Biology Reference
In-Depth Information
29. The temperature set point should be made in incremental
steps in order to avoid the increase to very high
temperatures.
30. The replacement of water by medium is made by removing the
water by the sampling system. First the filter of the exhaust
cooler is clamped to start pressurizing the vessel. Then the
sampling system clamp is open so the water flows through
the sampling bottle. The flask with medium is connected
to the bioreactor and the medium is introduced by gravity. All
the connections are made under sterile conditions.
31. Calibration requires the electrode polarization for about 6 h
(connect the pO
2
cable to the electrode). Make sure that the
“zero drift” and “slope” values obtained are inside the limits
of the electrodes used. If not, start the calibration again.
32. The pH is controlled by aeration with a CO
2
gas mixture and
1 M NaHCO
3
added by a pre-calibrated feed pump. The base
solution (1 M NaHCO
3
) is prepared in MilliQ water and is
sterilized by filtration (Autoclaving thermally degrade the
solution). Other base solutions are sometimes also applied
(KOH, NaOH, NaHCO
2
, or Na
2
CO
2
) depending on the buf-
fer system of the culture medium.
33. For larger scales, since the inoculum corresponds normally a
dilution 1:6 (0.5 × 10
6
-3 × 10
6
cells/mL) of the starting cul-
ture, the vessel inoculation can be performed directly without
centrifugation.
34. The inoculation should be gentle to avoid cell damage and the
flask should be agitated in order to avoid deposition of cells. If
inoculum is not being transferred through gravity pump for
2-3 s air through the filter inlet of inoculation flask. After
inoculation, the jacket temperature normally increases consid-
erable to compensate the decrease in temperature inside the
reactor. To overcome this problem, add cooling water manu-
ally by pressing the switch for water supply. Alternatively, turn
off the temperature control during inoculation and turn back
on afterwards controlling the temperature back to 37 °C in a
slow rate.
35. Harvesting time depends on the downstream process purifica-
tion and corresponding working volume. Low-volume work-
ing volumes should be harvested up to 48 hpi, and centrifuged
to concentrate viruses by collecting only intracellular fraction.
Alternatively, if downstream process is managed to high work-
ing volumes, culture bulk can be harvested latter, without this
concentration step.
36. As a guide to calculate the settle volume and medium entrap-
ment by microcarriers, 1 g of Cytodex™, for example, has a
volume of 15-18 mL.
