Common Cell Culture Problems: Precipitates

Precipitates in culture: an overview

When contamination is ruled out, turbidity in cell culture media is often explained by the precipitation of metals, proteins, and other media components. Precipitates can be harmful to cell health, as they may alter media composition by removing nutrients and other desirable components by processes such as chelation. Precipitates are also visible by microscopy as artifact, and may therefore interfere with assays that rely on imaging.

Purposeful precipitaton. Although precipitates are generally undesirable in cell culture, precipitation may be exploited as a purification strategy for proteins in the supernatant. For example, ammonium sulfate precipitation is used to purify antibodies from hybridoma supernatant or serum in a technique sometimes referred to as ‘salting out’.

Causes of Precipitation

• Temperature shift: Temperature is one of the main causes of precipitation in cell culture. High-molecular weight plasma proteins can fall out of solution when exposed to extreme shifts in temperature. Heat inactivation and freeze/thaw cycles can promote protein denaturation and precipitation. Because liquid or reconstituted media is kept refrigerated between uses, salts may precipitate out, particularly from 10x or other concentrated stock.
  
• Water loss-induced concentration change: If media is allowed to evaporate, the concentration of media components, including salts, will increase. Crystal precipitates may form particularly readily on culture surfaces.
  
• Calcium salts: When preparing serum-free media, the order in which components are added can cause the formation of insoluble molecules. Calcium salts in particular are prone to precipitation. For example, CaCl₂ and MgSO₄ react in solution to form CaSO₄ crystals. Autoclaving and pH instability can exacerbate this issue.
  
• Metal supplements: Copper, iron, and zinc metals are essential for cell growth, and are important supplements to serum-free media. The absence of other serum components can cause these metals to precipitate in culture, creating a toxic environment for cells. Copper and zinc are furthermore prone to precipitation under oxidative conditions.
  
• Contamination: Culture turbidity may be attributed to bacterial or fungal contamination (read more).

Troubleshooting Precipitates in Cell Culture

Cause of Precipitation	Solution
Temperature	Refer to manufacturer’s guidelines for optimal storage and handling of culture media. Avoid repeated freeze/thaw cycles.
Calcium Salts	Dissolve CaCl2 in deionized water separately before adding other media components one at a time.
Humidity	Ensure that flasks and plates are not experiencing evaporation. Monitor incubator humidification, and seal cultureware to prevent desiccation.
Other Metals	Inclusion of the iron-binding protein transferrin can prevent iron precipitation in culture.
Contamination	Discard contaminated cells and disinfect culture hood. Follow good laboratory practices to avoid contamination (read more).

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