| 1. |
Cultures to
be cryopreserved should be healthy, free from contamination, and
should be maintained in log phase growth for several days before
freezing.
|
| 2. |
Grow attaching
cell culture to late log phase, trypsinize and centrifuge. If freezing
suspension cells, only centrifugation is necessary. |
| 3. |
Resuspend cells
in sterile serumcontaining culture medium containing 10% v/v dimethylsulfoxide
(DMSO).Work should proceed quickly to minimize the length of time
the cells are exposed to DMSO in the liquid state. The highest purity
DMSO should be used and, preferably, it should come from a bottle
that has not been previously opened or exposed to light for long periods
of time. |
| 4. |
Place the appropriate
volume and cell number into cryopreservation ampules – usually 2 x
105 to 5 x 106 cells/1 mL ampule. Plastic or glass ampules may be
used. However, plastic ampules with external silicone seals function
best when kept above liquid nitrogen temperature (i.e. in the vapor
phase). Immersion into the liquid nitrogen phase can result in liquid
nitrogen entering the ampule and the contents of the ampule spraying
out during the defrosting procedure. If storage in liquid nitrogen
is preferred, plastic ampules with internal O rings perform satisfactorily.
Glass ampules offer the best results due to the secure seal and the
rapidity with which the ampule can be defrosted, thereby allowing
for higher culture viability. However, they can be inconvenient to
use due to the requirements of flame sealing. |
| 5. |
Place the ampules
in a controlled-rate freezer and cool at a rate of 1°C/minute. If
a controlled-rate freezing apparatus is not available, adequate
results can be obtained by:
|
| |
| a. |
Placing
the ampule inside a one-inch foam-insulated box and keeping
the box at -70°C for 12 hours.
|
| b. |
Cooling
the ampules in the liquid nitrogen phase using a liquid nitrogen
canister insert. |
| c. |
Placing
the ampules in an isopropanol bath that is subsequently cooled
in a -70°C freezer. Nalgene offers a convenient container for
this method (Cat #5100-0001). |
| d. |
Placing
the ampules directly into a -20°C freezer for several hours
and then transferring to a -70°C for further cooling. |
| e. |
Placing
the ampules directly into a -70°C freezer.
|
|
| |
The last two
methods (d and e) are not ideal since the culture viability can be
affected and result in the loss of sensitive populations. These methods
should only be used when no other options are available. |
| 6. |
After freezing,
the ampules should be transferred to a liquid nitrogen-fill storage
vessel. Prolonged storage at temperatures above -135°C will result
in decreased viabilities. |
|
 |
