Thawing of frozen cells to initiate a fresh cell culture

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Cryopreserved animal cells in the liquid N2 can be thawed anytime for further applications. Thawing and harvesting cells is important for reviving cells after long time storage.

The principle aim of this method is thawing healthy, cryopreserved, and integrated cells for future application.

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Sample: Frozen animal cells in a cryovial (confluent stage).

Apparatus: Water bath,

15ml centrifuge tube,

Serological Pipette,

Centrifuge with rotor.

Chemicals: 1X HBSS,

Cell culture media,

0.5% trypsin solution,



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Culture media:






450 ml

50 ml

500 µl

Take out one cryovial containing cells from liquid N2 and hold the cap and keep the cryovial half in the warm water to thaw the cells for 1-3 min (turn on the water bath before 30 min to initiate the experiment). Immediately add 500 µl of culture media and mix it and collect in 15 ml centrifuge tube, add 8 ml of culture media and mix it gently (DMSO is toxic for the cells so process it fast). Centrifuge it at 300x g for 3-5 min and discard the supernatant. Add culture media 5-10 ml and harvest the cells in to two sterile 60mm culture plates. Place the plates in the incubator.1

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In this experiment, we harvested animal cells after thawing a cryovial containing animal cells from the liquid N2.

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  1. Kumar A, Mishra HK, Dwivedi P. Subramaniam
    Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Annals of neuroscience. 2015;22(2):97-106.