Biology Reference
In-Depth Information
9. MDCK-E1 cells growing in microcarriers, after 2 days of inoc-
ulation, with final concentration of 1 × 10
6
cells/mL.
10. Optipro™ SFM supplemented with 4 mM glutamine (
see
Note 2
).
11. Purified first generation CAV-2 with GFP transgene or other
gene of interest (
see
Note 6
).
3
Methods
Cell manipulations are performed under sterile conditions in a
laminar flow wood and cell-culture reagents are pre-warmed to
37 °C prior to use.
3.1
Cell Culture
HEK 293 cells suspension adapted are maintained in 500 mL
Erlenmeyer flasks (80-100 mL working volume) and are split twice
a week when attaining 2-3 × 10
6
cells/mL. The cells are kept inside
an incubator at 37 °C with 8 % CO
2
in air in an orbital shaker at
130 rpm (
see
Note 9
).
1. Take 0.5-1 mL of cell suspension sample from an Erlenmeyer
with cells in culture for 3-4 days to an eppendorf tube.
2. Determine cell concentration (
see
Subheading
3.2.1
).
3. Calculate the volume of cells necessary to inoculate an
Erlenmeyer flask at 0.4-0.5 × 10
6
cells/mL with 80-100 mL.
4. Add the volume of cells suspension to a prefilled Erlenmeyer
with the necessary volume of fresh Ex-Cell
®
293 serum-free
medium to attain the final working volume and resuspend well.
5. Place Erlenmeyer in incubator until next cell passage (3-4 days).
3.1.1
HEK 293 Cells
Culture
MDCK-E1 cells are maintained in 150 cm
2
T-flask in an incubator
at 37 °C, 5 % CO
2
in air. After reaching ~90 % confluence (twice a
week), cells are passed to a new T-flask with a splitting ratio of 1:20.
3.1.2
MDCK-E1 Cells
Culture
1. Remove culture medium from the T-flask.
2. Rinse monolayer with 5 mL of D-PBS.
3. Remove D-PBS and add 5 mL of Trypsin-EDTA 0.25 %.
4. Place the T-Flask in the incubator until cell detachment
becomes evident (~10 min).
5. Tap the side of the T-Flask to detach all cells.
6. Add 5 mL of Optipro™ SFM medium and resuspend cells.
7. Transfer cells suspension to a centrifugation tube and centri-
fuge the corresponding inoculum volume at 300 ×
g
for 10 min
at room temperature.
8. Fill a new T-flask with 25 mL of fresh Optipro™ SFM medium.
9. Discard the supernatant of the centrifugation.
