Submitted by Lauren French (download pdf)



  • At the step in passaging cells, instead of adding media and replating, add 2 mls of freezing solution (Recovery Cell Culture Freezing Media,Cat #12648-010) and triturate to loosen the pellet.
  • Split the 2 mls of freezing solution into 3 equal quantities and place in 3 separate cryovials.
  • If the cells are grown on a P10 and not too confluent, use 1 ml and transfer the entire 1 ml to a 2 mlcryotube to freeze.  If the cells are grown on a T75 and are quite confluent, use 2 mls of freezing media and transfer 1 ml to each of 2 cryotubes.
  • Place the cryovials in a -20°C freezer for a quick freeze for ~1 hour, preferably in a cryovial tabletop freezer that has been stored at -20°C.
  • Remove from the -20ºC freezer and immediately place in a -80ºC freezer overnight (or up to 1 week).
  • Transfer cells from -80ºC freezer into a container with dry ice for transfer into liquid nitrogen.
  • Place cells in liquid nitrogen.


Note: Any thawing in the freezing media may result in cell death when trying to thaw the cells in the future.


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