Biology Reference
In-Depth Information
4. Optional: Add polybrene to a final concentration of 9 μg/mL
to facilitate the interaction between cells and adenoviral parti-
cles. It is highly recommended when the IU/PP ratio of your
pre-stock is worse than 1/100.
5. At the optimal harvest time collect cells and supernatant in
50-mL tubes.
6. Centrifuge for 5 min at 300 ×
g
. Pool the pellets and resuspend
it in 15-18 mL of supernatant.
7. Perform three freeze/thaw rounds (−80 °C/37 °C).
8. After the last thaw, centrifuge the cell lysate for 5 min at
4,500 ×
g
and recover the supernatant.
9. Purify the virus as described in Subheading
3.2
.
3.7 Production in
Suspension 211BS
Cell Cultures
There are some adenoviral serotypes whose productivity per cell is
very inefficient [
10
,
11
]. Therefore classical standard amplification
procedures must be avoided and more efficient systems like
large-scale production in cell suspension are highly recommended.
This is the case of the enteric adenoviruses as the Human
Adenovirus 40 serotype (HAdV-40), which due to their inefficient
productivity are also known as
fastidious virus
. Interestingly,
chimeric Adenovirus 5/40S (HAdV-5/40S) carrying the HAdV-
40 short fiber on the HAdV-5 capsid preserves the enteric tropism
of the HAdV-5/40S [
12
] but also the inefficient productivity.
To address this issue we have developed a protocol (
see
Fig.
2
)
based on the addition of polybrene during the amplification steps
in combination with the use of 211BS cells (expressing constitu-
tively HAdV-5 fiber) as producer cells [
9
]. First, polybrene inter-
acts with negatively charged adenoviral capsids facilitating their
interaction with the cell membrane [
13
,
14
], which increases virus
transduction. Second, 211BS cells allow to generate mosaic virions
containing both F5 and F40S fiber proteins. The fiber mosaicism
improves the infectivity of the chimeric virions during the amplifi-
cation cycles by facilitating an efficient cell entry mediated by the
CAR-F5 interaction, which together with the addition of poly-
brene reduces the number of amplification cycles and the duration
of the process. Finally, to further facilitate production of the chi-
meric vectors, 211B cells are grown in suspension, thus allowing to
easily up-scale the production process in bioreactors.
The procedure described here has been designed to use a single
amplification step in a single flask of 211BS. However, variations in
the number of flasks, or the number of amplification steps can be
adapted easily.
3.7.1 First Viral
Amplification Step in
211BS from Pre-stock
with Polybrene
