Endotoxins are small bacterially-derived hydrophobic lipopolysaccharide (LPS) molecules that can easily contaminate labware and whose presence can significantly impact both in vitro and in vivo experiments. Their presence is detected by the limulus amebocyte lysate (LAL assay) which can detect down to 0.01 endotoxin units (EU)/mL. Endotoxins are approximately 10 kDa in size, but readily form large aggregates up to 1,000 kDa. Bacteria shed endotoxin in large amounts upon cell death and when they are actively growing and dividing. A single Escherichia coli contains about 2 million LPS molecules per cell. Endotoxins have a high heat stability making it impossible to destroy them under regular sterilizing conditions. They are amphipathic molecules that carry a net negative charge in solution. Because of their hydrophobicity, they are likely to have strong affinities for other hydrophobic materials like plastic products used in the laboratory. For this reason, carryover contamination from laboratory beakers, stirbars, and other labware is common.
Figure 1.Structure and physical properties of bacterial endotoxin. Endotoxins are complex lipopolysaccharides (LPS) present in the outer cell membrane of gram-negative bacteria. Endotoxins consist of a core polysaccharide chain, O-specific polysaccharide side chains (O-antigen) and a lipid component, Lipid A, which is responsible for the toxic effects.
Both are potentially hazardous to cell cultures. Exotoxins are toxic substances, usually proteins, secreted by bacteria and released outside the cell. Whereas endotoxins are bacterial toxins consisting of lipids that are located within a cell wall of the bacteria. Exotoxins are usually heat destroyed whereas endotoxins cannot be destroyed by high temperature. Exotoxins have high antigenicity and elicit an immune response whereas endotoxins do not.
The limulus amebocyte lysate assay (LAL assay) is the most commonly used test for endotoxin. LAL (derived from the horseshoe crab) reacts with bacterial endotoxin lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, to form a gel-clot which can be quantified. Endotoxin is measured in endotoxin units per milliliter (EU/mL). One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. Currently there are three forms of the LAL assay, each with different sensitivities. The LAL gel clot assay can detect down to 0.03 EU/mL while the LAL kinetic turbidimetric and chromogenic assays can detect down to 0.01 EU/mL.
Figure 2.LAL assay test principle. The cascade of endotoxin detection in Limulus amoebocyte lysate (LAL) assay starts when the endotoxin LPS reacts with Factor C in and turn, activates the factor B. Factor B turns the gel-forming pro-enzyme into an enzyme. The resulting clotting enzyme is responsible for anchoring the two peptide units in the coagulogen, forming an insoluble gel. Other compounds which also cause the gelation in the amebocyte lysate of the horseshoe crab can interference in the test of bacterial endotoxins. Some of these compounds are (1,3)-β-D-glucans, which lead to false positives in the LAL test.
Thorough cleanliness in all labware, media raw materials, and proper lab techniques are essential to substantially reduce endotoxin levels in the cell culture lab. It is also recommended to filter sterilize all media using a filtration device such as Stericup® filter unit which can eliminate all potential endotoxins before introducing to cells.
Endotoxins affect both in vitro and in vivo cell growth and function and are a source of significant variability. In vitro, there is increasing evidence that endotoxin cause a variety of problems for cell culture research. Among the effects documented were the stimulation of leukocyte cultures to produce tissue factors, the induced production of IL-6 in equine macrophages, and the inhibition of murine erythroid colony formation by very low levels (less than 1 ng/mL) of endotoxin. In vivo, endotoxins elicit an inflammatory response in animal studies. The presence of endotoxin in products for injection (vaccines and injectable drugs) can result in pyrogenic responses ranging from fever and chills to irreversible and fatal septic shock.
Due to the serious risks associated with endotoxin contamination, the US Food and Drug Administration (FDA) has set limits on concentration of endotoxin for medical devices and parenteral drugs that researchers should be aware of. Current FDA limits require eluates from medical devices to be less than 0.5 EU/mL unless the device comes into contact with cerebrospinal fluid where the limit is then 0.06 EU/mL.
It is essential to use endotoxin tested reagents, supplements and media from a trusted reagents supplier. It is also important to user proper aseptic techniques and thoroughly rinse and sterilize all cell culture plasticware and consumables like pipettes and conical tubes before culturing cells.
It is recommended to discard all reagents and cells if cultures become contaminated with endotoxin and start with new reagents and cells. However, if sample cannot be discarded reagents can be used to eliminate them. These endotoxin removal solutions rely on the micellar properties of Triton X-114 to remove LPS endotoxins from samples.