Bacterial endotoxin is a complex of lipopolysaccharide (LPS) and protein on the cell wall of Gram-negative bacteria, which is released in large quantities when the bacteria are lysed. A sufficient amount of endotoxin in the whole body can cause fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation, multiple organ failure, and sometimes even human death. The reason for endotoxin-induced fever is that it acts on macrophages, neutrophils, etc. in the body to produce cytokines such as IL-1, IL-6, and TNF-α, which further affect the body temperature of the host hypothalamus. Increase body temperature and generate heat by regulating the central nervous system. In scientific research experiments, endotoxin is also a factor that cannot be ignored, which will interfere with the experimental results. Endotoxin will have different effects on different cells, greatly affect the transfection efficiency, and may also activate the non-specific reaction of immune cells, resulting in false positives in the experiment. Therefore, the cell or tissue culture system needs to avoid endotoxin contamination, minimize the chance of endotoxin contamination at the source, and ensure the smooth progress of the experiment.
Figure 1. Cell culture sample in sterile hood.
It is generally believed that serum, culture medium, and water used for external drug preparation are the sources of endotoxin contamination in cell or tissue culture. The impact of endotoxin on cells will vary depending on the type of cell line, and its impact mainly includes the following aspects:
In addition, endotoxin can also affect cell adhesion, reduce the binding ability of liver cells to insulin, and reduce the loss of potassium ions in cells. Endotoxin will accelerate cell aging, apoptosis, and premature cell death, which will affect the experimental results and cause fluctuations in experimental data. Excessive endotoxin content in serum is more likely to lead to "sub-healthy" cells, resulting in inaccurate data, poor cell status, aging, or even death, resulting in test interruption failures. Therefore, cell culture assays must be performed using endotoxin-tested reagents, supplements, and media.
During cell lysis for plasmid DNA preparation, endotoxin molecules are released into the lysate. Endotoxin, which is amphiphilic and negatively charged, will be extracted with plasmid during plasmid extraction because of its amphiphilicity and negative charge. In cell transfection experiments, endotoxin can compete with plasmid DNA for transfection reagents, preventing plasmid DNA from entering the cell membrane and nuclear pore complex. At the same time, the cell permeability increases during the transfection process, and the endotoxin enters the cells, resulting in a decrease in cell activity, affecting the transfection efficiency, and even causing cell death. For in vitro transfection of immune cells, endotoxin can activate non-specific immunity, resulting in false positives. In addition, commonly used transfection techniques are based on the principle of charge attraction. If there are other impurities in the plasmid DNA, such as salt ions, proteins, metabolic pollutants, etc., it will affect the formation of transfection complexes, thereby affecting the transfection efficiency.
