Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory

Podsumowanie

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.  

Streszczenie

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it’s important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime. 

Wprowadzenie

Cell culture has many uses in the research laboratory. Since the origins of cell culture in the early 20th century, cell lines have helped advance science. Cell lines have several advantages; various cell lines can help researchers study cell biology, produce baculovirus for further studies, or produce large quantities of a protein of interest, to name a few¹. Some additional uses include studying tissue growth, helping to advance vaccine development, toxicology research, studying the role of genes in healthy organisms and diseased models, and the production of hybrid cell lines^2,3. Cell lines can also enable drug production³. Proper aseptic techniques are necessary when working with cell lines; the practices and techniques outlined in this manuscript are applicable to research laboratories where cell culture work is performed. Other laboratory settings are not discussed.

Contamination is often the primary concern when performing cell culture work. In the context of this paper, contamination generally refers to fungi and bacteria. The overall goal of the method outlined in this paper is to thoroughly describe the best practices for avoiding contamination. All lab members should adhere to these practices when working in a research laboratory’s cell culture room. Laboratories should ensure all workers are active participants in using these best practices to prevent contamination. The knowledge of the correct practices and techniques will help ensure cell cultures remain viable, healthy, and free from contamination. The development of this technique is based on research of the literature, seven years of experience working with cell cultures, and the need for a method that both novices and experienced cell culture workers can refer to on an annual basis.

There is a need for a clear, standardized technique that all research cell culture laboratories should follow. Much of the literature on cell culture contamination discusses the detection of mycoplasma, aseptic techniques, sources of contamination, elimination of contaminants, and prevention by use of antibiotics and regular testing^4,5,6,7,8. While this information is helpful, there are no videos present in the literature that demonstrate the proper cell culture techniques one should follow. The advantage of the practices presented over alternative techniques is a focus on preventing contamination before it happens, rather than detecting and correcting mistakes later. Moreover, a thorough demonstration of aseptic techniques, a discussion about preventing fungi and bacterial growth, and information regarding biosafety cabinet airflow are valuable for both novice and experienced cell culture workers alike.

Bacteria and fungi are the two most common types of contaminants. Within the bacteria category, mycoplasma is a major concern due to its small size and ability to proliferate while remaining unnoticed. They are self-replicating organisms with no rigid cell wall that rely on eukaryotic cells to grow. They have reduced metabolic capabilities and can multiply greatly while remaining unrecognized in the routine visual inspection of cell cultures and regular microscopic analysis, although transmission electron microscopy can detect mycoplasma^9,10. Moreover, they can pass through microbiological filters¹⁰. Cell culture medium provides mycoplasma with nutrients, although unfortunately supplementing media with antibiotics does not affect mycoplasma¹⁰. One should note that, in general, it is not necessary to supplement media with antibiotics; proper techniques should suffice in keeping contamination at bay. Infection with mycoplasma does not lead to immediate cell death, but it is concerning to researchers as it affects data reproducibility and quality. 

All lab personnel should strictly adhere to good cell culture practices. Cultures should be tested for mycoplasma after they’ve been newly purchased, while they are currently being grown, before cryopreservation, and when thawed from liquid nitrogen^2,10. Different tests are available on the market using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), or immunostaining.³ The literature indicates that “human isolates represent a large percentage of the mycoplasma contaminants found in cell culture”⁵. Although more than 200 mycoplasma species have been described, about six of these account for most infections. These six species are M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, and Acholeplasma laidlawii¹⁰. As with other types of contamination, air and aerosols bring these into cell cultures⁵. This is echoed in other papers since the “human operator is potentially the greatest hazard in the laboratory”⁷. Although this is done through human error, the risk can be eliminated if a standard procedure is followed. Shedding from personnel is not restricted to only mycoplasma contamination; cell cultures in one lab are usually infected with the same mycoplasma species, indicating that contamination spreads from one flask to another due to improper cell culture techniques¹⁰.

