Six steps to take before, during, and after cell culture experiments.
Cell culture contamination, whether it is from microbes or due to the presence of other cell lines, is a serious problem affecting experimental reproducibility.
Aseptic technique is a collection of laboratory methods and processes intended to eliminate cell culture contamination. The guiding principle is to sterilize anything that comes in contact with or is in the same environment as your cells including incubators, water baths, media, pipettes, and clothing.
Whether you are a beginner or a seasoned cell culture expert in need of a refresher, the tips below will help you to ensure the proper use of aseptic technique.
1. Select and set up a designated space for cell culture.
You will need an enclosed room devoted solely to cell culture. For basic cell culture, you will need the following equipment: laminar flow hood, incubator, water bath, microscope, hemocytometer or automated cell counter, a centrifuge, squeeze bottles for disinfecting the hood, and a timer—useful when trypsinizing cells or performing other time-sensitive protocols.
Consumables will vary based on the type of research, but basic items include: serological pipettes and pipettors, micropipettes and tips, sterile filter systems, flasks, plates, media, serum, antibiotics, cell culture-grade PBS and water, Trypan Blue or another stain for cell counting, trypsin for adherent cells, ethanol for sterilizing surfaces, and bleach for cleaning cell waste.
2. Lay some ground rules for conduct in the cell culture room.
Good personal hygiene minimizes the chances of microbial introduction into the cell culture area. Make it a lab rule that all scientists and technicians must wear a clean lab coat in this room and dedicate specific lab coats for cell culture purposes only. Tie back long hair as it is a major source of microorganisms, especially yeast.1 Because we harbor in our mouths certain types of Mycoplasma, a serious and largely invisible source of microbial contamination, limit lab discussions and socializing near the cell culture area.1 Do not forget to wash your hands before and after performing culture work and before leaving the general lab area.
3. Decontaminate the cell culture area.
Remember that everything that comes in contact with the cells must be sterile. If the cell culture laboratory has not been used recently, wipe surfaces with a 70% ethanol + 2% phenol solution to ensure full decontamination.2 Allow the solution to work on the surfaces for several hours or overnight.
If the cell culture lab is used frequently, disinfect the flow hoods, bench tops, and all surrounding surfaces with a 70% ethanol solution (this is where the squeeze bottle comes in handy) before and after handling cells.
4. Prepare reagents.
Before using a fresh bottle of media, run it through a 0.2 micron vacuum filter—in the hood to maintain sterility—to remove viruses or Mycoplasma that may have entered your media from fetal bovine serum (FBS) or other sources.2
Treating multiple cell lines with the same media stocks increases the chance for cross-contamination, regardless of good technique. Dedicate one media (or other reagent) bottle to a single cell line and user per lab. Aliquot reagents such as trypsin, FBS, and antibiotics into single-use tubes and store in the freezer until needed.
5. Bring on the cells!
Give yourself plenty of time to prep reagents and the flow hood. Make sure to have all reagents in place before taking cells out of the incubator.
Twenty to thirty minutes before handling cells, place media and other reagents that will come in contact with cells in a water bath to bring them up to 37°C (or as high as 45°C, depending on your application). Sterilize the hood by running the UV lamp for at least 20–30 minutes before opening. Then, activate the airflow and let it run for ten minutes to establish proper airflow.
Spray gloved hands with 70% ethanol and then spray and wipe down the hood interior with a sterile cloth. Spray and wipe down all reagent bottles and equipment before bringing them into the hood, making sure bottle caps are screwed on tightly to avoid ethanol contamination. Open sterile packaging and reagent bottles only inside a prepped flow hood.
Handle one cell line at a time to avoid cross-contamination. When handling reagents, use individually packaged serological pipettes and sterile micropipette tips and do not put the pipette tip back into the media after it has been immersed in a culture plate or flask. Avoid pouring reagents from one container to another as it produces aerosol contaminants that are easily transferred to cultured cells and the surfaces of gloves and lab coats, significantly increasing the potential for microbial and cross-contamination.1
6. Take care of your equipment.
The hot, humid conditions inside a water bath and incubator are ideal growth environments for microorganisms.
Weekly cleaning will curb water bath contamination: Drain the bath, then wipe it down with 70% ethanol and allow it to dry. Refill the bath halfway with autoclaved distilled lab-grade water and make sure the temperature of the bath is set to 37ºC. Ensure the lid is on properly to maintain the set temperature and keep out contaminants.
A three-point system is best for curbing incubator contamination:
Weekly exchange of humidity pan water
Discard the water, wipe down the pan with 70% ethanol, and let it air dry. Refill the pan with autoclaved distilled lab-grade water and, with gloved hands, place it back into the incubator.
Weekly incubator decontamination using ethanol or UV irradiation
Each week, wipe down the incubator with 70% ethanol or run the incubator’s UV decontamination cycle overnight. Make sure to move cells to a second incubator during this process.
Bi-weekly to monthly incubator decontamination using heat or H2O2
Run the incubator’s decontamination cycle at least once a month. The incubator will typically use either heat or hydrogen peroxide (H2O2) to get the job done, but regardless of the method it is important to follow manufacturer recommendations to ensure full decontamination. To prevent the spread of contamination or loss of incubator parts, exercise caution when disassembling and reassembling heat-sensitive shelving components.
This guide represents the basics of aseptic technique, but more stringent protocols may be required for your specific applications. While certain aspects of aseptic technique can be tedious or seem excessive, the threat of bacterial invasion is constant and it is important not to let your guard down when culturing cells.
Carl Radosevich ([email protected]) is a product specialist at Panasonic Healthcare Corporation of North America. To read more recommendations on cell culture, incubators, and sample storage, visit Panasonic Healthcare’s Education Portal at http://us.panasonic-healthcare.com/education-portal/.
1 John M. Davis, K. L. (2013). Cell Culture, Aseptic techniques. In M. C. Flickinger (Ed.), Upstream Industrial Biotechnology: Expression Systems and Process Development (pp. 49–69). John Wiley & Sons, Inc.
2 Hans G. Drexler, C. C. (2002). Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39, 75–90.