Cell culture is one of the most important tools used in cell and molecular biology, providing an excellent model system for studying normal physiology and biochemistry (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on cells As well as the effects of cell mutagenesis and carcinogenesis. It is also used in drug screening and development, and in the large-scale production of biological compounds (e.g. vaccines, therapeutic proteins). A major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained by using a batch of clonal cells.
cell culture
Cell culture refers to the removal of cells from animals or plants and their growth in a favorable artificial environment. Cells can be removed directly from the tissue and dissociated enzymatically or mechanically prior to culture, or they can be derived from established cell lines or strains
1. Primary training
Primary culture refers to the stage of culture after cells have been isolated from tissue and propagated under appropriate conditions until they occupy all available substrate (i.e., reach confluence). At this stage, the cells must be subcultured (i.e., passaged) by transferring them to a new container with fresh growth medium to provide more room for continued growth.
2. Cell lines
After the first subculture, primary cultures are called cell lines or subclones. Cell lines derived from primary cultures have a finite lifespan (i.e., they are finite) and when they are passaged, cells with the highest growth potential predominate, resulting in a degree of genotypic and phenotypic uniformity in the population sex.
3. Cell lines
A cell line becomes a cell strain if a subpopulation of the cell line is actively selected from culture by cloning or other methods. Cell lines often acquire additional genetic changes after the parental line was initiated.
4. Limited cell lines and continuous cell lines
Normal cells typically divide only a limited number of times before losing their ability to proliferate, a genetically determined event called senescence; these cell lines are called finite. However, some cell lines become immortal through a process called transformation, which can occur spontaneously or be induced chemically or virally. A finite cell line becomes a continuous cell line when it undergoes a transformation and acquires the ability to divide indefinitely.
Continuously cultured cell lines are prone to genetic drift, finite cell lines are doomed to age, all cell cultures are susceptible to microbial contamination, and equipment failure can occur in even the best-run laboratories. Since established cell lines are a valuable resource whose replacement is costly and time-consuming, it is essential to cryopreserve them for long-term storage.
Once a small number of remaining cells are obtained from subculture, they should be frozen as a seed stock and protected from general laboratory use. Working stocks can be prepared and replenished from frozen seed stocks. If the seed stock is depleted, then the cryopreserved working stock can be used as a source for preparing fresh seed stock, yielding minimal increase in quantity from the initial freeze.
The best way to cryopreserve cultured cells is in liquid nitrogen, in complete medium, in the presence of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol. Cryoprotectants lower the freezing point of the medium and also reduce the rate of cooling, greatly reducing the risk of ice crystal formation, which can damage cells and lead to cell death.
DMSO is known to facilitate the entry of organic molecules into tissues. When handling reagents containing dimethyl sulfoxide, equipment and practices appropriate for the hazards of this material should be used. Dispose of reagents according to local regulations.
Grow cells from frozen working cell stocks
Make sure to fill in the mobile banking logbook
Determine the number of passages, e.g. if the previous cells were frozen and they were thawed three times, the thawing process would be the fourth passage.
Add growth media (minimal media, calf serum, and antibiotics) to T75 flasks labeled with cell line, passage number, and date
Place the flask horizontally in a CO incubator at 37 °C and 5% CO for at least 15 min. This will warm up the medium and bring it to its normal pH (7.0-7.6)
Thaw frozen vials with cell lines in a 37 °C water bath with gentle agitation. To reduce the risk of contamination, keep the o-ring and cap above the water level (2-5 minutes).
Place the vial in a biological safety cabinet (BSL-2). To avoid contamination, wipe down with 70% ethanol before opening the vial.
Transfer the frozen cell flask contents to a T75 flask containing growth medium. Mix and shake the flask gently to evenly distribute the cells over the surface of the flask.
Incubate the flask overnight in a CO2 incubator at 37 °C with 5% CO2. Allow cells to attach overnight
changing medium of growth
Incubate the flask for an additional 2-4 days at 37 °C, 5% CO2 and monitor until the cells grow
Once the cells reach approximately 90% confluency, the cell stock can be expanded using a T150 flask
