Basics of Cell Culture

Cell culture is one of the most important tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. 

Primary culture

Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Cell Line

After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

Cell Strain

If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.

Finite vs Continuous Cell Lines

Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite. However, some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.

Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated for senescence, all cell cultures are susceptible to microbial contamination, and even the best-run laboratories can experience equipment failure. Because an established cell line is a valuable resource and its replacement is expensive and time consuming, it is vitally important that they are frozen down and preserved for long-term storage.

As soon as a small surplus of cells becomes available from subculturing, they should be frozen as a seed stock, protected, and not be made available for general laboratory use. Working stocks can be prepared and replenished from frozen seed stocks.  If the seed stocks become depleted, cryopreserved working stocks can then serve as a source for preparing a fresh seed stock with a minimum increase in generation number from the initial freezing.

The best method for cryopreserving cultured cells is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethylsulfoxide (DMSO) or glycerol. Cryoprotective agents reduce the freezing point of the medium and also allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death.

DMSO is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with local regulations.

Cell Culture from a Frozen Working Cell Stock

• Make sure to fill out the cell bank log
• Determine the passage number, e.g. if prior cells were frozen, they were passed three times the thawing process will be the 4th passage.
• Add growth media (basic media, calf serum and antibiotic) to a T75 flask labeled with the cell line, passage number and date. 
• Place the flask horizontally in a CO2 incubator at 37oC and 5% CO2 for at least 15 minutes. This will allow medium to warm and reach its normal pH (7.0-7.6)
• Thaw the frozen vial with the cell line by gentle agitation in a 37oC water bath. To reduce contamination risk keep the O-ring and cap above the water level (2-5 minutes).
• Place the vial in biosafety cabinet (BSL-2). To avoid contamination, before open the vial wipe it with 70% ethanol.
• Transfer frozen cell vial content to the T75 flask containing growth media. Mix gently and rock the flask to distribute the cells evenly on the flask surface.
• Incubate flask overnight in a CO2 incubator at 37oC, 5% CO2. Allow cells to attach overnight
• Change growth media
• Incubate flask at 37oC, 5% CO2 for additional 2-4 days and monitor until the cell growth
• Once the cells have reached about 90% confluence, the cell stocks can be expanded using T150 flasks