Some ATCC cell, are shipped as growing cultures in culture vessels. These vessels are seeded with cells, incubated to ensure cell growth and then filled completely with medium for shipping.
If the cells are attached and growing in a monolayer:
- Aseptically remove all but 5 mL to 10 mL of the shipping medium. The shipping medium can be saved for reuse and should be stored at 4°C.
- Incubate the flask at the temperature and CO2 concentration recommended on the Product Sheet (37°C with 5% CO2 for most cell lines) until the cells are subcultured.
If the cells are not attached or are growing in suspension:
- Aseptically transfer the entire contents of the flask to a centrifuge tube.
- Centrifuge at 125 × g for 5 to 10 minutes.
- Remove all but 10 mL of the shipping medium supernatant and resuspend the cells. Store the remainder of this medium at 4°C for later use.
- Aseptically transfer the resuspended cells to a 25-cm2 flask or 75-cm2 flask, depending upon the cell line (see the Product Sheet).
- Incubate the cells at the temperature and CO2 concentration recommended on the Product Information Sheet until cells are subcultured.
Most cell lines begin as primary cultures originating from a piece of minced or enzyme-dispersed tissue. Primary cultures, as mixtures of several cell types, retain the characteristics of their source tissue.
After a period of time, primary cultures will reach confluency, the state when all available space of the culture vessel is covered due to cellular expansion. At this point, the culture will need to be disaggregated (usually with proteolytic enzymes like trypsin) into individual cells and subcultured (split, passaged, or transferred). Following this first passage, the culture is generally referred to as a cell line. With each subsequent subculture, the cellular population becomes more homogeneous as the faster growing cells predominate. Cells with desired properties can also be selected out of the culture by cloning.
Diploid cell lines rarely progress beyond a few population doublings. They have a finite replicative capacity and begin to slow down and eventually stop dividing after 20 to 80 population doublings.1 Recent evidence suggests that some of the observed cellular senescence in cell culture may be due to inappropriate culture conditions as opposed to a predetermined replicative senescence.2 Still other data support replicative senescence for the cells of some species (notably human) even when grown in improved culture conditions. This senescence is mediated by the shortening of the ends of the chromosomes (telomeres) with each cell division.3
In contrast, continuous (or immortalized) cell lines have infinite replicative capacity. These lines are derived from cell lines through immortalization or transformation by any one of a number of means. Many continuous cell lines were derived from tumor tissue. Most of the cell lines in the ATCC collection are continuous, though a few, such as CCD-1117Sk human skin fibroblast (ATCC® CRL-2465™) or CCD-18Co human colon (ATCC® CRL-1459™) are finite. For more information about ATCC immortalized cell lines see the website.
As noted in the section on culture vessels, cell lines grow either attached to a surface (anchorage dependent) or in suspension (anchorage independent). As cells grow and divide in a monolayer or in suspension, they usually follow a characteristic growth pattern composed of four phases: Lag, log or exponential, stationary or plateau and decline.
- Lag phase — Immediately after seeding of the culture vessel, the cells grow slowly while recovering from the stress of sub-culturing.
- Log or exponential phase — The cells enter a period of exponential growth that lasts until the entire growth surface is occupied or the cell concentration exceeds the capacity of the medium.
- Stationary phase — Cell proliferation slows and stops.
- Decline phase — If the culture medium is not replaced and the cell number is not reduced, the cells lose viability and their number decreases.
To ensure viability, genetic stability, and phenotypic stability, cell lines need to be maintained in the exponential phase. This means that they need to be subcultured on a regular basis before they enter the stationary growth phase, before a monolayer becomes 100% confluent or before a suspension reaches its maximum recommended cell density. Generating a growth curve for each cell line is useful to determine
the growth characteristics of the cell line. (See: Figure 1)
For detailed information on the growth and propagation of any ATCC cell line, see the specific cell line Product Sheet on the ATCC website or contact ATCC Customer Care for more information.