Cell Culture Guides
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One of the major advantages of cell culture is the ability to manipulate the environment in which cells grow. In most cases, the culture environment is controlled by the cell culture media chosen. A better understanding of your media is necessary in order to know its impact on your cells. In addition to the nutritional components of a media, phenol red, buffering compounds, and inorganic salts are included to balance pH and osmolality. These additional factors are important in creating an optimal culture environment for your cells.
Factors to consider for creating an optimized cell culture environment for your cells:
These factors are often overlooked but essential in media optimization. Here we provide a general overview of the importance of PH, buffers, and osmolality in your media.
As cells grow and proliferate, the culture media progressively becomes more acidic as the cells consume nutrients and produce metabolites. Some cells are particularly sensitive to this pH change, and phenol red, a pH indicator, is often added to media to enable researchers to visualize a shift in pH. As the media becomes more acidic, phenol red will change from pink to orange and yellow over time. It’s important to pay attention to this color shift to maintain a suitable pH for the cultures. As the cells in culture become more dense, they deplete the nutrients in the culture more rapidly, and media exchanges are key to maintaining a suitable growing environment. A pH shift toward yellow is a clear indicator that the media needs to be changed, regardless of a pre-determined feeding schedule.
Does phenol red affect the cells? In most cases, phenol red in culture media has no effect on the cells, and it’s use as a pH indicator is very beneficial to maintaining healthy cultures. However, there are some instances where a medium formulated without phenol red is preferred or necessary. For instance, some sensitive cells or assays may be affected by phenol red in the media, as it can act as a weak estrogen compound due to similarities in chemical structure. Another reason is that phenol red may interfere with some flow cytometry applications. Phenol red-free versions of media formulations are especially important for such applications.
Cell culture media must contain a buffer system in order to maintain a consistent pH (generally between 7.2 and 7.4 for many cell types). There are two main types of buffers normally used in cell culture media – carbonate and HEPES (pronounced “HEP-eez”).
Sodium bicarbonate (NaHCO3) is a natural, non-toxic buffer. Bicarbonate-buffered media is used in a 5% CO2 incubator, and functions when the CO2 atmospheric concentration is between 4 – 10%. Not only does the bicarbonate counteract the increase in pH from the CO2 gas, but it also provides some nutrition to the cells.
Sodium bicarbonate buffering of CO2
H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3¯
NaHCO3 ↔ Na+ + HCO3¯
HEPES is a stronger organic buffer than sodium bicarbonate, and does not require a CO2 incubator for buffering, expanding its use to molecular biology as well. HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) is a zwitterionic buffer that is most useful for pH in the range of 6.8 to 8.2. In cell culture media, HEPES can become toxic to some cells if used at high concentrations.
Inorganic salts in the media, including sodium and calcium, perform important functions in the cells in culture. Calcium chloride, for example, is known to improve overall cell attachment. Importantly, salts in the media also maintain osmotic balance. Osmotic pressure drives osmosis – it’s the force that pulls waters from areas of low solute concentration to high solute concentration.
Osmolality is the “saltiness” of the media. Osmolality of the media should be as close as possible to the osmolality of the inner cell. If the media does not contain enough salts (hypoosmotic), the cell will swell with water. If the media is too salty (hyperosmotic), the cell will shrink – and both states are damaging to the cells. Measuring the osmolality of a final media formulation is essential to ensure that the salt balance falls within the optimal range for the cells in culture.
Osmolality is the number of osmoles of solute per kilogram of solvent (Osm/kg), and is measured by an osmometer. An osmole is the unit of osmotic concentration – specifically the number of moles that a solute contributes to the osmotic pressure of a solution. Osmolality is often confused with osmolarity, which is the number of osmoles of solute per liter of solution (Osm/L). Osmolarity is calculated from osmolality, though at physiological concentrations, the relative difference is not very great between these two.
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