FREEZING HUMAN CELLS GROWN IN MONOLAYER CULTURES

It is sometimes desirable to store cell lines for future study. To preserve cells, avoid
senescence, reduce the risk of contamination, and minimize effects of genetic drift, cell
lines may be frozen for long-term storage. Without the use of a cryoprotective agent
freezing would be lethal to the cells in most cases. Generally, a cryoprotective substance
such as dimethylsulfoxide (DMSO) is used in conjunction with complete medium for
preserving cells at −70°C or lower. DMSO acts to reduce the freezing point and allows a
slower cooling rate. Gradual freezing reduces the risk of ice crystal formation and cell
damage.

Materials
Log-phase monolayer culture of cells in petri plate
Complete medium (e.g., supplemented DMEM, APPENDIX 2A)
Freezing medium: complete medium supplemented with 10% to 20% (v/v)
FBS and 5% to 10% (v/v) DMSO, 4°C
Benchtop clinical centrifuge (e.g., Fisher Centrific or Clay Adams Dynac)
with 45°C fixed-angle or swinging-bucket rotor
1. Trypsinize cells from plate (see Basic Protocol, steps 1 to 4).
It is best to use cells in log-phase growth for cryopreservation.
2. Transfer cell suspension to a sterile centrifuge tube and add 2 ml complete medium
with serum. Centrifuge 5 min at 300 to 350 × g (∼1500 rpm in Fisher Centrific rotor),
room temperature.
Cells from three or more dishes from the same subculture of the same source can be
combined in one tube.
3. Remove supernatant and add 1 ml of 4°C freezing medium. Resuspend pellet.
4. Add 4 ml of 4°C freezing medium, mix cells thoroughly, and place on wet ice.
5. Count cells using a hemacytometer (see Support Protocol 3). Dilute with more
freezing medium as necessary to get a final cell concentration of 106 or 107 cells/ml.
To freeze cells from a nearly confluent 25-cm2 flask, resuspend in ∼3 ml freezing medium.
6. Pipet 1-ml aliquots of cell suspension into labeled 2-ml cryovials. Tighten caps on
vials.
7. Place vials 1 hr to overnight in a −70°C freezer, then transfer to liquid nitrogen storage
freezer.
Alternatively, freeze cells in a freezing chamber in the neck of a Dewar flask according to
manufacturer’s instructions. Some laboratories place vials directly into the liquid nitrogen
freezer, omitting the gradual temperature drop. Although this is contrary to the general
recommendation to gradually reduce the temperature, laboratories that routinely use a
direct-freezing technique report no loss of cell viability on recovery.
Keep accurate records of the identity and location of cells stored in liquid nitrogen freezers.
Cells may be stored for many years and proper information is imperative for locating a
particular line for future use.