This work has been made possible thanks to funding from the Howard Hughes Medical Institute (HHMI). We wish to thank our head of lab, Jue Chen, for reading the manuscript and for her continued support, Donna Tallent for her helpful edits and comments, and Jeff Hennefeld from the Information Technology Department at The Rockefeller University for his help with the video component of this manuscript.

1. Preparations 
<ol>
	<li>General
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.&#160;The paper towel does not need to be sterile.</li>
		<li>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&#8217;s instructions for cleaning.</li>
	</ol>
	</li>
	<li>Inside the biological safety cabinet
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Working with adherent cell lines
<ol>
	<li>If a plastic flask is needed, spray the entire bag and place it inside the cabinet.</li>
	<li>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.&#160;</li>
	<li>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.</li>
	<li>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.</li>
	<li>Generously spray the gloves with 70% EtOH any time they become dry. Rub the hands together so the gloves are not dripping wet.</li>
	<li>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.</li>
</ol>
4. Checking and storing cells 
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Ensure caps on all the bottles are tightly closed before removing the bottles from the cabinet.</li>
	<li>Store cell culture media in the dark at 4 &#176;C when not in use since it is light sensitive.</li>
	<li>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.</li>
</ol>
5. Working with suspension cell lines
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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&#8212;do not touch it from the neck&#8212;once the foil is off. Use a 1 mL serological pipette to take a sample for cell counting.</li>
	<li>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.</li>
	<li>Ensure caps and aluminum foil are tightened before removing bottles or flasks from the biological safety cabinets.</li>
</ol>
6. Cell incubation 
<ol>
	<li>Use separate incubators for different cell types to prevent cross-contamination of the different cell types.</li>
</ol>
7. Liquid waste collection
<ol>
	<li>Collect liquid waste in a vacuum trap flask located outside the cabinet on the floor in a secondary container labeled &#8216;Waste&#8217;. 
	NOTE: The hose is connected to a high-efficiency particulate absorbing (HEPA) filter, which is replaced on a monthly basis.</li>
</ol>
8. Cleanup
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
9. Organization 
<ol>
	<li>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.</li>
	<li>Label any plastic bottles used to harvest cells and reused in cell culture afterward as &#8216;For Cell Culture Use Only&#8217;. Use the designated cell culture bottles for one specific cell type. Store the bottles in the cell culture room for easy access.</li>
</ol>
10. Identifying bacteria, fungi, and mycoplasma contamination 
NOTE: Not following the workflow above can lead to bacterial, fungi, and mycoplasma contamination.
<ol>
	<li>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.
	<ol>
		<li>Visually identify mold contamination by noting the appearance of round, fuzzy balls floating in the media (see Figure 2).</li>
		<li>Visually identify bacterial contamination if media is cloudy, white or turbulent (see Figure 2).</li>
		<li>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 &#956;L, 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.</li>
	</ol>
	</li>
</ol>

