What Do You Know About Endotoxin?
Cell culture contamination has been a major concern for researchers since cell culture was introduced in the early 1900's. One particular villain to the field of in vitro research has been endotoxin. But what do we really know about it?
What Is Endotoxin?
Endotoxin is a complex lipopolysaccharide (LPS), a toxic substance found in the outer membrane of most gram-negative bacteria. For example, one E. coli cell can contain up to 2 million LPS molecules. The bacteria releases the endotoxin into the culture environment while growing and when dying, possibly affecting the culture.
Studies of endotoxin in vitro have shown that they may affect the growth or performance of cultures and are a major source of experimental variation, although endotoxin does not affect all cells equally. Old cell lines that have been grown in culture for many years may have developed a resistance from long-term exposure before testing was common practice. CHO, 3T3, WI-38 and HeLa, for example.
Where Does It Come From And How Do I Stop It?
- Sera - Historically a major culprit for harboring endotoxin, but improved, automated screening has significantly reduced the risks. Many manufacturers offer Low-Endotoxin varieties of FBS, but this is not necessary for many cell lines.
- Water - High-purity water is a must in any lab. The purification system should be well-maintained and storage conditions should pristine. If possible, sterile water should be used promptly after filtration to avoid contamination during storage.
- Media and Supplements - Finished media should be tested for endotoxin introduced by either the water used to dissolve the powder media or by the other media components.
- Glass and Plasticware - Glassware, plastic tubing and fittings should be pyrogen-free, and reusables should be rinsed with pyrogen-free or low-endotoxin water. Plastic bottles should be sterilized by gamma irradiation.
- The User - The proper aseptic technique while handling cell cultures, sera and media is necessary to minimize the risk of introducing endotoxin into a system.
How Do You Test For Endotoxin?
An in vitro test was developed in the 1970's which showed the Lysate of a horseshoe crab (Limulus polyphemus) to clot in the presence of small amounts of endotoxin. Innovative tests for endotoxin using the Limulus Amebocyte Lysate (LAL) kinetic chromogenic methodology that measures a color intensity that provides reliable, quantitative results, using a FDA-licensed test device that provides reliable, quantitative results, eliminating the possibility of subjective evaluation. This method of of testing detects endotoxin in a range from 0.5 EU/mL down to as little as 0.005 EU/mL.