Cell Culture Procedures and Guidelines

Transcription

1 Cell Culture Procedures and Guidelines (November 2007 revision) Protective Clothing While these measures may seem extreme, they are our best chance of minimising contamination, protecting both ourselves and our cell lines. Disposable yellow lab coats must be worn. Disposable gowns are provided and can be found in the cell culture facility. Do not wear the gown outside of the cell culture facility. Replace the gown with a fresh one at least every week. Do not enter a cell culture facility with a gown from outside of the facility. Safety glasses and nitrile gloves must be worn at all times when using safety cabinets. Laminar Flow Hoods Swab the tray with 70% ethanol before and after each use UV irradiate for 20 minutes between cell types and strains and when finished. Use the online booking system ( to book the safety cabinet in the facility that you have been allocated to. You can make a maximum booking of 4hours. Be fair to others and plan your work properly. Full protective clothing, safety glasses and nitrile gloves must be worn at all times when using hoods. Clean spills of culture or media immediately. Prepare your Supplies Before commencing work, ensure you have adequate stocks of serological pipettes (transfer 10ml and 25ml), flasks of each size, 15ml and 50ml Falcon tube, filter tips, yellow gowns. There should 1

2 also be disposable inoculating loops, cryovials, microfuge tubes, syringes, needles and syringe filters. The aim is to minimise the possible. Solid Waste Empty the bin (a wet bag within an autoclave bag) when 80% full. Sharps and needles must be placed in a sharps container. Serological pipettes must be placed in a small autoclave bag. Liquid Waste If you are decanting waste into a waste bottle use a fresh receptacle for each hood session. When decanting waste from the receptacle into the main waste bottle, use an autoclaved polypropylene funnel in the top of the bottle to minimise aerosol formation. Decant waste into main storage bottle when finished, take ¾ full bottles to liquid autoclave waste buckets. Aspirated Waste using a Vacuum Pump Suction waste shall be collected in a side arm flask. A 0.2µm in line filter is to placed between to pipette and collection vessel. One hundred mls of a 1% iodine solution is to placed in the vessel prior to collecting waste and the waste should be removed when the volume reaches 1 litre (ie: 0.1% final concentration of iodine) Incubators You should regularly check the CO 2 levels on the cylinders, notify Michael Johnson or Philip Lawrence when the levels are low Dispose of unwanted flasks in the appropriate waste stream ASAP, to minimise clutter and contamination risk. 2

3 Media Fridges Space is limited, please keep bottles and tubes to a minimum, and discard unwanted media regularly. Ensure all media is clearly labelled with your name, date and components added to base. Room Irradiation Rooms are equipped with overhead UV lights for whole-room treatment. A warning sign must be posted on the entrance door to the room when the UV light is active. Leave on for a minimum of 1 hour. Benches should be clean and as clear as possible for maximum effectiveness. Laminar Flow Hood Cleaning Ideally, hoods should be cleaned thoroughly approximately every 6 weeks or less. Swab interior with 70% ethanol Remove tray and grills, wash with disinfectant/detergent using Chuxtype disposable cloths Wash interior of cabinet with disinfectant/detergent, including inside of glass panel, roof, walls and under wall rims Replace front cover and UV irradiate cabinet interior (without tray and grills) for 20 minutes. Replace tray and grills, swab interior again with 70% ethanol. Replace front cover and UV irradiate for a further 20 minutes. The benches, window sills and tops of incubators in each room require wiping down with disinfectant/detergent followed by 70%ethanol. 3

4 General The aim is to keep each room as an independent unit, to minimise sharing of equipment and supplies. Wait for the current user to finish their session before entering the room Keep the room tidy, take your lab books etc with you Put pipettes, tips, tubes away each time, benches should be kept as clear as possible. Return Neubauer counting chambers to rooms ASAP after use Wipe down benches with 70% ethanol regularly New Cell Lines It is good practice to assume that any new cell line brought into the lab, from whatever source (INCLUDING the ATCC) may be a source of mycoplasma contamination. For this reason, new cell lines should be established in isolation, and a portion grown for a minimum of 2 passages in antibiotic free media for Mycoplasma testing. Only after the cultures have tested negative should they be allowed to share incubators and hoods with existing cell lines. If you are starting up a new cell line please see Michael Johnson or Phil Lawrence to arrange for Mycoplasma testing. 4

5 Mycoplasma Testing Since the relocation to Building 4 in 2006, two PCR test kits (VenorGEM Mycoplasma Detection Kit [16SrRNA gene ] distributed by Biocene Pty Ltd (02) ) and the Sigma Lookout PCR kit (MP0035) have been used for the determination of the Mycoplasma status of cell lines in the cell culture facilities. The kits are used as per manufacturers instructions with the exception that genomic DNA is purified from the cell culture sample via an ultraclean 15 DNA clean up kit prior to PCR. Testing occurs on a 6 monthly basis and the results are kept with the Professional Officer (Technical) Michael Johnson. Working with Infectious Oocysts of Toxoplasma gondii All work to be done in a class 2 biohazard hood with the exception of the centrifugation steps. Tubes must always be sealed when moving in and out of the class II hood for the centrifugation steps and microscopic steps. The outside surface of the tubes are to be wiped and/or sprayed with 70% ethanol. All work must be contained within cell culture room This organism can cause birth defects and or miscarriage in pregnant women. Women of child bearing age must be fully informed of the additional risks of the handling of oocysts. The Operations Manager and/or Professional Officer must be advised when work is to proceed, so reasonable warnings can be provided to other researchers. A Sign saying Warning Infectious Organisms is to be placed on lab door when research occurring. The researcher should wear a face mask, double gloves (changed frequently) in addition to normal laboratory protective clothing. 5

6 Benches must be covered with benchcote. A metal tray lined with absorptive material should be used at all times to house tubes in a rack (when they contain oocysts). In case of spills this can be autoclaved. All liquid waste to be collected in a bottle (with a screw cap) which will be autoclaved before disposal. All solid waste to be placed in a wet bag within an autoclave bag (ie double containment) for autoclaving. The working surface of biohazard hood is to be sprayed with disinfectant (25% ammonia for 30mins) 1,2, and the solid waste is to be disposed of through biological waste bag listed above. The hood is to be UV irradiated for a minimum of 30 min. Material safety data sheet o Vet Parasitol Dec 9;126(1-2): Organisation of Cell Culture Room Usage Prior to starting cell culture, you will be allocated by into an appropriate facility by the cell culture facility manager. The principle of is to consolidate similar work types and allow for quarantine action to be taken quickly and effectively when needed. The facilities and the current work types are described below 6.02 Diabetes - Established mammalian lines 6.04 Diabetes - Established mammalian lines + viral vectors 6.14 Virology - Established Mammalian lines + viral vectors 6

7 6.17a Physiology - Established Mammalian lines (non-gmo) 6.46b Toxinology cell culture (non-gmo) 6.16 Established mammalian lines 6.22 Established mammalian lines 6.24 Parasitology established mammalian and parasite lines (including Infectious Toxoplasma Oocysts) 6.30 Parasitology - established mammalian and parasite lines 6.38 Immune System Therapeutics (IST) - established mammalian and parasite lines 6.40 Primary cell culture and Hybridoma preparation this room has been allocated for non-continuous cell lines and animal gross tissue samples. There is no guarantee that work in this room is Mycoplasma free. Cell lines and samples must not be transferred from this facility to any of the other cell culture facilities. 7