53
Phase
I
Selection
Use cell strainer whenever
clumping is observed
Day 0 Selection (48 hr post-transfection): Perform a complete medium
exchange and seed two T-flasks at 5 x 10
5
viable cells/mL
1 – 10P/100M
2 – 20P/200M
Day 7: Is viability ≥ 30%?
Place T-flask back in incubator,
and sample 3–4 days later. Is
viability above the previous value?
Perform a complete medium
exchange, transfer cells to shake
flask at 3 x 10
5
viable cells/mL,
and passage every 3-4 days.
At each subsequent passage: is
viability ≥ 85%?
• Passage cells every
3-4 days at 3 x 10
5
viable cells/mL.
• Add drugs based
on the volume of
fresh medium.
• If dilution is less
than 2X, centrifuge
for a complete
medium exchange.
• Perform a complete
medium exchange to
seed two shake
flasks at 4 x 10
5
viable
cells/mL for Phase II
selection (30P/500M,
50P/1,000M).
• Freeze at least 3 vials
of Phase I pool.
• Perform productivity
assessment.
Centrifuge cells for a complete
medium exchange and seed at
3 x 10
5
viable cells/mL (reduce
volume if needed). After day 10-11,
a complete medium exchange is
required once a week until the
culture is moved to shake flask.
Drugs 10P/100M 20P/200M
Puromycin 1 L/mL 2 L/mL
MTX (0.1 mM stock) 1 L/mL 2 L/mL
NO
NO
NO
YES
YES YES T-flask Shake flask
Each time cells are handled, if clumping is observed, strain cells before counting.
YES YES
FAQ Sections
Figure 9. Freedom
®
CHO-S
®
Phase I selection flowchart.
Life Technologies
™
| Freedom
®
CHO-S
®
Kit FAQ
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