The prevention of cross-contamination is also another reason why proper cell culture techniques should be followed. It is noted that at least 15%–18% of cell lines worldwide may be cross-contaminated or misidentified^11,12. In addition to testing cell lines for mycoplasma contamination, they should also be tested for cross-contamination¹⁰. For human cell lines, cell line authentication by an inexpensive DNA-based technique called short tandem repeat (STR) profiling is the current international reference standard, as it’s an easy way to confirm cell line identity^2,10,13,14. STR can identify mislabeled or cross-contaminated cell lines, but it cannot detect incorrect tissue origin^10,13,14. The validity of research data can be compromised if cell lines are mislabeled, wrongly identified, or contaminated¹³. Similar to other types of contamination, cross-contamination can occur due to poor technique causing aerosols to spread, mistaken contact leading to the wrong cell type entering a flask, or using the same media bottle and reagents with different cell lines¹⁰. No sharing of media bottles should occur; sharing one bottle of media between two different cell lines can allow those cell populations to be mixed, leading the faster growing cell type to completely take over the flask. This replacement is not noticeable and leads to mislabeling and misidentification². A cell line can also be mistaken for another if cultures are confused during handling or labeling¹⁰. Careful attention should be paid to keeping reagents, media, and flasks separate from one another. Each lab member should have their own media bottles; no sharing should occur between lab members. Cell lines themselves should be purchased from a qualified cell bank and provider. Laboratories should not share cells. Studies show that, although STR and mycoplasma tests are regularly used, many research papers in the literature have already used misidentified or contaminated cell lines¹⁵. Sifting through the research to find these problematic papers and retroactively inform readers about this matter is cumbersome. Prevention is the best way to ensure this problem does not occur in the first place.

The simple action of spraying items with 70% EtOH can kill organisms; 70% EtOH works by denaturing proteins and dissolving lipids in the most commonly contaminating organisms, including bacteria and fungi¹⁶. Studies have shown that 70% is the most effective concentration; surface proteins do not coagulate rapidly with 70% EtOH so they can enter the cell, while the water it contains is necessary for the denaturing process of proteins. Due to the concentration difference of water and alcohol on either side of the cell wall, 70% EtOH enters the cell to denature both enzymatic and structural proteins. If mold growth is observed in flasks, the entire incubator must be decontaminated by first spraying it with 70% EtOH and wiping it dry, followed by a 16 h overnight incubation at 60 °C¹⁷. This kills most mold and any bacteria.

The main advantage of prevention practices over alternative techniques of eliminating contamination after it occurs is that by preventing contamination early on, laboratory workers can be sure their cell cultures are healthy and there will be no high costs associated with the decontamination of incubators or discarding of cell cultures. The elimination of mycoplasma contaminants after, for instance, is not efficient⁷. Taking the time early on to ensure laboratory personnel are properly trained, the cell culture room is self-contained, and a standard procedure is used will save time and money.

Protokół

1. Preparations

1. General
   1. Wear a clean lab coat designated to be worn only in the cell culture room and no other parts of the laboratory.
      NOTE: The lab coat does not need to be sterile.
   2. Wear new gloves that have not touched any other surfaces. Make sure the gloves fit tightly. Nitrile, powder-free gloves are best.
      NOTE: The gloves do not need to be sterile.
   3. To prepare for work, spray the gloves, lab coat sleeves, and interior of the biological safety cabinet with 70% EtOH. Wipe the working surface and glass panel dry with a lint-free paper towel.
      NOTE: Using 70% EtOH kills bacteria with the highest efficiency¹⁶. The paper towel does not need to be sterile.
   4. Keep water baths inside the cell culture room and only use these for warming culture media or thawing cells. Drain and wash the water baths once a week, following the manufacturer’s instructions for cleaning.
2. Inside the biological safety cabinet
   1. Limit the number of items brought into the cabinet. Do not interrupt the airflow inside the biological safety cabinet by blocking the front or back grills.
      NOTE: See Figure 1 for an explanation of how air flows inside a cabinet.
   2. Spray all the items placed inside the cabinet with 70% EtOH and wipe them dry. Begin by spraying the top of the media bottle and working down. Similarly, with a clean paper towel, wipe it dry, progressively working the way to the bottom. Do not go back up toward the cap.
   3. If the cabinet is large enough to accommodate serological pipettes, they can be placed inside, otherwise they can be stored in a receptacle mounted on the outside of the cabinet. Check either side of the encased pipette for holes, tears, or punctures in the packaging before use. Do not rip off the wrapping. Instead, gently peel the ends of the wrapping, insert the serological pipette into a pipette aid, and remove the wrapping in one fluid motion.
   4. Do not hover over open bottles or flasks; reach over open bottles or flasks, or open items over the tops of already opened items in the biological safety cabinet.
      NOTE: The airflow inside the cabinet pushes down on the work surface, so any contamination present on the sleeve, for example, may enter the cell cultures.
   5. Do not pour liquids. Instead, add them using a serological pipette. After supplementing the media, mix the contents thoroughly and initial the bottle. Also, ensure to include a label for what the media was supplemented with.

