Adapting Sf9 Cells to Serum-free Media
Direct Adaptation:
1.
2.
3.
4.
5.
Cells growing in 5-10% FBS supplemented medium are transferred
directly into new media which has been prewarmed.
Cell inocula should be ~2.5 X 105 cells/ml.
When cell yield reaches 1-3 X 106 cells/ml (4 to 6 days post planting),
subculture the cells to an original cell density of ~2.5 X 105 cells/ml.
When the cells are fully adapted to the new media, they will reach cell
density in excess of 4 X 106 cells/ml after about 7 days in culture.
Stock cultures of the adapted cells should be should be subcultured in
the serum-free media every 4 to 5 days when the cell yield reaches 1 to
3 X 105 cells/ml with at least 90% viability.
Sequential Adaptation: (weaning procedure)
1.
Subculture the cell suspension grown in conventional media with 510% serum into a 50:50 ration of serum-free media to conventional
media. Allow cell density to reach in excess of 5 X 105 cells/ml.
2.
Subculture above cell suspension in equal volume of serum-free
media
5
to obtain a cell density of about 2.5 X 10 cells/ml.
3.
Continue to subculture the cell suspension in serum-free media until
the concentration of serum decreases to 0.1% with about 85% viability,
each time allowing the cell density to achieve at least 5 X 105 cells/ml.
4.
Subculture the cell suspension into serum-free media at a cell inocula
of 2.5 X 105 cells/ml.
5.
Pass the cells in serum-free media and after approximately 4 to 6 days
post planting, when the cell yield reaches 1 to 3 X 105 cells/ml with 90%
viability, subculture again to an original cell inocula of 2.5 X 105
cells/ml.
6.
After several passages, the yield should exceed 4 X 106 cells/ml after
about 7 days in culture. At this stage, the cells are adapted to serumfree media.