THAWING FROZEN CELLS
References:
1. Freshney, Culture of Animal Cells, 4th edition, page 303.
Materials and Reagents
Sterile:
Culture flask – T75
Growth medium
Pipettes – 1mL and 10mL
Non-sterile:
Gloves
Water bath at 37°C
70% alcohol
Protocol
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5.
Collect all materials, prepare the medium, and label the culture flask. Medium should contain
10% FBS and 1% antibiotic/antimycotic.
Retrieve the ampule of frozen cells from the liquid nitrogen, and place in water bath at 37°C.
When ampule is thawed, check the label to confirm identity of the cells; then swab the ampule
thoroughly with 70% alcohol.
Transfer contents of the ampule to a culture flask.
Add medium slowly to the cell suspension. 15mL over about 2 minutes to a T75 flask, gradually
diluting the cells and preservative. This gradual process is important with DMSO as a sudden
dilution can cause severe osmotic damage and reduce cell survival by half.