2. Working with adherent cell lines

1. If a plastic flask is needed, spray the entire bag and place it inside the cabinet.
2. Use autoclaved glass pipettes or disposable sterile plastic pipettes to aspirate the media or washing solutions. Carefully remove the metal cap from the storage container. Isolate one glass pipette by gently shaking the container at an angle. When reaching into the container, avoid touching any other pipettes.
   NOTE: Handle the chosen pipette from one end only. 
3. Quickly replace the caps on the bottles as soon as possible. Place the caps on the work surface upside down so that the rim does not touch the work surface. Do not grab the cap from the top or bottom; instead, touch the caps from the sides.
4. When aspirating liquids, use a vacuum trap flask located outside of the cabinet in a secondary container on the floor.
   NOTE: Do not throw any liquid waste in biohazard waste bags as the bags will leak. Waste will be created while working inside the cabinet. Moving hands in and out of the cabinet too often will interrupt the airflow. Leave any waste inside the cabinet temporarily. Place it off to the side so it will not interrupt the work.
5. Generously spray the gloves with 70% EtOH any time they become dry. Rub the hands together so the gloves are not dripping wet.
6. If a serological pipette mistakenly touches something in the cabinet, do not hesitate to throw it out. Start anew with a clean serological pipette instead of using one that may be contaminated.

4. Checking and storing cells

1. Before placing cells in the incubator, check to see how they look under the microscope. If the cells have been thoroughly suspended, single cells should be observed.
2. Do not speak, sneeze, cough, or breathe heavily into the incubators. Quickly open and close the incubator doors. Leaving doors open for longer than necessary may allow contaminants present in the air to enter the incubators.
   NOTE: Wearing a mask while working in the cell culture room can help since mycoplasma may be present in the human mouth. Avoid the use of cell phones in the cell culture room, as talking is not recommended.
3. Ensure caps on all the bottles are tightly closed before removing the bottles from the cabinet.
4. Store cell culture media in the dark at 4 °C when not in use since it is light sensitive.
5. Spray the interior of the cabinet with 70% EtOH again after the cell culture work is complete and wipe the surface dry with a paper towel. Empty the biohazard trash waste bags. Repeat this process and replace the gloves when switching to a different cell line.

5. Working with suspension cell lines

1. For suspension cells grown in glass flasks, ensure the gloves are sprayed thoroughly with 70% EtOH, then touch the aluminum foil with the wet gloves and spray only the bottom of the flask before placing it inside the cabinet.
2. When taking a sample for cell counting, remove only one 1.5 mL tube from its container. Do not touch any other tubes. Place the cap upside down on the working surface. Do not touch the inside rim. Handle it with care from the sides and replace it once finished.
3. Carefully remove the double-folded piece of aluminum foil that covers the entire neck of the flask. Handle the glass flask from the bottom only—do not touch it from the neck—once the foil is off. Use a 1 mL serological pipette to take a sample for cell counting.
4. Do not let media drip down the side of the flasks; if it does, spray a paper towel with 70% EtOH and clean it up right away.
5. Ensure caps and aluminum foil are tightened before removing bottles or flasks from the biological safety cabinets.

