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 ERDC TN-DOER-C30
February 2003
a) Aseptically transfer these reagents in a sterile 15-mL centrifuge tube:
9.0-mL supplemented MEM (90 percent)
1.0-mL DMSO (10 percent)
NOTE: For a larger volume of freezing medium, these ingredients can be
increased proportionally.
b) Filter sterilize the 10-mL medium through a 0.22- m syringe filter. Keep the
filtered medium cool at 4 C prior to use.
III.
CELL CULTURE
Subcultures of the HepG2-EcR cells are frozen in liquid nitrogen as a secondary source while a
working culture is kept for routine testing. Whenever the cells are thawed, they are cataloged as
"passage 0." Cells that undergo 25 or more passages or consistently show poor viability are
terminated.
A.
STARTING THE CULTURE FROM FROZEN PERMANENTS
1) Place supplemented MEM in 37 C water bath before thawing cells.
2) When warmed, dry outside of container with paper towel and wipe with paper
towel moistened with 70-percent alcohol.
3) Transfer about 12 mL of MEM into a 15-mL sterile centrifuge tube.
4) Remove a cryovial of HepG2-EcR cells from the liquid nitrogen storage Dewar.
CAUTION: Always wear a protective full-face mask, cryogenic gloves, and lab coat
during the retrieval or storage of cryovials from liquid nitrogen Dewar.
5) Thaw cryovial contents quickly (within a minute) in a 70 percent alcohol solution
bath.
6) Aseptically transfer all the thawed cell suspension into the 12 mL of medium. Mix
the cell suspension thoroughly to dilute the DMSO in the freezing medium.
7) Centrifuge the cell suspension at 1,000 RPM for 5 min.
8) Siphon off the supernatant medium using a Pasteur pipet attached to an aspirator.
This step is taken to remove any traces of DMSO.
9) Resuspend the cell pellet with 20 mL of fresh medium.
10) Transfer the cell suspension into a new T-75 flask.
11) Add 30 l each of 100-mg/mL Hygromycin B and Zeocin solution into the flask.
12) Place flask in the CO2 incubator to allow the cells to attach overnight.
13) Siphon off the medium and transfer 15 mL of fresh medium and 30 L each of
Hygromycin B and Zeocin the next day. Incubate cells.
14) Check cell growth daily. A successful thawing of frozen permanents will show
confluent growth of cells within a week.
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