6. Calculate the cell concentration and viability according the
manufacturer’s
instructions.
Dispose
trypan
bluecontaminated equipment according to local, state and federal
regulations.
3.1.4 Cell Counting
and Cell Viability
Determination via Crystal
Violet
The cell numbers attached on the microcarrier are determined by
the counting of nuclei [86].
1. Centrifuge 1 mL of microcarrier cell suspension sample at
300 Â g for 5 min, RT.
2. Discard 0.9 mL of the supernatant.
3. Incubate the pellet with 0.9 mL 0.1% crystal violet in 0.1 M
citric acid at RT for at least 24 h (alternatively: 30 min, 37
C,
500 rpm in a thermomixer).
4. Determine the dyed nuclei by means of the Neubauer counting
chamber under the incident light microscope.
5. Calculate the cell density according to the manufacturer’s
instruction of the Neubauer counting chamber.
3.1.5 Bioreactor
Preparation
1. Install and connect the following tubes and probes to the STR:
(see Note 5).
(a) pH probe: calibrate (before autoclaving) according to the
manufacturer’s instructions.
(b) pO 2 sensor: calibrate (after medium fill-up) according to
the manufacturer’s instructions.
(c) Dielectric spectroscopy: connect to amplifier and signal
transformation box.
(d) Temperature sensor.
(e) Exhaust condenser including sterile filter.
(f) Connect the sterile air filter to the gas inlets.
(g) Acid and base tubes.
(h) Tubes for sampling.
(i) L- sparger for oxygen supply.
2. Calibrate the pumps for acid and base, if necessary, according
to the manufacturer’s instructions.
3. Cover all probes and plastic parts (e.g., sterile air filter) with
aluminum foil.
4. Autoclave the STR (see Note 6) and prepared bottles (1 Â
medium, 1 Â inoculation, 1 Â base).
5. Let the STR cool down to RT after autoclaving (see Note 7).
6. Fill the medium into the reactor under the sterile working
bench:
(a) Prepare a sterile medium bottle with a bottom drain tubing with Luer lock lids.
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Keven Lothert et al.
manufacturer’s
instructions.
Dispose
trypan
bluecontaminated equipment according to local, state and federal
regulations.
3.1.4 Cell Counting
and Cell Viability
Determination via Crystal
Violet
The cell numbers attached on the microcarrier are determined by
the counting of nuclei [86].
1. Centrifuge 1 mL of microcarrier cell suspension sample at
300 Â g for 5 min, RT.
2. Discard 0.9 mL of the supernatant.
3. Incubate the pellet with 0.9 mL 0.1% crystal violet in 0.1 M
citric acid at RT for at least 24 h (alternatively: 30 min, 37
C,
500 rpm in a thermomixer).
4. Determine the dyed nuclei by means of the Neubauer counting
chamber under the incident light microscope.
5. Calculate the cell density according to the manufacturer’s
instruction of the Neubauer counting chamber.
3.1.5 Bioreactor
Preparation
1. Install and connect the following tubes and probes to the STR:
(see Note 5).
(a) pH probe: calibrate (before autoclaving) according to the
manufacturer’s instructions.
(b) pO 2 sensor: calibrate (after medium fill-up) according to
the manufacturer’s instructions.
(c) Dielectric spectroscopy: connect to amplifier and signal
transformation box.
(d) Temperature sensor.
(e) Exhaust condenser including sterile filter.
(f) Connect the sterile air filter to the gas inlets.
(g) Acid and base tubes.
(h) Tubes for sampling.
(i) L- sparger for oxygen supply.
2. Calibrate the pumps for acid and base, if necessary, according
to the manufacturer’s instructions.
3. Cover all probes and plastic parts (e.g., sterile air filter) with
aluminum foil.
4. Autoclave the STR (see Note 6) and prepared bottles (1 Â
medium, 1 Â inoculation, 1 Â base).
5. Let the STR cool down to RT after autoclaving (see Note 7).
6. Fill the medium into the reactor under the sterile working
bench:
(a) Prepare a sterile medium bottle with a bottom drain tubing with Luer lock lids.
232
Keven Lothert et al.