6. Cell incubation

1. Use separate incubators for different cell types to prevent cross-contamination of the different cell types.

7. Liquid waste collection

1. Collect liquid waste in a vacuum trap flask located outside the cabinet on the floor in a secondary container labeled ‘Waste’.
   NOTE: The hose is connected to a high-efficiency particulate absorbing (HEPA) filter, which is replaced on a monthly basis.

8. Cleanup

1. Remove the biohazard waste bag and wash the glass flasks as soon as possible. Keep a glass-washing protocol by the sink. See Supplementary File 1 for the washing protocol.
2. Autoclave the cell culture glassware instead of sending it to a glass washing facility. Keep it separate from glassware in the main area of the lab.
   NOTE: SF-9 cells leave a rim of dead cells on the side of flasks if the glass is not scrubbed well. See Supplementary File 1 for the autoclave protocol.

9. Organization

1. Organize the cell culture room so that all the supplies are located in one area, thereby minimizing the need for lab members to leave the room in search of supplies.
2. Label any plastic bottles used to harvest cells and reused in cell culture afterward as ‘For Cell Culture Use Only’. Use the designated cell culture bottles for one specific cell type. Store the bottles in the cell culture room for easy access.

10. Identifying bacteria, fungi, and mycoplasma contamination

NOTE: Not following the workflow above can lead to bacterial, fungi, and mycoplasma contamination.

1. Always before beginning work, observe flasks for heavy turbidity, extra growth in the form of fuzzy balls, and dense accumulations of cells on the side, all of which are indicators of contamination.
   NOTE: An experienced eye can tell the difference between turbidity caused by microbial contamination versus the actual cells. While cells cause the normally clear media to appear cloudy, bacterial contamination causes heavy turbidity with white color.
   1. Visually identify mold contamination by noting the appearance of round, fuzzy balls floating in the media (see Figure 2).
   2. Visually identify bacterial contamination if media is cloudy, white or turbulent (see Figure 2).
   3. Identify mycoplasma contamination by doing a monthly mycoplasma test. One PCR-based test instructs users to take a 1.5 mL sample of cells, perform a cell count using 10 μL, dilute to 0.08 x 10⁶ cells/mL, spin down the cells, lyse the cells using the buffer contained in the kit, and spin down one final time. The supernatant is incubated with primers that amplify a range of mycoplasma species. Run a DNA gel to visualize the bands. No bands mean that mycoplasma is not identified.
      NOTE: This major contaminant can be present in the human mouth. It is good practice to test for mycoplasma contamination in cells after thawing them and before using them for experiments. Afterward, monitor the cells for mycoplasma contamination once a month. Many companies offer mycoplasma test kits. Pick one that is suitable.

Wyniki

If the proper cell culture techniques and practices outlined in this paper are not followed, contamination by fungi and bacteria may occur in the research cell culture laboratory. Figure 2 shows flasks containing contamination in both the suspension and adherent cultures.

When not following aseptic techniques, mold contamination may occur 2–3 days later. Round fuzzy balls floating in the media are noticeable in suspension cells, while mold growth in attached...

Dyskusje

While contamination is one of the primary concerns when performing cell culture work, the practices and techniques outlined in this manuscript will help mitigate the risks. The critical steps include wearing a clean lab coat, which is only used in the cell culture room, using clean, powder-free gloves that are sprayed with 70% EtOH often and which are changed when switching between cell lines, encouraging each individual to not share media bottles, cleaning the cabinet thoroughly prior to and after finishing work, neatly...

Materiały

Name	Company	Catalog Number	Comments
DPBS	Gibco	14-190-144
DMEM F-12 Media	ATCC	30-2006
Glass Baffled Flask	Pyrex	09-552-40
Glass Pipettes	Fisher	13-678-6B
Pipette Aid	Drummond	13-681-15A
Serological Pipette	Corning	07-200-573
T75 flask	Corning	07-202-004
Trypsin	Gibco	25-300-054
*Items may vary because this video is about general cell culture techniques