Problems and Solutions of Mammalian Cell Culture Contamination

1. What is mammalian cell pollution?

Contamination usually refers to the presence of unwanted microorganisms, unwanted mammalian cells, and various biochemical or chemical substances in the cell culture medium, thereby affecting the physiology and growth of the desired mammalian cells. Because microorganisms are ubiquitous in the environment, including specific parts of the human body, and because they reproduce much faster than the fastest growth rates of mammalian cells, microbial contamination poses a major challenge to mammalian cell culture. The most important microorganisms that contaminate mammalian cell cultures are bacteria, fungi (yeasts/molds), mycoplasma, viruses, and protozoa. While bacterial and fungal contamination is the most common and easy to see, mycoplasma and viral contamination is difficult to detect and cannot be confirmed with the naked eye.

2. Common types of pollution

(1) Microorganisms are the most common type of pollution. Bacteria and fungi grow very quickly in culture-filled petri dishes, and their presence in the medium slows or kills the growth of mammalian cells. Since the cultured cells have no defense or immunity, infection with a single bacterial or fungal spore can lead to complete contamination of the culture. The presence of microorganisms in the culture can be detected by examining the cells under a microscope or by changes in the color or turbidity of the medium.

(2) Cross-culture contamination is another type of contamination caused by the contamination of mammalian cell cultures by other mammalian cells. This type of contamination is a significant problem in the field of mammalian cell culture. A culture contaminated with different types of cells can be extremely difficult to detect, and researchers may not notice it for years, leading to incorrect interpretations of the experiment.

(3) Chemical contamination may be introduced by toxic decomposition products in serum, impurities or media, or even by impurities in water. Toxic chemical pollution can cause cells to grow poorly or die.

3. Mycoplasma contamination solutions

Mycoplasma is a common contaminant of animal cells in cell culture. About 25 species of mycoplasma and mycoplasma have been identified as cell culture contaminants, but the most common are Mycoplasma Hirschi, Mycoplasma oral, Mycoplasma arginine, and Mycoplasma Leighi. In cell cultures, these species are responsible for 85 percent of the pollution. Differences in nucleic acid metabolism between mycoplasma and mammalian cells, mycoplasma has nutritional requirements for nucleic acid precursors, which can be met by purine and pyrimidine bases or nucleosides. On the other hand, the amount of free pyrimidine in mammalian cells is small, so the amount of free bases (such as uracil) in contaminated cell cultures is used to detect mycoplasma. One of the base analogs that can be selectively bound to mycoplasma nucleic acids is 5-bromic acid (5-BrUra). Visible light induces cleavage of DNA10 containing 5-brura, and the combination of fluorescent dye 33258-Hoechst with DNA11 greatly enhances this photosensitivity. Its unusually high A+T content makes Mycoplasma DNA3 an excellent candidate for 5-BrUra, 33258-H and light-induced cleavage, as 33258-H has A high affinity for A−T base pairs and a higher affinity for A-Brura base pairs. Treating them in this way provides a practical and simple way to selectively kill contaminated mycoplasma; They are likely to die from DNA breakage.

4. Bacterial contamination phenomena and solutions

Bacterial contamination is easily detected by visual examination of the culture within a few days of infection, and infected cultures often appear cloudy, sometimes forming a thin film on the surface; There is also often a sudden drop in pH value of the medium; Under a low-power microscope, bacteria appear as tiny particles that move between cells, and the shape of individual bacteria can be distinguished when viewed under a high-power microscope.

Once there are signs of contamination, the color of the medium, pancreatic enzyme, PBS, etc., can be observed to see whether the clarification is normal. After determining the contamination, the media in the culture container can be immediately discarded, and the PBS can be used to moisten the culture container for 2 to 3 times. The pancreatic enzyme can be added to the treatment until the cell morphology changes slightly but the cell does not leave the container, and the PBS can be added to gently moisten the culture. Secondly, 20× double antibody should be added, the cells should be soaked for 3-5 minutes, and the double antibody should be discarded. Fresh complete medium (containing 10× double antibody) was added again for 12 hours, and then the medium was discarded and washed with PBS for 2 to 3 times for 12 hours, and the complete medium (containing 10× double antibody) was added. When passing, centrifugation was performed at a small rotational speed, and culture was still performed in medium containing 10× diantibody. If no bacteria can be found in the second generation under the mirror, the third generation can be cultured normally, and if there is no rebound after 48 hours of normal culture, the contamination can be considered cleared.

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