Investing in best practices and products at the beginning of any experiment is the most time- and cost-effective way to approach cell culture.
Walk into a lab and ask researchers if their cultures are infected, and they will tell you no. Walk into that same lab and ask them which microorganisms are infecting their cultures, and they will tell you what they are finding.
These seemingly contradictory responses reflect an unwitting acceptance that a certain level of contamination is unavoidable.
In a world thick with bacteria, from the lab floor to the lab tech’s eyelashes, truly sterile conditions are rare and fleeting. That is why, in an effort to mitigate the potential damage infections can cause, training sessions and work practices stress specific processes. Yet over time, work demands, personal idiosyncrasies, distractions and the tedium of performing repetitive tasks take their toll, and inconsistencies begin to appear in the processes of even the most well-run laboratories.
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Figure 1. Environmental conditions are interdependent and critical to the growth of many cells. (Source: BINDER) |
Investing in best practices and products up front is the most time- and cost-effective way to approach cell culture. The price of media and labware is modest when compared to the value of cell lines, some of which are literally irreplaceable. The time involved in utilizing proper technique is short when compared to the time needed to redo experiments that are ruined. And the sterility of cells and integrity of cultures is essential to the validity of research.
In short, we’ve come to tell you what you already know, and to point out some things you may not have considered.
Good practices are critical
It is common for scientists to learn cell culture technique from the person beside them at the bench, who learned it from someone with whom they worked. It is also common for the newest bench scientist to be given responsibility for routine tasks related to cell culture. This is at least partially because the work can be mind-numbingly repetitive and can take considerable amounts of time.
Maintaining proper technique is paramount, and it is important for researchers to audit their processes as they work, staying attentive to procedure and the steps they are taking. It is also important to take a break and refresh. When possible, share the workload.
When working in a hood it must be wiped down completely, both before and after any procedure. Hoods should not be used for storage—the only things in a hood should be those that are necessary to the current process. Wipe down all tools and containers before bringing them into the hood, and again when finished. Use the ultraviolet light to sterilize the hood when it is empty.
When you are dispensing, do not let anyone come into the lab. Put a “Do Not Disturb” sign on the door. Consider locks if the sign goes unheeded. This may seem extreme, but do not cave in to pressure from those who might consider your attention to detail excessive.
Leave lab coats in the lab. Wash your hands immediately upon entering the lab. When a service technician comes in, make sure they put on gloves and wipe down all tools and sensors before touching anything. The same rules apply for sales representatives or any other visitors. When moving cells, be sure the dishes are covered properly and minimize transport time.
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Figure 2. DIN standards are more rigorous than ASTM standards. (Source: BINDER) |
The incubator: A great place to grow
Incubators provide a synthetic environment, one in which living cells are encouraged to grow and thrive as they would
in vivo. And they do grow, often very well, as do their nemeses, mycoplasma and other infectious organisms.
Cleaning incubators, a job that no one likes, is, like other seemingly routine tasks, often assigned to the junior member of a team. Every nook and cranny of the unit, inside and out, must be thoroughly wiped down, using a good, non-bleach, non-corrosive CO2 incubator-cleaning agent. (Bleach, even in small amounts, will destroy stainless steel.)
Isopropyl alcohol is safe to use, in limited amounts, and only on work surfaces. Be aware though, that as long as alcohol can be smelled, it is killing things. In the incubator, the victims can include your cells.
Shared incubators present their own challenges. It is imperative that all researchers use proper techniques. Never let anyone touch an incubator who has not washed his or her hands and put on gloves. If an ungloved person touches an incubator, anyone who touches it subsequently, even those wearing gloves, will carry that contamination forward.
Before opening an incubator, stop and think about what you are looking for and where it is located. Standing with the door open and poking around is an invitation for environmental pathogens to enter. Make sure locations within the chamber are assigned to, and remembered by, those sharing the unit. Moving plates and flasks around and relocating other people’s cells while looking for your own culture will increase the risk of contamination.
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Figure 3. A minimum temperature of 160°C for a period of at least two hours is a requirement for sterilization. (Source: BINDER) |
If you find—or even suspect—infection, inform everyone who shares the incubator immediately. Whether or not your colleagues will be able to save their cultures, they will at the least be able to recognize problems sooner and act accordingly.
Preventative maintenance cannot be emphasized enough. If your unit has a HEPA filter, it needs to be changed after every major cleaning—and a major cleaning needs to be done after each experiment is completed and before the next experiment starts. It takes time to change HEPA filters. In some units they are difficult to get at, and there are disposal and replacement costs. But you don’t want the filter harboring infectious agents screened from your previous work to be used to safeguard your next experiment. Consider the manufacturer’s guidelines for filter changes to be the minimum requirement.
