Culturing Sf-9 Insect Cells
Thawing frozen cells
1. Place a bottle of Sf-900 II SFM medium (located in 4˚ C refrigerator) in the 37˚ C T.C.
water bath for about five minutes or so. It does not need to heat up to 37˚ C. Remove
bottle and allow medium to equilibrate to room temperature.
2. Prepare a bottle of Sf-900 II SFM (equilibrated to room temperature) plus 0.5X
penicillin/streptomycin antibiotics. For serum-free culture, never use more than 0.5X
antibiotics.
3. Remove a vial of cells from the liquid nitrogen tank and thaw quickly in 37˚ C T.C. water
bath. Immediately upon thawing, add thawed cells (using a pasteur pipet) to a 15 ml tube
containing 5 ml of Sf-900 II SFM. Spin in low speed centrifuge for 5 min.
4. While cells are being harvested, label 4x or 5x10 cm2 plates with the appropriate
information. Add 8 ml of medium (plus antibiotics) to each plate.
5. After cells have been harvested, remove supernatant by aspiration. Add 10 ml of medium
(plus antibiotics) to the cell pellet and resuspend cells by pipetting up and down.
6. Add 2 ml of cell suspension to each plate, shaking the plate around to promote even
distribution of the cells.
7. Incubate in the 28˚ C incubator (non-humidified, no CO2) for 4 days, checking cells each
day. Expect some cell death.
8. After 4 days, change medium* if cells are not at the desired density. For the first two
passages, it is important to allow cells to become almost entirely confluent.
9. Allow cells to incubate for another 3-4 days (or until they become confluent) before
splitting them.
*Remember to change medium every 3-4 days if cells are not confluent.
Splitting cells
When cells become about 80-90 % confluent, split them 1:5 (or less), depending on when you
want the plates to become confluent again. I have found that if cells are confluent one day
before you are planning to use them, you can split them 1:2 or 1:3 and still have good results.
1. Equilibrate the Sf-900 II SFM (plus antibiotics) to room temperature using the procedure
described above.
2.
Remove old medium from each plate to be split (do one plate at a time) and resuspend
cells in or 10 ml (or appropriate volume) of equilibrated medium. These cells do not
need to be treated with trypsin to remove from plate.
3. Prepare 5x10 cm2 plates (or appropriate number) by adding 8 ml of equilibrated medium
to each.
4. Add 2 ml of cell suspension to each plate, shaking plate to allow for even distribution of
the cells.
5. Incubate at 28˚ until use. Remember to change medium or split every 3-4 days.
Freezing cells
1. Grow cells to desired confluency. Determine viable cell count and calculate the amount
of cryopreservation medium needed to yield a final cell density of 1 x 107 to 2 x 107 cells
/ ml.
2. Prepare an appropriate amount of cryopreservation medium consisting of 7.5 % DMSO
in 50 % fresh Sf-900 II SFM and 50 % conditioned medium (medium from a 2-3 day old
culture; sterile filtered). Place on ice until ready for use.
3. Remove old medium from cells and resuspend in 10 ml of fresh medium. If freezing
more than one plate at a time, you may resuspend cells from all plates in just 10 ml total.
Add to a 15 ml tube and spin in low-speed centrifuge for 5 min.
4. While cells are spinning, prepare an appropriate number of 2 ml cryogenic tubes to yield
1 ml of cells per tube.
5. Resuspend cells in appropriate amount of cryopreservation medium to give a final cell
density of 1 x 107 to 2 x 107 cells / ml.
6. Add 1 ml of cells to each cryogenic tube. Place in -20˚ C freezer for about 20 min.
Remove and place in –80˚ C freezer overnight.
7. Remove cells from –80˚ C freezer and place in liquid nitrogen tank.