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P., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+growing+cells+as+model+systems+in+vitro.">Cell culture: growing cells as model systems in vitro.</a> Basic Science Methods for Clinical Researchers. , 151-172 (2017).</li><li>Drexler, H. G., Uphoff, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+contamination+of+cell+cultures:+Incidence,+sources,+effects,+detection,+elimination,+prevention.">Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.</a> Cytotechnology. 39 (2), 75-90 (2002).</li><li>Lincoln, C. K., Gabridge, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination:+sources,+consequences,+prevention,+and+elimination.">Cell culture contamination: sources, consequences, prevention, and elimination.</a> Methods in Cell Biology. 57, 49-65 (1998).</li><li>Nikfarjam, L., Farzaneh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+detection+of+mycoplasma+contamination+in+cell+culture.">Prevention and detection of mycoplasma contamination in cell culture.</a> Cell Journal. 13 (4), 203-212 (2012).</li><li>Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination.">Cell culture contamination.</a> Methods in Molecular Biology. 731, 79-91 (2011).</li><li>Young, L., Sung, J., Stacey, G., Masters, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Mycoplasma+in+cell+cultures.">Detection of Mycoplasma in cell cultures.</a> Nature Protocols. 5 (5), 929-934 (2010).</li><li>Barth, O. M., Majerowicz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+by+transmission+electron+microscopy+of+mycoplasma+contamination+in+sera+and+cell+cultures.">Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures.</a> Memorias do Instituto Oswaldo Cruz. 83 (1), 63-66 (1988).</li><li>Mirabelli, P., Coppola, L., Salvatore, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+are+useful+model+systems+for+medical+research.">Cancer cell lines are useful model systems for medical research.</a> Cancers. 11 (8), 1098 (2019).</li><li>A cell culture master class: What your cells wish they could tell you. Science Available from: <a target="_blank" href="https://www.science.org/content/webinar/cell-culture-master-class-your-cells-wish-they-could-tell-you">https://www.science.org/content/webinar/cell-culture-master-class-your-cells-wish-they-could-tell-you</a> (2020)</li><li>Langdon, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination:+an+overview.">Cell culture contamination: an overview.</a> Methods in Molecular Medicine. 88, 309-317 (2004).</li><li>Babic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-research:+Incidences+of+problematic+cell+lines+are+lower+in+papers+that+use+RRIDs+to+identify+cell+lines.">Meta-research: Incidences of problematic cell lines are lower in papers that use RRIDs to identify cell lines.</a> eLife. 8, e41676 (2019).</li><li>Visconti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+tandem+repeat+profiling+for+the+authentication+of+cancer+stem-like+cells.">Short tandem repeat profiling for the authentication of cancer stem-like cells.</a> International Journal of Cancer. 148 (6), 1489-1498 (2021).</li><li>Horbach, S. 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Processing and Safety. Food Safety Publications Available from: <a target="_blank" href="https://www.ars.usda.gov/ARSUserFiles/60701000/FoodSafetyPublications/p328.pdf">https://www.ars.usda.gov/ARSUserFiles/60701000/FoodSafetyPublications/p328.pdf</a> (2004)</li><li>Sterilizing Practices. Centers for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/sterilizing-practices.html">https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/sterilizing-practices.html</a> (2023)</li><li>Cot&#233;, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+technique+for+cell+culture.">Aseptic technique for cell culture.</a> Current Protocols in Cell Biology. , (2001).</li><li>Uphoff, C. C., Drexler, H. 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The author does not have any conflicting interests.

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...

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 lines2,3. 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&#8217;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 testing4,5,6,7,8. 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 mycoplasma9,10. 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.&#160;
All lab personnel should strictly adhere to good cell culture practices. Cultures should be tested for mycoplasma after they&#8217;ve been newly purchased, while they are currently being grown, before cryopreservation, and when thawed from liquid nitrogen2,10. Different tests are available on the market using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), or immunostaining.3 The literature indicates that &#8220;human isolates represent a large percentage of the mycoplasma contaminants found in cell culture&#8221;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 &#8220;human operator is potentially the greatest hazard in the laboratory&#8221;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%&#8211;18% of cell lines worldwide may be cross-contaminated or misidentified11,12. 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&#8217;s an easy way to confirm cell line identity2,10,13,14. STR can identify mislabeled or cross-contaminated cell lines, but it cannot detect incorrect tissue origin10,13,14. The validity of research data can be compromised if cell lines are mislabeled, wrongly identified, or contaminated13.&#160;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 &#176;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14-190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F-12 Media</td>
			<td>ATCC</td>
			<td>30-2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Baffled Flask</td>
			<td>Pyrex&#160;</td>
			<td>09-552-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipettes</td>
			<td>Fisher&#160;</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond</td>
			<td>13-681-15A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette</td>
			<td>Corning</td>
			<td>07-200-573</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>07-202-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25-300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>*Items may vary because this video is about general cell culture techniques</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell culture techniques and practices to avoid contamination by fungi and bacteria in the research cell culture laboratory

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&#8217;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.&#160;

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&#8211;3 days later. Round fuzzy balls floating in the media are noticeable in suspension cells, while mold growth in attached...

Watch this Scientific Journal Video about Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory at JoVE.com

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

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.&#160;&#160;

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 ...

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JoVE Journal

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13.1K Views.  The Rockefeller University. 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.  

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

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Title Cell Encapsulation by Droplets

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...