l
Perform a mass balance for the dsDNA content and the total
protein content. Again, consider the mass/volume of each fraction. In this case, the reduction of both parameters in the
retentate fraction is the value of interest. The higher the reduction, the better (see Note 22).
3.4 Transduction
of hMSC with BV
All steps must be done under sterile conditions.
3.4.1 Thawing
and Cultivation of hMSCs
l
Add 0.12 mL of growth medium per cm
2 growth area into a
T-flask (e.g., 3 mL growth medium in a T25-flask).
l
Incubate the T-flasks at 37
C and 5% CO 2 in an incubator to
adjust pH and temperature.
l
Thaw the cryovial with MSCs in a water bath (37
C).
l
When the cells have been thawed, pipet the complete cell suspension into the T-flask.
l
Incubate the cells at 37
C and 5% CO 2 in an incubator.
l
Monitor the growth microscopically every day.
l
If the cells are 80% confluent, remove the cell culture medium
with a sterile pipette and an aspiration system.
l
Wash the cells twice with 0.3 mL PBS per cm2 growth area (e.g.,
7.5 mL of PBS in a T25-flask) (see Note 23).
l
Add 0.012 mL/cm of 0.25% (w/v) trypsin–EDTA at 37
C
(e.g., 0.3 mL of trypsin solution in a T25-flask).
l
Incubate for 5 min at 37
C (see Note 24).
l
Add 0.12 mL/cm of growth medium and mix it by pipetting to
get a homogeneous cell suspension.
l
Take a 100 μL cell sample in a 1.5 mL tube and determine the
cell concentration.
l
Centrifuge the cell suspension (300 Â g, 5 min, RT, in a 15 or
50 mL centrifugation tube).
l
Remove and discard the supernatant (to remove residual
trypsin).
l
Resuspend the pellet in the appropriate volume of a fresh prewarmed complete growth medium.
3.4.2 Determination
of hMSC Concentration
and Viability
l
Use the manufacturer’s instructions to prepare the Neubauer
counting chamber.
l
Take the 100 μL cell sample from your culture in a 1.5 mL tube.
l
Dilute the cell suspension with PBS, if necessary (see Note 25).
l
Mix 50 μL of cell suspension with 50 μL trypan blue solution (see
Note 26).
Baculovirus-Mediated Gene Transfer to Stem Cells
381
Perform a mass balance for the dsDNA content and the total
protein content. Again, consider the mass/volume of each fraction. In this case, the reduction of both parameters in the
retentate fraction is the value of interest. The higher the reduction, the better (see Note 22).
3.4 Transduction
of hMSC with BV
All steps must be done under sterile conditions.
3.4.1 Thawing
and Cultivation of hMSCs
l
Add 0.12 mL of growth medium per cm
2 growth area into a
T-flask (e.g., 3 mL growth medium in a T25-flask).
l
Incubate the T-flasks at 37
C and 5% CO 2 in an incubator to
adjust pH and temperature.
l
Thaw the cryovial with MSCs in a water bath (37
C).
l
When the cells have been thawed, pipet the complete cell suspension into the T-flask.
l
Incubate the cells at 37
C and 5% CO 2 in an incubator.
l
Monitor the growth microscopically every day.
l
If the cells are 80% confluent, remove the cell culture medium
with a sterile pipette and an aspiration system.
l
Wash the cells twice with 0.3 mL PBS per cm2 growth area (e.g.,
7.5 mL of PBS in a T25-flask) (see Note 23).
l
Add 0.012 mL/cm of 0.25% (w/v) trypsin–EDTA at 37
C
(e.g., 0.3 mL of trypsin solution in a T25-flask).
l
Incubate for 5 min at 37
C (see Note 24).
l
Add 0.12 mL/cm of growth medium and mix it by pipetting to
get a homogeneous cell suspension.
l
Take a 100 μL cell sample in a 1.5 mL tube and determine the
cell concentration.
l
Centrifuge the cell suspension (300 Â g, 5 min, RT, in a 15 or
50 mL centrifugation tube).
l
Remove and discard the supernatant (to remove residual
trypsin).
l
Resuspend the pellet in the appropriate volume of a fresh prewarmed complete growth medium.
3.4.2 Determination
of hMSC Concentration
and Viability
l
Use the manufacturer’s instructions to prepare the Neubauer
counting chamber.
l
Take the 100 μL cell sample from your culture in a 1.5 mL tube.
l
Dilute the cell suspension with PBS, if necessary (see Note 25).
l
Mix 50 μL of cell suspension with 50 μL trypan blue solution (see
Note 26).
Baculovirus-Mediated Gene Transfer to Stem Cells
381