Cell Culture
Cell culture is the process by which cells are grown under controlled conditions in artificial growth medium. Cells can be maintained outside their natural environment in optimal growth medium for continued growth.
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Technical notes for standard Cell Cultures
In this post, you’ll learn troubleshooting tips and equipment information for culturing cells.
Developing a Primary Cell Cultures
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. Primary culture refers to the stage of the culture after the cells are isolated from the tissue of interest and are proliferated under appropriate conditions until they reach confluence or occupy all the available substrate. One method of acquiring cells for primary culture entails sampling from the tissue directly. Cells taken in this manner must then be disaggregated, using enzymatic or mechanical means, before they are placed on the substrate.
Primary culture is also acquired by deriving it from an established cull culture or cell strain. These are commonly immortalized cells, cells that have mutated and lost the ability to undergo senescence. These cells can continuously divide and are optimal cell lines for prolonged studies. The most common immortalized cell lines used for primary culture include HeLa cells and HEK 293 cells
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Developing Cell Lines
Over time, cells will undergo senescence, a gradual loss of the ability to procreate. These cells are known as finite cells. However, cells may undergo a transformation process to become continuous cells, capable of dividing indefinitely. Examples of these cells include the immortalized cell lines. Regardless of the cell line type, it is necessary to propagate the cell line by continuously passaging the cells into fresh media, a process known as splitting the cells.
To split cells, cell media is aspirated via a vacuum and the cells are washed in warmed Phosphate Buffered Saline (PBS) to remove residual media. If the cells are anchorage-dependent and rely on adherence to a surface, the cells can be treated with trypsin, a chemical that causes cells to lose their adherence temporarily. Trypsin, however, is toxic and should not be applied for long.Trypsin toxicity can be neutralized by adding growth media. For mammalian cells, this growth media is typically DMEM supplemented with Fetal Bovine Serum (FBS) and antibiotics. From this solution, the media and cells can be partitioned, or split, into new cell culture plates. A common practice is to always maintain at least one plate for further splitting and then split into any additional plates as needed that will be used for experiments.
While the above example is typical for anchorage-dependent mammalian cells, the premise applies to other types of cells including those in suspension and spore colony culture. In suspension culture, cells from a developed culture may be split into new suspension media filled with fresh growth media. In spore colony culture, spores or colonies may be taken from a developed plate and smeared onto fresh media plates.
Different cells require different growth conditions. While conditions may vary from cell type to cell type, most cells are grown in sealed incubators to maintain optimum growth conditions. Incubators have the ability to maintain an optimum temperature (37oC for most cells) and precise O2 and CO2 levels. Additionally, cells will require a growth medium that is unique to each cell type. Media contains the nutrients, hormones, and buffers for cells to grow and should be continually changed. The right media and incubation conditions are critical for cell growth.
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Maintaining Cell Lines
Successful cell culture depends on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses. Non-sterile supplies, media, reagents, airborne particles, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique is the best barrier to prevent contamination by invasive microorganisms and should always be maintained. Aseptic techniques include:
Only opening cell culture in a controlled environment such as a cell culture hood with working airflow.
Sterilizing the work area before and after each handling of the cells with proper cleaning reagents. 70% ethanol is the most common choice.
Maintaining good personal hygiene.
Using gloves, lab coats, and other PPE as needed to prevent contamination of the samples and maintain personal safety.
Only mixing and opening any media or solutions within the cell culture hood.
Opening and/or mixing all media and solutions within a cell culture hood.
All media and solutions should be opened and/or mixed within a cell culture hood.
Using only sterile glassware and disposable pipettes. Dispose of pipettes after each use.
Opening media and other containers only when ready to use.
However, even with proper aseptic techniques, contamination can happen. In these cases, the incubator and cell culture hood should be thoroughly cleaned to prevent other samples from being contaminated. If contaminated cells or media are critical to work, they may be treated with antibiotics to kill the contaminants - though care should be taken as antibiotics may cross-react with the cells of interest. In most cases, it is preferred the contaminated cells and media are thrown out and prepared anew.
Storing cells long-term is a useful way to backup any experiments relying on a certain subculture or strain of cells. The most common method is Cryopreservation. Cryopreservation require a surplus of cells be taken from cell culture and mixed with a protective agent, typically DMSO or glycerol, before being stored below –130°C.
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- Biolidics ClearCell® FX1 Run Kit (10 Runs/Kit)