| 1. |
Defrost the
vial in a 37°C water bath with constant, moderate agitation, until
ice in the ampule is no longer visible.
|
| 2. |
Continue to warm
the ampule in the water bath for 30 seconds with gentle agitation. |
| 3. |
Immediately disinfect
the vial with 70% ethanol. |
| 4. |
Working in a
hood, open the vial and transfer the contents to a sterile 15 mL tube.
(Note: if thawing a glass vial, open the vial by wrapping the disinfected
vial in a sterile gauze pad and breaking the neck of the pre-scored
ampule). |
| 5. |
Add 1.5 mL of
culture medium (serum containing medium) that has been prewarmed
to 37°C.
|
| 6. |
Allow to stand
for 5 minutes. |
| 7. |
Add 3 mL of prewarmed
culture medium and allow to stand for 5 minutes. |
| 8. |
Add 6 mL of
prewarmed culture medium and allow to stand for 5 minutes. |
| 9. |
Centrifuge the
suspended cells at 200 x g for 10 minutes. |
| 10. |
Decant the medium
and gently resuspend the cell pellet in 25 mL of culture medium and
transfer into one 75 cm2 culture flask. |
| 11. |
Observe the cells
microscopically to estimate cell viability and then place them in
an incubator. |
| 12. |
After overnight
incubation, the cells should be observed so that the viability may
again be evaluated. A trypan blue dye exclusion stain may be appropriate
when precise viability assay is desired. |
|
 |
