Top 5 tips for reducing Endotoxin Contamination in the lab

Chemical or biological contamination can lead to unreliable experimental results in diagnostic and research laboratories. Bacterial endotoxins are one of the most insidious contaminants that can confer inflammatory effects to the investigated samples and mask their authentic biological properties. Bacterial endotoxins (lipopolysaccharides) are pyrogens and components of the outer membrane of gram-negative bacteria. They shed in small amounts during bacterial growth and are released in large amounts after bacterial death and lysis.¹ The Limulus amebocyte lysate (LAL) assay is the most established test for endotoxin detection. It is based on the incubation of protein extracted from horseshoe crab blood with endotoxin, leading to a clotting reaction that can be quantified for an accurate endotoxin assessment.¹

Sources of endotoxin contamination in the laboratory

The most common sources of endotoxin contamination in the lab include water, cell culture medium and reagents, serum, and plastic-/glassware. Fetal bovine serum (FBS), cell culture media, E. coli–derived recombinant growth factors, hormones, and dissociation reagents are some of the reagents that can be contaminated with endotoxins. Notably, endotoxin contamination removal is challenging because endotoxins are resistant to many of the traditionally used methods for decontamination.

Consequences of endotoxin contamination in the lab

Endotoxin contamination can lead to erroneous readout of the gene expression or function of analyzed cells or samples. This phenomenon has been observed in many different cell types or materials and has been related primarily to the pyrogenic properties of endotoxins. For example, endotoxin contamination can confer inflammatory properties to nanomaterials, interfering with the readout of their authentic biological properties.² Thus, avoiding or removing endotoxin contamination in the lab is key to ensure high-quality experimental results.

Here are 5 steps to reduce endotoxin contamination in the lab:

1. Nonpyrogenic plasticware – Endotoxins have high affinity for plasticware. Even though laboratory plasticware is produced at high temperatures that generally destroy endotoxins, endotoxin contamination can be introduced during subsequent handling and packaging. Using commercially available nonpyrogenic plasticware will help avoid this potential source of endotoxin contamination.

2. Depyrogenated glassware – Endotoxins can also contaminate laboratory glassware. Notably, washing or even autoclaving glassware under standard conditions is not sufficient to remove endotoxin contamination. Depypogenation with dry heat at 250°C for 30 min³ or at 180°C for 4 h¹ has been demonstrated to remove endotoxin contamination.

3. High-purity water – Endotoxin contamination of water may occur due to insufficient water purification or storage under unsuitable conditions. Ultrapurification systems based on reverse osmosis or glass distillation can ensure that the water is free of endotoxin contamination. Moreover, high-purity water should be stored only in nonpyrogenic containers, and prolonged storage should be avoided.

4. Endotoxin-tested sera, reagents, and media – Cell culture media, reagents, and sera, such as FBS, can also be a source of endotoxin contamination. Endotoxin-tested sera, media, and reagents have been developed commercially to prevent endotoxin contamination.

5. Discarding or decontaminating endotoxin-contaminated materials – Whenever possible, endotoxin-contaminated reagents and cells should be discarded and replaced with endotoxin-free materials. Endotoxin decontamination can be performed for highly valuable materials that cannot be replaced. Affinity chromatography and Triton X-114–based solutions have used successfully for endotoxin decontamination.^4,5

Endotoxin contamination can lead to erroneous and unreliable experimental results both in diagnostic and research laboratories. Simple steps, such as endotoxin testing and use of nonpyrogenic plasticware, depyrogenated glassware, high-purity water, and endotoxin-tested cell culture reagents and media will prevent endotoxin contamination and ensure the reliability of the obtained results.

Literature sources:

1. https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens
2. Li Y, Boraschi D. Endotoxin contamination: a key element in the interpretation of nanosafety studies. Nanomedicine (Lond). 2016;11(3):269-87. doi: 10.2217/nnm.15.196. Erratum in: Nanomedicine (Lond). 2016;11(6):739.
3. Sandle T. A comparative study of different methods for endotoxin destruction. Am Pharm Rev. 2013;Supplement.
4. Schneier M, Razdan S, Miller AM, Briceno ME, Barua S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol Bioeng. 2020;117(8):2588-2609. doi: 10.1002/bit.27362.
5. Teodorowicz M, Perdijk O, Verhoek I, Govers C, Savelkoul HF, Tang Y, Wichers H, Broersen K. Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. PLoS One. 2017;12(3):e0173778. doi: 10.1371/journal.pone.0173778.