Therefore, consider limiting the number of times you continue to passage an individual cell culture. When passaging cells, it is important to use sterile technique and the appropriate reagents and equipment.
It is essential to use the appropriate media for the optimal growth and expansion of your cell line. Each cell line will require a specific growth supplement cocktail, however, most cell lines at minimum require supplementation with the following: Serum, such as Fetal Bovine Serum, which must be heat-treated to inactivate the bovine complement and which provides vital growth factors for the cells, antibiotics, like penicillin and streptomycin, which help limit contaminating growth in the culture, and other growth factors, like fibroblast growth factor, to help prolong the growth and expansion of the cell lines.
First, take note of the color of the tissue culture media. Fresh, cell culture media is rich in nutrients and appears a clear, orange color, partly due to the addition of the pH indicator, phenol red.
As cells begin to use up nutrients in the media, waste and acid begin to build up in the cell culture, lowering the pH. For this reason, many tissue culture medias contain phenol red, which turns the media from orange to yellow when cells turn the culture acidic. Change the media before it turns this color! When the phenol red turns the media a pinkish color, indicating that the pH has become too basic for healthy cell growth, it may be time to change your CO2 tank as well as your media.
Most adherent cell lines attach naturally to uncoated plastic. Therefore plastic cell culture plates, like petri dishes or 6 well plates, are frequently used for subculturing cells. Plastic cell culture flasks are also used.
The T25 cell culture flask, for example, is often used to expand small cell line populations or to start the growth of a slowly expanding cell line. T75 culture flasks are commonly used for cell lines that proliferate more quickly or to generate larger numbers of cells than can fit in a T25 flask. When removing adherent cells, the proteins that bind the cells to the plastic must first be cleaved.
For this purpose, the digestive enzyme trypsin is frequently used. It is important to carefully time the trypsin exposure, as treatment for too long can result in damage to other cell surface proteins.
Cell culture media can contain trypsin neutralizers. Therefore, phosphate buffered saline, or PBS, is often used to wash the cells before trypsinization. Before you begin, it is important to understand how frequently your cells should be monitored, which will depend on how fast your cells proliferate. Now that your media is a happy orange color, observe the other culture conditions, such as whether or not the culture is cloudy — possibly indicating contamination -- the size and density of the cell colonies, and the overall quality of the cells.
Now add trypsin to the cells and then incubate them at 37 C. After about 5 minutes, confirm that the cells have detached, and then stop the proteolysis by adding fresh tissue culture media. Transfer the cell suspension into a conical tube for centrifugation. Then, after spinning down the cells, carefully remove the supernatant without disturbing the pellet and resuspend the cells in fresh media.
As mentioned earlier, human embryonic stem cells are a type of cell that must be passaged. They are typically cocultured with mouse embryonic fibroblasts, or MEFs, which provide factors that help stem cells retain their pluripotent state. They can be selected by micropipette or by gentle scraping with a glass picking tool and then plated in a new culture for expansion.
A cell scraper can be used to gently remove the cells from the bottom of the culture plate. If the cells are particularly adherent, try moving a serological pipette in a firm scraping motion while rinsing the bottom of the cell container with media or another appropriate solution. Take care with this method, as delicate cells can be damaged by this mechanical method of dissociation. To more quickly and reproducibly scale up the expansion of your cells than can be achieved in single layer flasks, multilayer flasks, which can expand cell cultures fold compared to single layer flasks, can be used.
In this video we reviewed what a cell line is, how to subculture one, and some different applications of passaging cells. Thanks for watching and remember to keep your media fresh! Subscription Required. Please recommend JoVE to your librarian. Basic Methods in Cellular and Molecular Biology.
Passaging Cells. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team.
Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. If the confluent state is prolonged, cells change their phenotype to abnormal characteristics or cells may die. In order to maintain cells in culture, they need to be passaged. The timing of passage is determined based on cell density, culture duration, and cell distribution. This website uses cookies to improve your experience by anonymously collecting browsing data. Cookies can be disabled in your browser settings.
For further details, please check our Cookie Notice. To TOP page of Glossary passaging Passaging is the procedure of harvesting cells from a culture, transferring the cells to one or more culture vessels with fresh growth medium, and using those cells to start new cultures.
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