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This protocol is designed
to adapt High Five (BTI-Tn-4) cells to serum-free suspension culture in
HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately
two weeks and the resulting adapted cells have doubling times of 16-20
hours and minimal clumping in suspension culture. The key is to obtain
High Five cells which have not been adapted to any other serum-free medium.
| 1. |
Rapidly thaw
a 1 mL vial of High Five cells (Invitrogen Cat. No. B855-02) and
pipette into a T-75 cell culture flask containing 15 mL of pre-warmed
HyQ SFX-Insect. Incubate flask, cap loosened, in a humidified 28°C
incubator.
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| 2. |
On day 3 post-planting
(P.P.), when the monolayer culture is 80-90% confluent, subculture
flask 1:5 by adding 3 mL of cell suspension from the original Passage
X+1 culture into each of 5 x T-75's containing 12 mL of pre-warmed
media. Cells are easily detached from the monolayer by using 2.3 "wrist-snap"
shaking motions. |
| 3. |
On day 3 P.P.,
when the monolayers are at 80% confluent, detach cells from the passage
X+2 flasks, count, and set up 2 x 35 mL shaker flask cultures at 3
x 105 viable cells/mL (in 125 mL
Corning Erlenmeyer Shaker Flasks) and 5 x T-75's (split 1:5 as back-ups
in case the initial adaptation to suspension culture doesn't work).
Incubate shaker flasks, caps loosened, at 27.5-28.5°C with a shaking
speed of 150 rpm. |
| 4. |
Monitor cell
growth of the passages X+3 shaker cultures and subcultures on day
3 P.P. when the suspension cultures attain a density of 2.3 x 106
cells/mL. If the first attempt at suspension culture fails (minimal
growth and/or viabilities <80% on day 3-4 P.P.), reseed another set
of 35 mL shake flask cultures at 5 x 105
viable cells/mL using the cells in the back-up monolayer culture set-up
in Step 3 above. |
| 5. |
Subculture suspension
cultures every 3 days with seeding densities of 3 x 105
viable cells/mL. In three passages the cells will be completely adapted
to suspension culture in HyQ SFX-Insect with minimal clumping and
viabilities >95%. We use total culture volumes of 75 mL in the 250
mL Corning Erlenmeyer flasks, and 150 mL in the 500 mL Corning Erlenmeyer
flasks. We recommend maintaining the monolayer cultures as back-ups
until the adaptation has been successfully completed. Split monolayer
cultures every 3-4 days when they reach 80-90% confluency using split
ratios of 1:5 to 1:10. |
| 6. |
As the cells
are adapted to suspension culture, simultaneously scale-up a replicate
culture and freeze down a large number of High Five cells at passage
X+5 for use as your low passage master or working cell bank. Use HyQ
SFX-Insect (complete with L-Glutamine with HyQ SFX-Insect) supplemented
with 15% DMSO (final 7.5% DMSO) and 50% spent medium from the culture
in which the cells were grown, prior to cryopreservation. |
*High
Five is a trademark of Invitrogen Corp.
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