Biology Reference
In-Depth Information
6. Monitor cell growth by sampling immediately after inocula-
tion and at least every 24 h until the desired cell concentration
to infect is reached. Cell infection can be performed when
HEK 293 cells attain 1 × 10
6
cells/mL in batch mode.
7. Prepare infection flask with the corresponding amount of
purified vectors (
see
Note 6
) and fresh medium in order to
infect cells with an MOI of five infectious particles per cell.
8. Connect the infection flask to the bioreactor and infect cells.
9. Sample the culture at least every 24 h for cell and virus analy-
sis. Virus samples can be stored at −80 °C until analysis.
10. Harvest the culture bulk at 36-72 hpi (through the sampling
system) creating pressure inside the vessel (
see
Note 35
).
Considering that MDCK-E1 cells are strictly adherent, microcarri-
ers were used to develop a scalable stirred culture system. Thus, the
production of CAV-2 vectors requires (1) silanization of glass
material and (2) microcarriers preparation before (3) bioreactor
inoculation and (4) cell infection and harvesting.
Glassware (bioreactor vessel, addition flasks, etc.) must be silanized
to prevent microcarrier/cell adherence to the glass surface.
3.3.3
CAV-2 Production
1. Fill the glass container with the washing solution and wait
for at least 3 h and remove the solution (
see
Note 7
).
2. Wash the glass container with water and let it dry completely
(to evaporate the remaining water). If necessary, leave the
glass container at 37 °C or overnight.
3. Add a small volume of dimethildichorosilane to the glass
container(s) and rotate the container(s) to ensure the contact
with the entire surface. Remove the excess solution (
see
Note 7
).
4. Wash the glass container with a small volume of toluene and
let it dry in the air protecting the container to avoid dust
accumulation.
5. To ensure that the glass materials have been properly
silanized, place a drop of water in the container: If the drop
sticks to the glass, all the procedure must be repeated other-
wise the material can be used.
6. Autoclave the container before use.
Next steps are designed to prepare microcarriers for proper
cell inoculation. In order to facilitate inoculation procedures,
microcarriers are directly prepared in inoculation flask, used
to insert cell inoculum inside bioreactor.
7. Weight 6 g of microcarrier beads (3 g/L) and transfer them
to a silanized inoculation flask.
8. Suspend the microcarrier beads at 20 g/L in PBS and auto-
clave at 121 °C for 15 min, liquid cycle.
