1

Department of Microbiology

SOP No:

NML-SOP-XX

Category:

Activity

Title: Mammalian Cell culture using Bioreactor

Effective Date:

01/04/2014



Revision No:
Revision Date:

2
01/04/2017



Pages

7

1.0 BJECTIVE
This SOP outlines procedures to provide guidance on how to perform mammalian cell
culture using bioreactor safely and maintain it in a correct manner.

2.0 SCOE
The procedure is applicable to onl y t he l ab personnel of NUS staff/students, external
staff/students, visiting academics of the Flavivirology lab, who intended to use mammalian
expression using bioreactor. Those staffs and students are to adopt these practices in the
procedure.


3.0 RESPONSIBILIY
Principal Investigator
Principal Investigators (PI) is responsible for ensuring that this SOP is disseminated to
all laboratory personnel and that they are aware of the procedures for safe handling of
bioreactor. The PI is responsible for providing or arranging the appropriate training of
personnel, supervising and providing on-the-job training for all the research staff and
students in her research group; and preparing specific SOPs when required for selected
research protocols and equipment use.

LaboratoryPersonel
The operat or who intended to use this bioreactor must be aware of potential hazards, and
must be trained and proficient in the practices and techniques required to handle the
equipment safely.

4.0 Usage and
TrainingRequirements:

The safety instructions in the operating manual, the safety, accident prevention, and
environmental protection regulations valid for the location of use of the unit must also be
observed.

In particular, the following apply:

During the entire period of use of the unit, the operator must check whether the
operating instructions correspond to the current state of regulations and adapt them as
needed.
The operator must clearly regulate and determine responsibilities for operation,
maintenance, and cleaning.
2
The operator may only allow trained and authorized personnel to work with the unit.
Trainees such as apprentices or auxiliary staff members are only allowed to work with
the unit under supervision of qualified technicians.
The operator must ensure that all employees involved with the unit are suitable in terms
of physical capacity, person and character to operate the unit responsibly.

The operator must also ensure that all employees are familiar with the basic regulations
concerning workplace safety and accident prevention, trained in handling the unit, and
have read and understood the operating manual.

4.1 Personal Protective Equipment
(PPE)
Gloves, laboratory coats are to be worn, and eye protection worn when
appropriate.

Other PPE should be used as appropriate, refer to OSHE website for more details.
https://wws.nus.edu.sg/osh/nus_manuals/guidelines/U_GL_01_PPE.pdf


4.2 Incident Reporting
a) All accidents and injuries must be reported immediately to your PI and lab safety officer.
Report to the Department Safety Health Officer, and to OSHE within 24 hours if deemed
necessary. More details can be obtained from
https://wws.nus.edu.sg/airs/report.aspx
b) In the event of an accident/incident, following investigation, the SOP and RA will be
reviewed and amended accordingly.

4.3 Chemical Waste Disposal
a) All wastes containing chemicals should be handled with appropriate personal protective
equipment. Used paper towels and gloves should be placed in a biohazard bag and
subsequently disposal of by incineration.
b) Ensure that the biohazard bag is double wrapped.
c) The chemical wastes should be disposed off via a licensed waste collector for special
chemical waste incineration. The list of NEA licensed waste contractor may be found
from the NEA website. http://app2.nea.gov.sg/.
d) Chemical wastes should be collected in sturdy leak proof waste container with a
secondary container that can withhold 20% of the volume of waste in case of leakage.
e) Flammable chemical waste that contains flammable solvents like methanol must be store
in a flammable safety cabinet in MD4 level 1 Chemcal room.
f) Waste labelling- A GHS waste sticker label with appropriate labelling must be affixed on
the waste container to identify its contents.
g) Refer to SOP on waste disposal of chemical wastes.
https://share.nus.edu.sg/corporate/procedures/safety_and_health/Chemical-Safety-
Manuals/Manual-chemical-safety.pdf
4.4 Biosafety
Also refer to departmental sop, MIC-SOP-HS-05 on Chemical Waste Disposal.
The need for precautions when experimenting with cells in culture depends upon the source and
nature of the biological material, the experimental procedure, and the laboratory/containment
3
conditions. Since every situation is different, the risks need to be identified and appropriate
precautions need to be taken before any work begins.

