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Many cultures obtained from a culture collection, such as ECACC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic above 4oC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

A schematic diagram of "Resuscitation of Frozen Cell Lines"

Materials

• Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and size of flask to resuscitation into.)
• 70% ethanol in water
• DMSO

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Glass Bottom Dishes
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• ELISA plates
• Ampule Rack
• Tissue

Procedure

1. Read Technical data sheet to establish specific requirements for your cell line.
2. Prepare the flasks as appropriate (information on technical data sheet). Label with cell line name, passage number and date.
3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.
4. Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37oC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet.
5. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (cell culture flasks prepared in Step 2).
7. Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere.
8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Key Points

1. Most text books recommend washing the thawed cells in media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks. See Protocol 7 for further details.
2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.
3. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.25m filter.
4. For some cultures it is necessary to subculture before confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 (Prod. No. 93061524) cells is lost if they are allowed to reach confluence repeatedly.

Scouce: ECACC Handbook Protocol 2