8. Resuspend the cell pellet in the prewarmed complete growth
medium.
9. Transfer the cell suspension into a 75 cm
2 T-flask. The preferred cell density is 5 Â 10
3 /cm
2 .
10. Place the T-flask into an 37
C incubator and grow the cells to
80–90% confluency before passaging.
3.1.2 Cell Passaging
Subculture cells when confluency reaches 80–90% respectively at
the log phase, or 2–3 per week. Work under aseptic conditions.
1. Prepare prewarmed (37
C) DMEM-HG growth medium,
supplemented with 10% (v/v) fetal bovine serum (FBS),
10 mM HEPES and 4 mM L -glutamine.
2. Cool PBS (w/o Ca
2+ and Mg
2+
).
3. Remove and discard the used medium.
4. Wash cells 2Â with 0.3 mL/cm
2 PBS.
5. Detach cells with 0.012 mL/cm
2 0.25% (w/v) trypsin at 37
C
for 8 min. Verify the detachment of the cells with an incident
light microscope (see Note 3).
6. Resuspend the detached cells in 0.12 mL/cm
2 complete
growth medium (see Note 4).
7. Take a 100 μL cell-sample in a 1.5 mL tube and determine the
cell concentration (see Subheading 3.1.3).
8. Centrifuge cell suspension at 300 Â g, 5 min, RT in a 15 or
50 mL centrifugation tube.
9. Remove and discard the supernatant (residual trypsin).
10. Resuspend the pellet in the appropriate volume of fresh prewarmed complete growth medium.
11. Seed out cells in an initial cell concentration of 5 Â 10
3 cell/
cm
2 into 75–175 cm
2 T-flask.
12. Incubate cells in an incubator at 37
C.
3.1.3 Cell Counting
and Cell Viability
Determination via
a Counting Chamber
1. Use manufacturer’s instruction to prepare the Neubauer
counting chamber.
2. Take a 200 μL sample from your culture in a 1.5 mL tube.
3. Prepare an appropriate dilution of the cell suspension with PBS
to obtain a final cell count of approx. 150 cells.
4. Mix 50 μL of cell suspension with 50 μL trypan blue 0.5%
solution. Wear protective gloves, clothing and eye and face
protection when working with trypan blue.
5. Rapidly transfer the stained cells to the prepared Neubauer
counting chamber and count the cells using the inverse light
microscope.
Viral Nanoplex Vaccine Production
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medium.
9. Transfer the cell suspension into a 75 cm
2 T-flask. The preferred cell density is 5 Â 10
3 /cm
2 .
10. Place the T-flask into an 37
C incubator and grow the cells to
80–90% confluency before passaging.
3.1.2 Cell Passaging
Subculture cells when confluency reaches 80–90% respectively at
the log phase, or 2–3 per week. Work under aseptic conditions.
1. Prepare prewarmed (37
C) DMEM-HG growth medium,
supplemented with 10% (v/v) fetal bovine serum (FBS),
10 mM HEPES and 4 mM L -glutamine.
2. Cool PBS (w/o Ca
2+ and Mg
2+
).
3. Remove and discard the used medium.
4. Wash cells 2Â with 0.3 mL/cm
2 PBS.
5. Detach cells with 0.012 mL/cm
2 0.25% (w/v) trypsin at 37
C
for 8 min. Verify the detachment of the cells with an incident
light microscope (see Note 3).
6. Resuspend the detached cells in 0.12 mL/cm
2 complete
growth medium (see Note 4).
7. Take a 100 μL cell-sample in a 1.5 mL tube and determine the
cell concentration (see Subheading 3.1.3).
8. Centrifuge cell suspension at 300 Â g, 5 min, RT in a 15 or
50 mL centrifugation tube.
9. Remove and discard the supernatant (residual trypsin).
10. Resuspend the pellet in the appropriate volume of fresh prewarmed complete growth medium.
11. Seed out cells in an initial cell concentration of 5 Â 10
3 cell/
cm
2 into 75–175 cm
2 T-flask.
12. Incubate cells in an incubator at 37
C.
3.1.3 Cell Counting
and Cell Viability
Determination via
a Counting Chamber
1. Use manufacturer’s instruction to prepare the Neubauer
counting chamber.
2. Take a 200 μL sample from your culture in a 1.5 mL tube.
3. Prepare an appropriate dilution of the cell suspension with PBS
to obtain a final cell count of approx. 150 cells.
4. Mix 50 μL of cell suspension with 50 μL trypan blue 0.5%
solution. Wear protective gloves, clothing and eye and face
protection when working with trypan blue.
5. Rapidly transfer the stained cells to the prepared Neubauer
counting chamber and count the cells using the inverse light
microscope.
Viral Nanoplex Vaccine Production
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