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Direct Adaptation
Maintain stock cells
in classical medium with serum supplementation.
Upon subsequent passage,
following trypsinization, seed two new T-flasks. One flask will be maintained
at the current serum concentration, while the other will be reduced to
half that serum concentration. This serum depletion process is repeated
until reaching a concentration of 2.5% FBS, at which time cells will grow
readily in HyQSFM4HEK293.
| 1. |
A bottle of
DMEM is supplemented with 10% FBS.
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| 2. |
Cells cultured
in T-flasks are passaged, using trypsin, during logarithmic phase
growth and 2 new flasks (A & B) are seeded. |
| 3. |
Flask A is seeded
at appropriate density (4.0 x 104
cells/cm2) and fed DMEM with serum
supplementation of 10% (or appropriate level). |
| 4. |
Flask B is seeded
at the same density (4.0 x 104 cells/cm2)
and fed half the serum concentration of flask A. |
| 5. |
Cultures are
incubated for 3.5 days then observed for growth. Cultures should be
at least 70% and not more than 95% confluent. If these parameters
are attained within the 3.5 day interval proceed to the next step,
otherwise passage cultures and maintain the same serum concentrations,
repeating steps 3 through 5. |
| 6. |
Flask A is passaged
as above, but serum concentration is reduced to that of Flask B. |
| 7. |
Flask B is passaged
as above, but serum concentration is reduced by one half (from 10%
to 5%, and so forth). |
| 8. |
Repeat steps
5 through 7 until serum concentration in flask B is 2.5% and cells
are growing well. This process may take approximately two weeks. |
| 9. |
Maintain flask
A as a backup during the proteinfree adaptation. Upon passage of logarithmic
phase culture in flask B, feed again DMEM/2.5% and allow cells to
attach and spread during incubation for 24 hours. Following the 24
hour incubation remove the serum-containing medium from flask B and
replace with SFM4HEK293. |
| 10. |
Maintain a passage
interval of 3.5 days. Pass cells in SFM4HEK293 without trypsin if
possible (cells will not attach firmly and may aggregate in static
cultures, pipette gently to disaggregate before counting). If trypsin
must be used (very unlikely), inhibit with soybean trypsin inhibitor
and/or centrifuge to remove all trypsin/inhibitor complex, or trypsin
alone. Seed into new T-flasks to ensure residual trypsin is minimized.
The complete removal of trypsin is very important in protein-free
cultures. |
| 11. |
Maintain the
cells in static culture (T-flasks) using SFM4HEK293 for approximately
3 passages before initiating shaken or stirred cultures. |
Cryopreservation
Cells maintained in
HyQ SFM4HEK293 during logarithmic phase growth with viabilities greater
than 90% may be cryopreserved using the following procedure:
| 1. |
Centrifuge cells
at 100x g to produce a cell pellet. Remove supernatant and store
for later use.
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| 2. |
Gently resuspend
pellet at a concentration of 10 x 106
cells/mL in the conditioned medium. Slowly add an equal volume of
fresh SFM4HEK293 supplemented with 20% DMSO to adjust cell density
to 5 x 106 cells/mL. Transfer this
suspension to plastic Nalge 1.8 mL cryovials at a volume of 1.0 mL.
Cells should be frozen at a rate of -1.0°C/minute to reach a final
temperature of -70°C. |
| 3. |
Once the vials
have reached the final temperature transfer them to gaseousphase liquid
nitrogen storage tanks. Cells should not remain at -70°C for longer
than 24 hours. |
| 4. |
To thaw cells
remove vials from liquid nitrogen storage and place immediately into
a 37°C waterbath with gentle agitation, allowing vial to warm to 25°C. |
| 5. |
Disinfect the
vial using 70% isopropyl alcohol. Transfer the cell contents to a
15 mL centrifuge tube, adding an equal volume of fresh, prewarmed
(25°C) SFM4HEK293 drop-wise to the suspension. |
| 6. |
Allow the tube
to stand for 5 minutes, then add 3 mL of fresh, pre-warmed SFM4HEK293
drop-wise to bring the total volume to 5 mL. |
| 7. |
Again, allow
the tube to stand for 5 minutes. |
| 8. |
Finally add an
additional 5 mL of fresh, prewarmed SFM4HEK293 drop-wise to bring
the volume to 10 mL at an expected cell density of 5 x 105
cells. Centrifuge the cells at 100x g for 10 minutes, pelleting the
cells to remove the cryoprotectant. Remove the supernatant and gently
resuspend the cells in 10 mL of fresh, prewarmed SFM4HEK293 medium.
A small sample of the suspension may be counted to determine actual
cell density with the realization that viability assessment will not
be accurate for at least 24 hours. |
| 9. |
Place the cell
suspension into a 25 cm2 T-flask
and incubate at 37°C, 5% CO2. Shaken or stirred cultures may be initiated
within 3.5 days, or when average viability is >90%, and a normal population
doubling time (24-36 hours) is observed. |
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