The following article originally appeared on April 20, 2020 in Biocompare here.
Endotoxins are complex lipopolysaccharides that perform important structural and functional roles in the outer membrane of most gram-negative bacteria. They are shed in small amounts when bacteria are actively growing, and in large amounts when they die, and they are widely recognized for their detrimental effects on in vitro cell growth. Potential sources of endotoxins in cell culture include the water used to wash glassware and prepare media; commercially sourced media and sera; media components and additives; and laboratory glassware and plasticware. By taking steps to avoid endotoxin-induced cell culture problems, researchers can be more confident in experimental results. This article looks at best practices to reduce the risk of endotoxin contamination.
- Use high-purity water
Cell culture laboratories rely on high-purity water for preparing media and solutions, and also for washing glassware. However, poorly maintained water purification systems, water storage containers, and associated tubing can harbor significant levels of endotoxin-producing bacteria and should always be tested if endotoxins are suspected or discovered in cell cultures. Testing can be achieved using a simple Limulus amebocyte lysate (LAL) assay, with sensitive formats now available to detect as little as 0.001 endotoxin units/mL (EU/mL). If the laboratory water is found to be a source of endotoxins, and the problem cannot be fixed, nonpyrogenic water for injection (WFI) can be used to prepare media and essential solutions.
- Consider choosing premium FBS
Development of the LAL assay in the 1970s greatly increased awareness of endotoxins in sera, with a study performed around that time showing that of 111 lots screened, 23% contained >1ng/mL endotoxin1. This finding led sera manufacturers to handle the raw materials under more aseptic conditions, and many now offer high-quality, low endotoxin (<1ng/mL) FBS in addition to standard FBS products. Although not all cell cultures will be impacted by the more standard endotoxin level, premium FBS can be a sensible choice for researchers concerned that higher endotoxin levels might cause issues for their specific cultures.
- Check that media and additives have been tested for endotoxins
Like standard FBS, most commercially prepared media are certified to contain <1ng/mL endotoxin. For many cultures, this level is suitably low to avoid adverse effects on in vitro cell growth, yet it is important to be aware that any reagents added to media after it has been filtered can also harbor endotoxins. Indeed, the previously referenced study showed some 1M amino acid solutions to have endotoxin levels as high as 50ng/mL. Where possible, researchers should ask manufacturers to confirm the endotoxin levels of their products. When in doubt, the media endotoxin levels should be tested by LAL assay both before and after adding any new components.
Where media is prepared in-house, the endotoxin level will be determined primarily by that of the water used to dissolve the media components, making routine testing of the water source by LAL assay essential. The frequency of this testing will depend largely on the nature of the research being carried out.
- Follow the correct autoclaving procedures for glassware
Because endotoxins have a high heat stability, standard laboratory autoclaving procedures are ineffective at removing them from glassware. To destroy any contaminating endotoxins, glassware should be subjected to 250oC for >30 minutes or 180oC for 3 hours; this has the added benefit of sterilizing the glassware.
- Use certified plasticware
Although the high temperatures used in producing laboratory plasticware will usually eliminate any contaminating endotoxins, these can subsequently be reintroduced during the handling associated with assembly and packaging. By choosing plasticware that is certified by the manufacturer as having been endotoxin-tested and proven to be below a defined level (typically <0.1 EU/mL), researchers can be confident that plastics are not a source of endotoxins that can impact cell growth.
Although much remains unknown about the mechanisms through which endotoxins interact with in vitro cell cultures, it is clear that endotoxin contamination remains a major source of concern. Whenever endotoxins are present, there will always be a level of doubt over the validity of results. Even if you are sure that endotoxins have no effect on your cultures, following the precautionary steps outlined here will help to prevent unwanted surprises.
Corning offers a wide range of cell culture media, sera, vessels, and surfaces that are certified as low endotoxin. More information about endotoxins and cell culture can be found here, while product solutions can be found here.
1. Case Gould MG. Endotoxin in Vertebrate Cell Culture: Its Measurement and Significance. In Uses and Standardization of Vertebrate Cell Lines. Tissue Culture Association, Gaithersburg, MD. 1984. pp. 125–136.