Dr. Anne Sloan, Senior Scientist, discusses Endotoxins and how these complex lipopolysaccharides (LPS) introduce spurious results in cell culture ― and the innovative solutions for resolving them.
Contamination of cell culture has always been a serious problem for researchers, focused typically on avoiding biological contaminants such as bacteria, mycoplasma, yeast and fungi. There is increasing evidence that endotoxin can create variable results for researchers using cell culture.
Endotoxin is a complex lipopolysaccharide (LPS) which is the major component of the outer membrane of most gram-negative bacteria, such as E. coli. Bacteria shed endotoxin in small amounts when they are growing, and in large amounts when they die. LPS consists of a very hydrophobic lipid group, Lipid A, which anchors LPS to the bacterial membrane and is responsible for most of its biological effects. Endotoxins are amphipathic molecules characterized by a net negative charge in solution, high heat stability and form very large aggregates. Because of their hydrophobicity, they have strong affinities for other hydrophobic materials, especially plastic products used in cell culture and parts of protein molecules.
Cell lines that have been grown in culture for many years (CHO, 3T3, WI-38, HeLa, etc.) may have been naturally selected for resistance to endotoxin by long-term, inadvertent exposure to high levels of endotoxins that could be found in media, sera, and culture additives before endotoxin testing became widely used. Based on a review of the literature, it is clear that endotoxins do not affect all cultured cells equally. Some cell cultures, perhaps lacking appropriate endotoxin receptors may only be sensitive to very high levels of endotoxin. Unless you are sure endotoxin has no effect on your cultures and is not a potential source of variability in your experiments, you should reduce the possibility of endotoxin-related problems by taking some basic precautionary steps such as using high purity water, cell culture media, sera, and plasticware certified by their manufacturers to be nonpyrogenic.
Endotoxins are an unavoidable by-product in E.coli expressed recombinant proteins, and due to their stability and affinity to proteins, difficult to completely remove. Currently endotoxin levels in recombinant protein products below 1 EU/mg (< 0.1 ng/mg) is accepted as safe limit, however there has long been evidence to suggest the level of endotoxin that will not result in an adverse response varies between cell types, with unwanted effects observed below this accepted limit.
Barley expressed growth factors and cytokines are endotoxin free.
There are now endotoxin-free reagents coming from barley grains. It has really helped in specific areas of research, where concentrations of endotoxins as low as 2 pg/ml have been reported to elicit very strong responses (i.e., pro-inflammatory cascade). The ability of utilizing growth factors and recombinant proteins that are virtually endotoxin free helps to eliminate these as a source of contamination.
Who do you recommend these lines of products to?
In the span of biomedical / life science research – these products are currently used for specific types of research with stem cells or some cancer research. Though this is just one more option that is available to all researchers. I will say that as issues of reproducibility of research are capturing headlines and top line discussions in research circles – these products will continue to be adopted. There is always a fine balance that researchers find to be able to perform their research, be able to reproduce their research, and to have that research be able to move successfully onto a further phase of development. These products and these new tests are a part of providing tools that researchers now need to find that balance.
Learn more about Endotoxin-Free Stem Cell Growth Factors & Cytokines Expressed in Barley Increase Reproducibility and contact our technical support team with any questions.