Biology Reference
In-Depth Information
20. Harvest the culture bulk at 36-72 hpi (through the sam-
pling system) creating pressure inside the vessel (
see
Note
35
). Alternatively separate intracellular and extracellular
fractions by letting microcarriers settle-down and collecting
supernatant to other container.
Prior to bioreactor disassembling and cleaning, sterilization is per-
formed to inactivate any remaining viruses.
1. Switch off all the controllers.
2. Disconnect the cables and tubings connected to the control
unit.
3. Remove the motor.
4. Prepare the bioreactor for sterilization to inactive viruses
(
see
Note 26
).
5. Autoclave the bioreactor.
6. Clean and store pO
2
and pH electrodes.
7. Remove the tubings installed on the top-plate and leave them
overnight into lab disinfectants dissolved in warm water.
8. Wash the vessel and all components, rinse everything with
70 % ethanol and let it dry.
9. Store everything in the appropriated place until next use.
3.3.4 Bioreactor
Disassembling
4
Notes
1. HEK 293 cells can be easily adapted to suspension to
Ex-Cell
®
293 serum-free medium or other commercially
available cell culture media following media suppliers'
instructions (directly or sequentially). MDCK-E1 cells were
adapted sequentially to Optipro™ SFM medium following
supplier instructions.
2. GlutaMAX™ media (Gibco) can be used in replacement to
glutamine. It is a standard cell-culture media that contain a
stabilized form of l-glutamine, the dipeptide l-alanyl-
l-glutamine, that prevents degradation and ammonia build-
up even during long-term cultures. Glutamine is normally
used when the metabolism of the cells during growth and
production is necessary to be evaluated since most metabo-
lite analyzers have sensors for glutamine and not
GlutaMAX™ media.
3. Several cell counting methods can be used including direct
counting with hemocytometer or automatic cell counters. The
method described herein uses Trypan blue, a dye that enters
the cells whose membrane is damaged. In this way, when
