1–5 × 10⁶ cells/mL for most lines, 1 mL per vial in internally threaded cryovials.
- Move vials to liquid-nitrogen vapour after 24–48 h at −80 °C.
- Programmable freezers can hold a seeding step near the freezing point to avoid supercooling.