Recalibrate CO2 sensors as recommended by the manufacturer. If you wait until cells are dying to recalibrate the results can be catastrophic. Put the sensor in the chamber and let it read for a few hours, to allow complete equilibration. This will provide an exact measurement and indicate whether changes to the controller are needed.
Consider the lab environment. Look for the location of heating and air conditioning ducts and locate your incubator and hood anywhere but under them. Pull incubators out and clean underneath them. They can be heavy, and this can be hard, but units that sit in one place for years are traps for contaminants lurking just outside the incubator door.
Humidity, or lack of humidity, in the lab should be considered. Ultra-dry, heated winter air will suck all of the humidity out of an incubator when the door is opened. Keep incubators out of the direct sun, especially in warmer climates.
Cold rooms and freezers
The cold room can be an undetected source of contamination. Do not store media in cardboard boxes, which absorb moisture and can host contaminants. Make sure to regularly clean moldings.
Some labs go to great extents to isolate the cold room as a source of contamination. One person enters the room and hands needed supplies to a co-worker outside the room. The person who is in the cold room is not allowed back in the lab; because of this, the task is often planned for the end of the day. This example is worth imitating.
Opening and closing of freezers should be minimized, just as it is with incubators. Before opening the door, know what you are looking for and where it is located. Do not move tubes around or linger in front of an open freezer. When freezers are moved or unplugged, check them carefully upon restarting, and watch them for a few days to make sure that performance has not been affected. Be certain that alarms are maintained and functioning.
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Figure 4. Incubator interior. The absence of tight corners, seams and other spots for infectious agents to accumulate reduces the possibility of cross-contamination and facilitates cleaning. (Source: BINDER) |
Incubator selection
Models currently on the market offer a variety of features that are designed to promote the growth of cultures and to keep cells safe from contamination. Is the unit air-jacketed or water-jacketed? Does it provide active humidification or utilize a water pan?
Temperature, pH, CO2, and humidity levels are all interrelated—measurement and maintenance of proper levels is critical to healthy cell growth. What kinds of sensors are included, and where are they located?
What kind of cleaning cycle is necessary in your work? Decontamination is the temporary elimination or inactivation of infectious agents; disinfection is the elimination or irreversible inactivation of all pathogenic germs; sterilization is the inactivation or elimination of all vital microorganisms. Be sure you know the level of cleaning offered by the incubators you use or are considering.
Labware and media—quality and consistency are key concerns
All major manufacturers provide labware of comparable quality. It is made of polystyrene, which is naturally hydrophobic, then treated to form the hydrophilic surface that is necessary for cell growth and sterilized using cobalt radiation.
There is some variation in the kinds of treatment used to create the hydrophilic surface, and some variation in the way quality control procedures are performed. While most cells will basically grow equally well on all top line products, cell lines sometime seem to acclimate to a particular type of plate or flask, so use the same product unless production issues create shortages.
Labware costs can add up (some big labs can spend as much as $100,000 a year on labware) and pressure to cut costs can be great. It remains cost effective to buy quality products from mainstream manufacturers, or products that are comparable in all ways. Make sure all products are certified to be free of RNase and DNase. Do not, under any circumstances, reuse labware.
Externally threaded cryotubes are preferable. Whenever there is even the slightest residue on the tube, threads, or cap, wipe it down.
Culture media should be produced using high quality components that are prepared in consistent amounts and are sterile. Concerns about quality and consistency are important with serum as well. It is often possible to have a manufacturer store serum from a specific lot for an individual lab and ship from that batch as needed over time.
Summary
Attention to detail, rigorous adherence to standards and processes, and investments in quality consumables, work spaces and apparatus offer long-term benefits. Scientists have lost months, even years, of work because routine precautions were ignored or corners were cut. When the potential ramifications of infection are taken into account, the effort and expense taken to prevent them seem a modest price indeed.
About the Authors
Ron Breuer is business development manager, and David Craig is senior regional sales manager, with BINDER Inc. Located in Great River, NY, BINDER Inc. is the Americas subsidiary of BINDER GmbH, a German manufacturer of environmental simulation chambers. BINDER CO2 Incubators utilize a 180° C Hot Air Sterilization Cycle.
Bob Sherman is a regional sales representative with Greiner Bio-One, a Monroe, North Carolina manufacturer of labware, liquid handling apparatus, and other equipment.
This article was published in Bioscience Technology magazine: Vol. 34, No. 3, March, 2010, pp. 26-29.