5.0 PROCEDURES
5.1 Materials
Culture vessels containing your suspension cells
Shaker flasks without baffles or spinner bottles
Complete growth medium, pre-warmed to 37C
37C incubator with humidified atmosphere of 5% CO2
Magnetic stir plate (if using spinner flasks), roller rack (if using roller bottles), or
shaking platform (if using conventional culture flasks or petri dishes)
Reagents and equipment to determine viable and total cell counts (e.g., Countess
Automated Cell Counter, Trypan Blue and hemacytometer, or Coulter Counter)

5.2 Equipment
The device is made up of the following components.
Control unit
Control unit in single model
Aeration module CC (BIOSTAT B-CC) for enriching the air with oxygen,
decreasing the O2 content by supplying N2 and supplementing CO2 for regulating pH,
e. g. for tissue cell cultures containing animal cells in the suspension culture
Temperature control modules with associated fittings (temperature control by water or
heating blanket and cooling finger)
Cooling water circuit for exhaust cooler and or exhaust filter heater
Peristaltic pump modules
Exhaust Cooler

Culture vessels
Culture vessel volume (2 L)
UniVessel, double-walled (jacketed)
Equipment components for microbial cultures and cell cultures
Vessel tripod
Cover plate with ports | retainers for sensors, supply media, sampling, aeration
Supply bottle with bottle holder
Stirrer driver
Top drive with direct stirrer drive motor
Drive with magnetic coupling between motor and stirrer
6-blade disk impeller or 3-blade disk impeller
The standard stirrer shaft is sealed with a rotating mechanical seal. The optional
magnetic coupling is also sealed with a rotating mechanical seal; the motor coupling on
the outer side, however, is sealed in an enclosure and attached to the driver motor by
means of a magnetic coupling.

External Cooling Equipment

You can connect a laboratory cooling water circuit or cooling device to the cooling
water inlet and outlet.
For the external cooling devices, following specifications apply:
Water pressure max. 2 barg
Flow rate quantity max. 4 l/min
Temperature min. = 4 C
4
Depressurized outlet
Nozzle connection | outer diameter = 10 mm
Make sure that the inlet and outlet are connected in the right order:
Connect it from the outlet of the external circuit or cooling device to the
device inlet.
Connect it from the outlet of the device to the laboratory return pipe or the
inlet of the cooling device.
The cooling device or external cooling circuit must be operated at ambient pressure.
Prevent the cooling medium from flowing back into the devices outlet.

5.3 Gas Supply
The gas supply for the device category BIOSTAT B CC includes the following gases:
Air Air
Oxygen (O2) Oxygen (O2)
Nitrogen (N2)
Carbon dioxide (CO2)

Aeration modules CC are used to supply up to 4 gases. These are by default:
Supply of air
N2 for decreasing the O2 content, or O2 for increasing the oxygen content;
Sparger outlet for supplying gas to the culture medium.
CO2, for regulating the pH value or as a source of C.

Air and CO2 can be fed both into the medium in the culture vessel (sparger) and into the
headspace (overlay), while the other gases are fed into the supply line for the culture medium
(sparger) by default. These modules are designed for tissue cell cultures, e.g. suspension
cultures that contain animal cells. They are also suitable for use with cultures with special
aeration requirements (if CO2 needs to be used as the carbon source, e.g. in anaerobic bacteria
or algae cultures).
Regulates N2 and O2 by means of 3/2-way solenoid valves that are controlled by the
DCU system pO2 controller.
Regulates CO2 flow by means of a solenoid valve that is controlled by the DCU system
pH controller.
Operating modes available for selection in the controller operating menu: man, auto, off
Gas volume can be adjusted at the variable area flow meter or by means of optional
mass flow controllers.
Sparger outlet for supplying gas to the media and overlay for supplying gas to the
headspace in the culture vessel.
Up to four optional mass flow controllers.

Aeration During Process

After autoclaving, connect the culture vessel to the aeration module outlets (hose
nozzle, .. = 6 mm).
Configure the laboratory gas supply points for supplying gas to the equipment during
the process. To calibrate, supply gas to the pO2 sensor and pO2 (and pH, where
relevant) controller during the process.
When using the Additive Flow aeration module, calibrate the CO2 supply (pH
control).


5
Flow Rate Measurement

Flow rate meters for gases are calibrated for standard conditions. The specified values can be
found on the manufacturers ID label of the glass test tube. The following information is given
on the manufacturers ID label:
Parameters
Flow rate meter
Model
Gas type Air
Standard temperature 20 C = 293 K
max. pressure, Nitrogen (N2)=1.5 barg (22 psig)

When gases with deviating pressures pass through the meter, higher or lower values can be
displayed. These must be recalculated to evaluate the flow rates.

