Basic Techniques - The "Do's and Don'ts" of Cell Culture
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12.1 Basic Techniques - The "Do's and Don'ts" of Cell Culture
Given below are a few of the essential "do’s and don'ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas. However some of them are specific to tissue culture.
The Do’s
Use personal protective equipment, (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.
Always use disposable caps to cover hair.
Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
Keep all work surfaces free of clutter.
Correctly label reagents including flasks, medium and ampules with contents and date of preparation.
Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
Wherever possible maintain separate bottles of media for each cell line in cultivation.
Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.
Quality Control all media and reagents prior to use.
Keep cardboard packaging to a minimum in all cell culture areas.
Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
Test cells for mycoplasma on a regular basis.
The Don’ts
Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for commercial purposes.
Don’t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.
Don't have too many people in the lab at any one time.
Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
Avoid keeping cell lines continually in culture without returning to frozen stock.
Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet.
Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added.
Avoid water baths from becoming dirty by using Sigma Clean (Prod. No. S5525).
Don’t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.
12.2 Protocol 1 - Aseptic Technique and Good Cell Culture Practice
Aim
To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines.
Materials
Chloros / Presept solution (2.5g/l)
1% formaldehyde based disinfectant e.g.Virkon,Tegador
70% ethanol in water (Prod. No. R8382)
Equipment
Personal protective equipment (sterile gloves, laboratory coat, safety visor)
Microbiological safety cabinet at appropriate containment level
Procedure
Sanitize the cabinet using 70% ethanol before commencing work.
Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work.
Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.
Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.
Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped with tissue soaked with 70% ethanol prior to use.
Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.
After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.
Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.
Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.
More on Aseptic technique
Lauffenburger Tissue Culture SOP
Rev: 11/26/07
Background:
Tissue culture refers to the three-dimensional culture of tissue and/or a culture derived from dispersed cells taken from original tissue. Investigations of intracellular activity, intracellular flux, environmental interaction, cell-cell interaction, genetics, and cell products and secretion can be performed through tissue culture. The two major advantages of tissue culture are control of the physiochemical environment and the ability to maintain constant physiological conditions. Limitations of tissue culture include the need for a high level of expertise, the need for stringent environmental control, genetic and phenotypic instability, and differences in cell behavior in vitro. There are three main methods of initiating a culture: 1) organ culture, 2) primary explant culture, 3) and cell culture.
This protocol describes techniques to be used for tissue culture in the Lauffenburger lab (56-378, 56-286). These guidelines must be followed- failure to do so can result in damage to your and the experiments of others. To protect the overall lab, you (and any collaborators you work with in our facilities) will need to be certified by the Tissue Culture Czars before beginning work. In addition, repeated failures to follow the guidelines can result in loss of privileges.
Lab Setup:
You will be assigned a space in a tissue culture incubator. You must use the hoods and incubators in the room you were assigned; in no case should you use those in a separate tissue culture room (though other areas of each room may be used: microscope, centrifuge etc.). The only exception to this rule is for lab members who use the 2nd floor tissue culture area - they may use either hood and backup incubator on occasions where they need to do an experiment on the 3rd floor. If you need space in the liquid nitrogen container for archival of cell lines, contact Megan Palmer.
The doors to the tissue culture rooms are to be kept shut at all times. When in the tissue culture room, you can wear a disposable lab coat. This coat MUST NOT be used for general lab work; conversely, your general lab or radiation lab coats can not be used in the tissue culture room.
Dealing with Biohazards:
Although most cell lines are reasonably safe to work with, be aware that you are working with or around potentially dangerous materials (you should have already taken Blood Borne Pathogen training since we use human lines in our facilities). To protect everyone’s safety, follow these guidelines:
1) Label EVERYTHING. Minimal labeling includes your initials, the date, and the cell line nomenclature. For cell passages, it is usually best to include passage # and split ratio for your own information.
2) For long term experiments update the plate with the last date you changed media, etc. In general, cells that haven’t been observed in several days should be disposed of to minimize contamination risks.
3) If you have a lot of tubes, etc that are not for long term use (e.g. ViCell vials, falcon tubes), you can avoid labeling them all, but ONLY IF you dispose of properly immediately after you are done. At no time can you leave unlabeled materials in the lab when you are not in the lab.
4) Anything that has touched cells should be bleached and placed in autoclave waste, not general waste. Media/serum that has not been used on cells can be disposed of in the sink.
Aseptic Technique:
Contamination by microorganisms is a major problem in tissue culture. Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions and many other sources. Proper aseptic technique seeks to eliminate these contaminants. It is important to establish and maintain aseptic techniques particularly given the importance of cell culture to everyone’s experiments in the lab. Contamination can be minor and confined to one or two cultures, can spread among several cultures and infect a whole experiment, or can be widespread and wipe out your entire stock. Catastrophes can be minimized if 1) cultures are checked carefully under a microscope, 2) cultures are maintained without antibiotics for at least part of the time to reveal cryptic contaminations, 3) reagents are checked for sterility, 4) bottles of media are not shared with other people, and 5) the standard of sterile technique is kept high at all times. All materials that will come into direct contact with the culture must be sterile. Aseptic technique is a combination of procedures designed to reduce the probability of infection. Most cell culture work is done in a laminar flow hood.
General Rules:
1. Wear gloves – but note that these are to protect you, and are not inherently clean!
2. Keep the work surface clean (clean with 70% ethanol and keep the work area clear enough to allow work without reaching over an item and preventing inadvertent brushing of a sterile tip against another object.)
