Establishing cultured cells begins by surgically removing cells from an organism and placing them into a suitable culture environment. For that, a suitable vessel is needed, made of glass or treated plastic, and a culture medium containing nutrients to help cells grow. The medium contains a buffer to maintain an appropriate pH (often pH 7.0–7.4) and culture vessels are maintained at a suitable constant temperature (usually 37°C), often within an incubator designed to provide an atmosphere of 5% CO2 . After a short period, cells will attach to the surface or a substrate layer on the surface of the culture vessel, then divide and grow to form a primary culture. There are two primary routes toward achieving this objective, as listed below.
• Explant cultures. Small pieces of tissue are attached to a glass or plastic vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant onto the culture-vessel surface where they begin to divide and grow.
• Enzymatic dissociation. In this widely used method, proteolytic enzymes (trypsin, collagenase) are first used to dissociate tissue fragments. The ensuing suspension of single cells is placed into culture vessels containing culture medium, and cells are allowed to grow and divide.
After cells in the primary culture vessel have grown to fill up all of the available culture substrate, the cells must be subcultured to give them room for continued growth. Cells are gently removed from the substrate using proteolytic enzymes (to break protein bonds attaching the cells to the substrate), and the suspension of recovered cells is then subdivided and placed into new culture vessels.
Two basic cell culturing methods are employed, according to whether or not cells are able to adhere to a glass or treated plastic substrate. If they are able to do so, monolayer cultures are established, and different vessels are possible: tissue culture treated dishes, T-flasks, roller bottles, multiple-well plates, and so on. If, instead, the cells float freely, they are often kept actively suspended in the medium (suspension cultures—the cells are grown in magnetically rotated spinner flasks or in shaken flasks).
Cells may be maintained by passaging (or splitting): a small number of cells from a previously grown culture are seeded into fresh culture medium in a new culture vessel. If cells are passaged regularly, cell senescence (associated with high cell density) can be avoided and the cells can be cultured for longer.
Cell line storage and testing
Cell lines may be stored in a frozen state after adding some kind of cryopreservative agent—often DMSO (dimethylsulfoxide)—to the cell medium. As described in the following section, the most useful cell lines are immortalized cell lines that can be cultured indefinitely (they can be temporarily stored frozen in liquid nitrogen; thawing a frozen sample yields a viable culture). Certain major centers serve as cell line repositories—see Table 1 for some examples. There is a need to periodically check the authenticity of cell lines (by DNA fingerprinting) because proximity of other cell cultures in laboratories often leads to overgrowth of cultured cell lines by other, faster-growing cell lines.
Table1. EXAMPLES OF MAJOR CELL LINE REPOSITORIES
