The cells' environment

Cells in culture need conditions that imitate the body: the right temperature, osmolality, pH, gases, a surface to grow on, and protection from stress.

Temperature: 37 °C, the body temperature of humans and of the warm-blooded animals most cultured cells come from. The flasks are kept in CO₂ incubators.

Osmolality. If the medium is too dilute, cells swell and may burst; too concentrated, and they shrink. Glucose and salts set the osmolality; commercial media are about 300 mOsmol, measured with an osmometer.

pH must stay near 7.4. Two kinds of buffer keep it there:

• A natural buffer: the medium contains bicarbonate, and the incubator air contains 5-10% CO₂. The CO₂ in the air balances the CO₂/bicarbonate in the medium. More CO₂ lowers the pH (makes it more acidic). This buffer is cheap and non-toxic. Adding pyruvate makes the cells produce CO₂ themselves.
• A chemical buffer: HEPES, a zwitterion, buffers well at pH 7.2-7.4 without needing a controlled gas atmosphere. But it is costly, and toxic at high levels.

Phenol red is added as a pH indicator, so the state of the medium can be seen at a glance:

• Yellow: too acidic (below 6.8), often because cells have grown too much or microbes have contaminated the culture.
• Bright red: just right, about 7.4.
• Pink to purple: too alkaline (above 8.2).