3. Use sterile reagents and media and work to keep them that way (do not reuse tips, clean the outside of reagent bottles with 70% ethanol, autoclave or sterile filter as appropriate)
4. Keep bottles, flasks and tubes covered as much as possible.
5. Remove liquid from containers at an angle.
6. Clean with 70% ethanol often (surfaces and gloves).
7. Remember that it is the inside of containers (autoclaved bottles, centrifuge tubes, etc) that is sterile.. treat the outside as dirty and clean with EtOH.
The major advantage of working in a laminar flow hood is that the working environment is protected from contamination by a constant flow of filtered air. A vertical flow hood is where air blows down from the top of the hood onto the work surface. Follow these guidelines when using the laminar flow hood. Keep this direction of flow in mind when setting up your work area.
Start-up in the hood
1) The hood must be operating correctly to be used- be sure UV light is OFF, turn on regular light if needed. Sash should be opened to upper tape mark (approximately 8 inches of clearance). Hood blowers are not to be turned off- if they were turned off for any reason, run for 15-20 minutes to clean the air in the hood.
2) Remember that everything that goes INTO the hood must be cleaned with 70% EtOH. This includes your hands. Spray them down before starting.
3) Swab down the work surface liberally with 70% EtOH. Wipe with a Kimwipe, starting from the back of the hood and moving forward. Wipe off anything you intend to use in the hood (pipettor, vacuum line/valve, Pasteur pipet box, etc).
4) Bring stuff into the hood that you intend to use – pipets, tips, etc. Each of these items needs to be cleaned with 70% EtOH. In addition, if you plan to use tips or microcentrifuge tubes, be sure to keep an autoclaved stock that is kept for hood use only.
5) Dry media bottles thoroughly if they have been taken out of the water bath (this water is a great source of contamination). Swab with 70% EtOH, especially at the neck and bottom before placing in hood.
6) Ironically, the greatest source for contamination comes from the cell culture flask itself. If you notice that you have dripped media onto a flask, either replace the lid or carefully wipe down with 70% EtOH before returning to the incubator.
Working in the Hood
1) As stated above, everything that goes into the hood must be wiped with 70% EtOH before putting it onto the bench in the hood. This means your hands, new boxes of tips, new beakers of tubes, the exterior of bottle-top filter units, etc. Everything.
2) Keep the air intake (the vent in the bottom of sash, underneath your elbows) clear of all items at all times. If this vent is blocked, it allows room air to enter the hood, which is great source of contamination! Also, keep items from directly blocking the vent in the back of the hood.
3) Bring only the items you need for a particular procedure into the hood to prevent cluttering your working space. Having a clear working space will significantly reduce the chance of contamination! Ensure easy access to items in the hood and maintain plenty of clear space in the center of the hood to work in.
4) This is a vertical laminar flow hood, which means that air flows straight down from the top of the hood. Do not work directly over any open vessels, or contaminants from your hands could be blown into your vessel. Always work at an angle, off to one side.
5) Watch what you are pipetting! The replaceable filters in the air pipettors are expensive. Ensure that you don’t suck fluid up inside them. Especially watch the 1 mL pipettes...they fill really fast!
6) If you spill anything in the hood, clean up immediately to prevent cross-contamination and damage to working surface. Stop what you are doing and wipe up the spill – salts in particular can corrode the metal if left. Wipe the area with 70% EtOH before returning to work.
7) Styrofoam is not recommended in the hood – it often flakes and is difficult to keep out of the vents. Use plastic racks instead (if you need more racks, be sure to clean with EtOH before putting into the hood).
Using the UV Light
Although not recommended for routine sterilization of the hood itself, each hood has a UV light which can be used to sterilize surfaces, etc.
1) Put in surface to be sterilized. Turn off blower and shut sash.
2) Turn on UV light for desired time (10-15min is usually sufficient).
3) Turn off UV light, open sash to upper marked height and turn on blower.
4) Let blower run for ~20 minutes and proceed with normal EtOH start-up.
Incubators and Microscope
These are shared equipment, so they present a great method to spread contamination!
1) Before using the microscope, spray a Kimwipe with 70% EtOH and wipe down the stage. Do this also when you are done to prevent media spills from spreading between plates.
2) If you need counter space to set plates down on by the microscope, clean area with 70% EtOH.
3) The incubators are not technically sterile- however, every effort must be made to maintain their cleanliness to prevent contamination from spreading. If media has spilled in the incubator, clean the spill with EtOH. Spills into the water bath MUST be immediately taken care of- talk to either the lab manager or the tissue culture czars.
4) Put plates carefully into incubator- be sure not to bump other people’s plates, minimize stacking as much as possible, and keep your own plates set up to allow you easy access to what you need next.
When You Are Finished
1) Trash must be disposed of properly- sharps/pipettes in the sharps waste bins, non-sharps that have contacted cells must be bleached and disposed of in autoclave bins, and all other non-sharp/non-biohazard waste goes in regular trash.
2) Remove all of your media/solution bottles (tightly capped), tubes, etc from the hood.
3) Be sure the pipet-aid is returned to its rack to turn off pump. Close the vacuum line.
4) Restock Pasteur pipettes if empty with a new container of autoclaved pipettes– remember to clean the container with EtOH before putting in hood.
5) Wipe down the bench with 70% EtOH.
6) Check serological pipettes and restock if necessary.
7) All waste media and/or cell cultures should be bleached. Add bleach to a final concentration of 10% and let stand for 20 minutes. Then discard down the sink and put plates, etc into biohazard bins. ALL BIOHAZARD WASTE MUST BE PROPERLY DISPOSED OF BEFORE YOU LEAVE FOR THE DAY!