6.0 Protocol

The system must be properly installed and connected in accordance with the specifications.
You must also have gained familiarity with the safety instructions in the Operating Manual,
Chapter Safety instructions. Ensure that all required supply energies are connected to the
unit.
Turn the unit on at the main switch
Starting up and operating the bioreactor during the relevant fermentation process involves the
following main steps:

Set up the device as well as the other devices and equipment, supplementary to the
measures described in Operating Manual
Autoclave the culture vessels and the accessories to be attached [UniVessel, Operating
Manual]
Place the culture vessels in front of their supply unit in such a way that all of the lines
and peripheral devices can be easily connected. Connect the culture vessels and set up
the bioreactor at the site designated for the fermentation process

Test the pH sensor as recommended by the manufacturer using reference buffers.
Calibrate the pH sensor to zero point and slope of the sensors using the buffers in
accordance with the scheduled measuring range.

Test the pO2 sensor as recommended by the manufacturer and service it if required.
Replace the membrane and the measuring electrolyte solution. The pO2 sensor must be
calibrated after the culture vessels have been sterilized in readiness for the fermentation
process.

Prepare the bottles for acid, base, anti-foaming agent and nutrient solutions.

Fit the motors to the stirrer shaft couplings.
Connect the supply and drain lines of the temperature control system with the culture
vessel.
Fit the heating blanket to the culture vessel and connect the device to the power supply.
Connect the supply and return lines of the exhaust cooling to the ports of the exhaust
cooler at the culture vessel.
Fit the exhaust filter heater to one of the exhaust filters and connect the plug to the mains
6
supply.

Connect the culture vessel sensor cables to the associated female connectors.
Place the connection hoses of the correction medium bottles into the associated
peristaltic pumps of the device.
Configure the measurement and control parameter for the process at the DCU system.

pH sensor, feed pipe for acid and base, Anti-foam sensor, feed pipe for anti-foam agent
The culture process set points has to be set using the values recommended based on
the cell type. When setpoints were reached, the inoculum flasks were connected to the
bioreactors addition line, and contents were pumped.
The bottles have to be ready for use. Autoclave the corrective solution bottles together
with the culture vessel.

All solutions and equipment that come in contact with the cells must be sterile. Always use proper
sterile technique and work in a laminar flow hood.

Subculture cells when they are in log-phase growth before they reach confluency. When they reach
confluency, cells in suspension clump together and the medium appears turbid. The maximum
recommended cell density before passaging varies with cell lines; refer to the cell-specific product
insert or manual for details.
1. The inoculum was grown to a density of 2.03.0 10
5
cells/mL, with greater than 90% cell
viability.
2. One day before cells reached inoculation density, growth medium was warmed to 37C for
24 h in a CO
2
incubator; and the DO probe was connected to the bioreactor controller for =6 h
to enable polarization.
4. Culture medium vessel was connected to the bioreactor vessels inlet line, and medium was
pumped into the bioreactor. Connections to the controller, including sparge, overlay, RTD, pH,
and agitation were made.
6. Gases were introduced into the vessel headspace only through the overlay port at a rate of
0.30 L/min using 4-gas mixing to maintain pH and DO. On day 3, and for the remainder of the
run, 510 ccm gas were additionally directly sparged using a porous sparger and automatic gas
mixing into the bioreactor vessel.
7.Process data were continuously logged. A built-in sampling device enabled sterile sampling.
Daily off-line measurements of glucose and lactate concentration were read, and cell density
and cell viability were measured.








7
Note:

Only use lines and fittings approved for use with the bioreactor or whose suitability
has been confirmed in writing by Sartorius Stedim Biotech.
Only replace damaged components and wear parts with Sartorius Stedim Biotech
approved parts.
Sartorius Stedim Biotech does not accept any liability for operating faults and
breakdowns related to the use of equipment that has not been approved for use with the
bioreactor, as well as any secondary damage arising from this.
Risk of injury when handling heavy culture vessels! Fully equipped and filled culture
vessels are heavy, e.g. a UniVessel with an operating volume of 5 liters weighs > 18
kg.
The culture vessels must be handled with care. Always use suitable transport and
lifting equipment. Only lift the vessels at the handles provided for this purpose.
Information on installing and connecting the culture vessels to the device can be found
in the operating manual.
Equip the culture vessels only with those components that are needed for the process
[UniVessel Operating Manual].
Make sure that the vessel equipment is in perfect condition and clean before installing
it in the culture vessel.
Remove all residues, contaminations or microbes from the previous fermentation
process from the culture vessel and its fittings.
Carefully check all equipment, and glass culture vessels, seals and silicone hoses in
particular, for damage. Replace all damaged and worn out parts.