3

Methods

3.1

Cell Expansion

1. Vials of frozen cells are thawed and the cells are expanded to

have enough biomass to inoculate the fed-batch bioreactor.

Usually, expansion is performed using shake flasks or other

vessels of increasing size. Duration between passages is

2–4 days. Initial VCC (Viable Cells Concentration) for pas-

sages will depend on the passage duration and is typically in the

range of 0.3–1 million cells per mL (see Notes 10 and 11).

3.2

Probe Calibration

and Sterilization

1. When not using (pre-installed) single-use probes, pH probe

calibration is performed prior to sterilization (see Note 12).

2. For DO probes, it is advisable to confirm that they are in proper

working condition (typically by checking the electric signal in

air) before the sterilization (see Note 13).

3. Depending on the type of measurement system and/or biore-

actor used, the same might need to be performed for tempera-

ture- and other probes.

4. Probes are then packaged in autoclaving bags and sterilized (see

Note 14). In case of glass vessel use, probes could be inserted

before the sterilization of the bioreactor and sterilized with it

(see Note 15).

3.3

Bioreactor

Preparation

1. In case of single-use bioreactors, pre-sterilized probes need to

be inserted aseptically. Clamp all lines and remove protection to

insert both DO and pH probes under a laminar flow hood.

2. For both single-use and glass vessels, transfer the bioreactor

next to the control tower. Connect the overlay gas line to the

filter, same for the sparger line. Unclamp the exhaust gas line if

necessary. Add air (at a constant minimal flow) through the

overlay to maintain an overpressure inside the bioreactor.

3. Connect pH and DO probes to the control towers and confirm

proper signal transmission (see Note 16).

4. Put in place the motor (see Note 17), temperature probe, and

electrical heating blanket/heating system around the bioreac-

tor. Controlling the bioreactor temperature with a double-

jacket filled with pre-heated water is also possible for glass

vessels.

5. To avoid clogging of the exhaust filter due to humidity of the

exhaust gas, a filter heater or condenser around the exhaust

system needs to be applied.

3.4

Bioreactor

Setpoints

1. The temperature should be tightly controlled within the biore-

actor. Usually a setpoint of 37
C is used for CHO cells.

Modification of this setpoint during the culture (typically

Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines

7