HyStem™ Cell Culture

What is Endotoxin?

Brief Summary  |   Why Choose Ultrapure HyStem Hydrogels  |   Structure and Physical Properties
Consequences of Endotoxin Contamination  |   Detection and Measurement
Sources of Contamination  |   Avoid Contamination  |   References

Brief Summary

Endotoxin ImageEndotoxins are small, stable, bacterially-derived hydrophobic molecules which can easily contaminate labware and whose presence can significantly impact both in vitro and in vivo experiments.2 Their presence is detected by the limulus amebocyte lysate (LAL) assay which can detect down to 0.01 Endotoxin Units (EU)/ml. Thorough cleanliness in labware, raw materials, and in lab technique is required to substantially reduce endotoxin levels. Sigma’s ultrapure HyStem line of products is produced according to these guidelines, yielding a product that is suitable for demanding in vitro and in vivo experiments.

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Why Choose Ultrapure HyStem Hydrogels

Why choose ultrapure Hystem Hydrogels? There is growing concern that the presence of bacterial endotoxins in media and matrices used in cell culture, cell therapy, and tissue engineering applications may induce aberrant cell function and host immune reactions. In vivo endotoxins elicit a pyrogen-like response in proportion to their concentration. To minimize these concerns, it is essential to use reagents and biomaterials which are virtually endotoxin-free for both in vitro and in vivo biomedical research.

Glycosan BioSystems’ HyStem line of ultrapure, low-endotoxin hydrogels help researchers obtain more reliable results by minimizing cell or host response to these contaminants.

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Structure and Physical Properties

Endotoxin is a complex lipopolysaccharide (LPS) found 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 (see figure). Endotoxins are approximately 10 kDa in size, but readily form large aggregates up to 1000 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.2 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.3

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Consequences of Endotoxin Contamination

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 1ng/mL) of endotoxin.3 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.

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Detection and Measurement

Endotoxin is measured in Endotoxin Units per milliliter (EU/mL). One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. 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.2 A successful assay was developed to measure endotoxin concentration based on the observations of Fred Bang, an Marine Biological Laboratory scientist, that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab (Limulus polyphemus) to turn into a semi-sold mass. It was later understood that the lysate from horseshoe crab amebocytes would clot due to the presence of very low endotoxin. This reaction is the basis of the Limulus amebocyte lysate (LAL) assay which was approved by the FDA in 1970 for testing drugs, products and devices that come in contact with the blood.1 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.

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Sources of Endotoxin Contamination

Water is perhaps the greatest source of endotoxin contamination in the laboratory. High purity water is absolutely essential. While distillation and deionizing columns do not remove endotoxin, special columns or filtering systems have been shown to be effective.2 As previously mentioned, glassware, plasticware, and other laboratory equipment contribute greatly to contamination especially since endotoxin can adhere strongly to glassware and plastics. These items can be decontaminated by the inactivation of pyrogens (endotoxin) from a solution or a substance (depyrogenation). Another source of endotoxin is people’s fingers, necessitating care in handling water and containers with clean gloves. Chemical reagents, raw materials, and buffers are also all potential sources of endotoxin.2 These items should be thoroughly tested for endotoxin before use in the laboratory.

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How does Sigma® avoid Endotoxin Contamination?

Hystem chemists begin by using ultrapure water from the Millipore nanopure filtration system. Next, they use specially sourced, low endotoxin raw materials which are rigorously tested for endotoxin using the LAL gel clot assay. In addition, the Hystem production team uses extreme care in manufacturing products including the use of nitrile gloves and working exclusively in a certified chemical fume hood. All glassware is depyrogenated using a drying oven set at 180°C for 4 hours to guarantee the inactivation of endotoxin. Finally, the Hystem quality control team ensures low endotoxin of every outgoing product has been maintained by doing a final LAL gel clot assay before product release.

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References

  1. “Endotoxin.” Wikipedia The Free Encyclopedia. 7 Jan. 2009.
  2. Gorbet, M.B. et al. Endotoxin: The Uninvited Guest. Biomaterials. 26: 6811-6817 (2005).
  3. Ryan, J. Endotoxins and Cell Culture. Corning Technical Bulletin. (2008).

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