Biology Reference
In-Depth Information
9. Allow the beads to settle-down, aspirate and discard the
D-PBS and add the same volume of sterile D-PBS (final con-
centration 20 g/L). At this stage, and prior to use, microcar-
riers can be stored at 4 °C.
10. Prior to cell inoculation, aspirate and discard D-PBS, then
rinse the beads twice in small amount (approximately 20 % of
the final volume of inoculation flask) of culture medium.
At this point, forty T-Flasks of 175 cm
2
(or equivalent)
with 80-90 % of MDCK-E1 cells confluence are required
(
see
Fig.
4
), as well as the previously prepared microcarriers
and bioreactor unit.
11. Harvest cells from standard cultures (
see
Subheading
3.1.2
)
and determine cell concentration using the trypan blue
exclusion method (
see
Subheading
3.2.1
).
12. Centrifuge the corresponding volume of cells to ensure an
inoculum of 2 × 10
5
cells/mL at 300 ×
g
and during 10 min.
13. Suspend cells in inoculation culture medium
(
see
Subheading
2.3.3
) in order to bring the bioreactor vol-
ume to 100 % (2 L) of the working volume. Consider the
volume occupied by microcarriers when adding the corre-
sponding inoculation culture medium volume (
see
Note 36
).
14. Confirm cell concentration and transfer cells to inoculation
flask.
15. Connect the inoculation flask to the bioreactor and intro-
duce the cells into the bioreactor by gravity, opening the
clamps of the connected tubes (
see
Note 34
).
16. Monitor cell growth by sampling and counting cells
(
see
Subheading
3.2.1
). When cells reach a concentration of
1 × 10
6
cells/mL, or 2 days after inoculation, infect cells to
produce CAV-2 vectors.
Once the concentration of 1 × 10
6
cells/mL is reached,
cells can be infected. Culture medium is replaced during
infection to maximize production of CAV-2 vectors.
17. Prepare medium/infection flask with fresh medium
(Optipro™ SFM supplemented with 4 mM of glutamine) to
exchange medium at infection. Consider the volume that
can be removed without removing the microcarriers from
the bioreactor to fill the medium/infection flask.
18. Add the corresponding amount of purified vectors (
see
Note 6
)
to the fresh medium in order to infect cells with an MOI of 5.
19. Perform the medium exchange/cell infection: stop bioreac-
tor agitation, let microcarriers settle-down and remove
medium using the extra sampling tube. Add the fresh
medium with viral vectors and turn on the stirring.