42
General Methods
Procedure
Freezing 1. Prepare the cell suspension (for substrate-dependent cells, see chapter 4 subprotocoII-3).
2. Mix the cell suspension and autoclaved glycerol (or DMSO) in a ratio
of 9 : 1 or 10 : 1.
3. Distribute the mixture to cryotubes.
4. Put the tubes into a programmable freezer, and cool gradually at a
rate of 1°C/min down to -30°C. Then, transfer the tubes to an ultra
low-temperature freezer or a liquid nitrogen freezer to drop the temperature rapidly to either -80° to -150°C, or to -196°C, respectively.
If a programmable freezer is not available, put the tubes in an appropriately sized styrofoam box, and place the box in an ultra low-temperature
freezer at below -80°C. The cells will freeze gradually and their temperature will drop down to -80°C over a period of several hours. The cells
may be stored continuously in that freezer, or by transferring the tubes
to a liquid nitrogen freezer.
Thawing 1. Take the tubes out of the freezer or liquid nitrogen freezer.
2. Place the tubes immediately into hot water at a temperature greater
than 80°C.
3. Watch the tubes carefully, and remove them from the hot water just
before the ice disappears.
4. Transfer the cell suspension to a centrifuge tube.
5. Centrifuge at 150 g for 5 min.
6. Discard the supernatant.
7. Disperse the cell pellet in fresh culture medium.
8. Transfer the cell suspension to a culture vessel.
Results
Even if most cells die during storage, the cell population may recover
from a small number of surviving cells if there are any. Serum-free cultured cells are usually sensitive to freezing. If these cells cannot be recovered from freezing, it is best to add serum to the cell suspension that is to
be frozen at a concentration of approximately 10% before freezing, and,
after thawing, to change the medium to a serum-free medium when the
cell population has recovered. The velocity of cell population recovery
varies according to the cell line used.
Troubleshooting
When recovery of a cell population is poor or very slow after thawing,
nutrient-rich media should be used until the cells begin to multiply at
their proper growth rate.
General Methods
Procedure
Freezing 1. Prepare the cell suspension (for substrate-dependent cells, see chapter 4 subprotocoII-3).
2. Mix the cell suspension and autoclaved glycerol (or DMSO) in a ratio
of 9 : 1 or 10 : 1.
3. Distribute the mixture to cryotubes.
4. Put the tubes into a programmable freezer, and cool gradually at a
rate of 1°C/min down to -30°C. Then, transfer the tubes to an ultra
low-temperature freezer or a liquid nitrogen freezer to drop the temperature rapidly to either -80° to -150°C, or to -196°C, respectively.
If a programmable freezer is not available, put the tubes in an appropriately sized styrofoam box, and place the box in an ultra low-temperature
freezer at below -80°C. The cells will freeze gradually and their temperature will drop down to -80°C over a period of several hours. The cells
may be stored continuously in that freezer, or by transferring the tubes
to a liquid nitrogen freezer.
Thawing 1. Take the tubes out of the freezer or liquid nitrogen freezer.
2. Place the tubes immediately into hot water at a temperature greater
than 80°C.
3. Watch the tubes carefully, and remove them from the hot water just
before the ice disappears.
4. Transfer the cell suspension to a centrifuge tube.
5. Centrifuge at 150 g for 5 min.
6. Discard the supernatant.
7. Disperse the cell pellet in fresh culture medium.
8. Transfer the cell suspension to a culture vessel.
Results
Even if most cells die during storage, the cell population may recover
from a small number of surviving cells if there are any. Serum-free cultured cells are usually sensitive to freezing. If these cells cannot be recovered from freezing, it is best to add serum to the cell suspension that is to
be frozen at a concentration of approximately 10% before freezing, and,
after thawing, to change the medium to a serum-free medium when the
cell population has recovered. The velocity of cell population recovery
varies according to the cell line used.
Troubleshooting
When recovery of a cell population is poor or very slow after thawing,
nutrient-rich media should be used until the cells begin to multiply at
their proper growth rate.