Acknowledgements: CP2022 /Thawing/FTT/2010-11
Care must be taken when recovering cryopreserved animal cells as animal cells are shear sensitive. Incorret technique in thawing of the sample can lead to cell damage thus causing a low yield of viable cells.
Geral Rule of the Thumb: Slow Freezing, Fast Thawing.
Strict sterility must be observed throughout the process.
PRECAUTIONS TO TAKE
1. When removing cell line from liquid nitrogen, make sure all proper PPEs are worn as liquid nitrogen has a temperature of -190 degree celcius and can cause severe burns.
2. To prevent contamination to cell line, before cell-enumeration step, all procedures must be carried out in the Biosafety Cabinet ( Class II ).
3. Enumeration of cells to be done within 10 minutes to prevet cells from attaching to the plastic vessel.
Frozen adherent cell line in cryovial
Sterile complete RPMI- 1640 media + 10% Foetal Calf Serum ( FCS )
Water-Bath, 37 degree celcius for thawing
Neubauer haemocytometer for cell enumeration
0.2% - 0.4% w/v trypan blue in 0.85% w/v saline
Micropipettes P20, P200 and sterile tips
Sterile pipettes 1mL, 10mL.
1. An ampoule of cryopreserved cells was removed from the liquid nitrogen container and placed immediately into a water-bath pre-set to 37 degree celcius. Take notes of precautions.
2. The contents of the cryovial was thawed by shaking it at the same time keeping the bottom half of the vial submerged in the water-bath. Precaution taken not to let the water touch the cap of the cryovial. Optimal thawing time should be less than 1 minute.
3. As soon as the last trace of ice vanishes, the cryovial was removed quickly from the water-bath and the neck of the cryovial was sprayed with 70% ethanol.
4. The contents of the cryovial was transferred into a tube containing 9-10mL of cold complete RPMI-1640 medium ( RPMI-1640 + 10% FCS + 2mM L-Glutamine ). The contents were mixed gently and spinned at 1,500 rpm for 6 minutes.
5. The supernatant was decanted and cell pellet was resuspended in 5ml of fresh culture medium. A sterile aliquot of about 0.1mL of cell suspension was removed and placed into an Eppendorf tube for cell counting ( enumeration ). Sterility unnescessary after this step.
6. 2 drops of 15 micro-litre of dye was added onto a Nescofilm and the first drop was mixed with the cell suspension before loading the chamber of the Neubauer haemocytometer using a P20. The cells were enumerated and the viability of the recovered cells were accessed.
7. Seed at least 1,000,00 cells/mL in a total volume of 10mL with complete RPMI-1640 medium in a T-25 flask or 60mm petri-dish
8. Cell growth to be examined daily and fresh medium to be replenished every two days as necessary.