3.4 Suspension
Culture of Sf-9 Cells
This chapter comprises a brief description of the strain maintenance, seeding train, preparation, and initiation of an insect-cell
bioprocess in a bioreactor. The authors point out, that a closely
related and even more detailed description of the setup handling,
process initiation and sampling has previously been published in
Animal Cell Biotechnology—Methods and Protocols in 2019 by
K€ aßer et al., with a focus on the cultivation of stable insect cell
lines [8].
3.4.1 Thawing the Cells
1. Thaw a cryovial containing 2 Â 10
7 Sf-9 cells/mL in
your hand.
2. Gently add 9 mL of insect cell medium to the cells and mix
them properly by pipetting them gently up and down (see Note
8).
3. Centrifuge the cell suspension at 200 Â g for 5 min.
4. Aspirate the supernatant and therefore remove the DMSO (see
Note 9).
5. Resuspend the cells in 10 mL fresh medium.
6. Transfer the 10 mL of cell suspension into a 100 mL baffled
Erlenmeyer flask.
7. Cultivate the cells in the incubator on the orbital shaker at
28
C and a suitable shaking rate.
8. After 3–4 days, passage cells to a concentration of
0.5 Â 10
6 cells/mL (see Note 10).
3.4.2 Cell Counting by
Trypan Blue Staining
1. Assemble the counting chamber as recommended by the
manufacturer.
2. Take a sample (about 100 μL).
3. Dilute sample by mixing with PBS to obtain a concentration
suitable for counting (typical ratios are 1:1 to 1:20).
4. Mix 75 μL of diluted cell suspension with 75 μL trypan blue (see
Note 11).
5. Pipet the stained cell suspension immediately into the counting
chamber.
6. Count the cells and calculate the cell density according to the
manufacturer’s instructions (see Note 12).
3.4.3 Passaging the Cells
1. Count cells according to Subheading 3.4.2 using the Neubauer
chamber (see Note 13).
2. Calculate the volume of cell suspension and medium needed to
passage the cells to 0.5 Â 10
6 cells/mL (see Note 10).
3. Pipet the calculated volume of fresh medium into a new shake
flask and add the calculated volume of the cell suspension (see
Note 8).
108
Julie Harnischfeger et al.
Culture of Sf-9 Cells
This chapter comprises a brief description of the strain maintenance, seeding train, preparation, and initiation of an insect-cell
bioprocess in a bioreactor. The authors point out, that a closely
related and even more detailed description of the setup handling,
process initiation and sampling has previously been published in
Animal Cell Biotechnology—Methods and Protocols in 2019 by
K€ aßer et al., with a focus on the cultivation of stable insect cell
lines [8].
3.4.1 Thawing the Cells
1. Thaw a cryovial containing 2 Â 10
7 Sf-9 cells/mL in
your hand.
2. Gently add 9 mL of insect cell medium to the cells and mix
them properly by pipetting them gently up and down (see Note
8).
3. Centrifuge the cell suspension at 200 Â g for 5 min.
4. Aspirate the supernatant and therefore remove the DMSO (see
Note 9).
5. Resuspend the cells in 10 mL fresh medium.
6. Transfer the 10 mL of cell suspension into a 100 mL baffled
Erlenmeyer flask.
7. Cultivate the cells in the incubator on the orbital shaker at
28
C and a suitable shaking rate.
8. After 3–4 days, passage cells to a concentration of
0.5 Â 10
6 cells/mL (see Note 10).
3.4.2 Cell Counting by
Trypan Blue Staining
1. Assemble the counting chamber as recommended by the
manufacturer.
2. Take a sample (about 100 μL).
3. Dilute sample by mixing with PBS to obtain a concentration
suitable for counting (typical ratios are 1:1 to 1:20).
4. Mix 75 μL of diluted cell suspension with 75 μL trypan blue (see
Note 11).
5. Pipet the stained cell suspension immediately into the counting
chamber.
6. Count the cells and calculate the cell density according to the
manufacturer’s instructions (see Note 12).
3.4.3 Passaging the Cells
1. Count cells according to Subheading 3.4.2 using the Neubauer
chamber (see Note 13).
2. Calculate the volume of cell suspension and medium needed to
passage the cells to 0.5 Â 10
6 cells/mL (see Note 10).
3. Pipet the calculated volume of fresh medium into a new shake
flask and add the calculated volume of the cell suspension (see
Note 8).
108
Julie Harnischfeger et al.