OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
STGCL.SWP.73.1
School/ Divisional Unit
St George Clinical School
Initial Issue date
15/04/10
Current version
1.0
Current Version
Issue date:
15/04/2010
Next review date
15/04/2012
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title: Standard Cell Culture Techniques
Description: This technique is widely applied in a variety of assays which include cytotoxic assay, tube formation assay,
actin staining and RhoA activation assay.
Associated risk assessment title and location: Standard Cell Culture Techniques
Describe the activity or process
This SWP outlines the correct procedure to be followed for standard cell culture. Read the Risk Assessment
attached and the relevant MSDSs before carrying out this procedure.
Procedures:
Cell culture (Subculture):
i.
1A9 cells, a subclone of the A2780 human ovarian carcinoma cell line, are maintained in RPMI1640
(Sigma) media, supplemented with 10% foetal bovine serum (FBS) (JRH) and 1% penicillin/ streptomycin
(Sigma).
ii.
Human umbilical vein endothelial cells (HUVEC) are maintained in EGM Bulletkit (Lonza): M199 (Sigma)
(1:1 v/v) media, supplemented with 10% foetal bovine serum (FBS) (JRH).
iii.
The cells are grown as attached cultures in the humidified CO2 incubator at 37oC and 5% CO2, and are
subcultured upon reaching 70-80% confluence.
iv.
The spent medium is removed.
v.
The cells are washed with sterile PBS without Ca2+/Mg2+.
vi.
Sufficient amount of 0.05% (for 1A9)/ 0.5% (for HUVEC) Trypsin/ EDTA is added onto the flask.
vii. The flask is left to incubate in the humidified CO2 incubator at 37oC and 5% CO2 for < 5 minutes.
viii. The flask is examined under the inverted microscope to ensure that all the cells were detached and
floating
ix.
The cells are resuspended in fresh medium to inactivate the effect of Trypsin/ EDTA.
x.
The cell suspension is centrifuged at 1,100 rpm (200g) for 5 minutes at room temperature.
xi.
Supernatant is then discarded and the tube is replaced with fresh media.
xii. Cell quantification is performed using 0.4% Trypan Blue to differentiate the viable cells from the dead
cells.
xiii. Cells are then transferred to new flasks containing fresh medium and left to incubate in the humidified
CO2 incubator at 37oC and 5% CO2.
Remark: HUVEC revived from cryotubes are seeded onto flask pre-coated with 0.1% gelatin (Sigma).
Subsequent passages are seeded onto ordinary flasks.
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Page 1 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Equipments:
 Class II Biological Safety Cabinets
 Centrifuge
 Centrifuge tubes
 Drug vial
 Haemocytometer (with cover slips)
 Inverted microscope
 Micropipettes
 Pippette aid
 2ml/ 5ml/ 10ml/ 25ml serological pipettes
 Tips
 Water Bath
Chemicals:
 Culture media
 80% v/v ethanol
 Foetal Bovine Serum (JRH)
 Gelatin (Sigma)
 Penicillin/ streptomycin (Sigma)
 0.4% trypan blue (Sigma)
 0.5% or 0.05% trypsin/EDTA (Sigma)
 PBS
PPCE:
 Gloves
 Gown
List potential hazards and risk controls including
specific precautions required

Refer to relevant risk assessment form.
List emergency shutdown instructions
List clean up and waste disposal requirements
Before cell culture:
i.
Wear the correct PPCE at all times
ii.
Switch on the UV lamp for UV irradiation as part of the aseptic techniques (at least 15 minutes). Please ensure that
the protective shield is tightly attached to the biosafety cabinet.
iii.
The safety cabinet should be turned on about 10-20 minutes before being used.
iv.
Wipe down all surfaces with 80% ethanol (v/v) before each use.
v.
Keep the safety cabinet as free of clutter as possible because this will interfere with the laminar flow air pattern.
vi.
Thaw media, trypsin/EDTA and PBS to 37oC using the water bath.
After cell culture:
i.
Wipe down all surfaces with 80% ethanol (v/v) after each use.
ii.
Switch on the UV lamp for UV irradiation as part of the aseptic techniques (at least 15 minutes). Please ensure that the
protective shield is tightly attached to the biosafety cabinet.
iii.
Treat culture wastes with 1% hypochloride (total volume) and leave treatment for at least 30 minutes before transfer to
the culture waste container.
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Page 2 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List legislation, standards and codes of practice used
in the development of the SWP
 MSDS
 OH & S policies
Supervisory approval, training, and review
Supervisor: Peter Galettis
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Peter Galettis
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Researcher to attend trainings as follow:

Induction program

Laboratory Safety Awareness

PC2 Bio Training

Hazardous Substances

OHS Awareness for Employees

St George Hospital Annual Mandatory Training
SWP review date: 15/04/2010
Responsibility for SWP review: Steven Lim
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007