Primary cultures, cell lines and storing cells

Primary culture: cells taken directly from a tissue. They are a mixture of cell types, like the tissue they came from. They are obtained from small chopped pieces (explants), or by separating the cells with enzymes such as trypsin or collagenase (disaggregation).

Secondary culture: a primary culture that has been moved into new vessels (sub-cultured, or passaged). With each passage, the fastest-growing cells take over, and the population becomes more uniform.

Cell lines come in two kinds:

• Finite cell lines, from normal cells, can divide only a limited number of times; then they stop and age (senescence).
• Continuous cell lines arise when cells transform, spontaneously or because of chemicals or viruses. They are immortal, can form tumours, stick less to surfaces, grow faster, and need fewer nutrients.

How cells grow:

• Adherent cells are anchorage-dependent: they must attach to a surface, and they spread into a monolayer, one cell thick (fibroblasts, epithelial cells).
• Suspension cells float freely in the medium (blood-forming stem cells, tumour cells).

When adherent cells cover the whole surface (confluence), they stop dividing. They are then detached, with trypsin, trypsin with EDTA, or a scraper, and split into fresh flasks. The passage number counts how many times a culture has been sub-cultured.

Storing cells (cryopreservation). Cells are stored in liquid nitrogen, at −180 to −196 °C, with cryoprotectants that stop ice crystals from tearing them: a freezing medium of 90% serum and 10% DMSO, or glycerol. Healthy cells from the log phase are cooled slowly, to −80 °C first. To revive them, they are thawed rapidly in a 37 °C water bath. The rule: freeze slowly, thaw fast.