Once the cells we culture in

cell factory are contaminated, most of them are difficult to handle. If the contaminated cells are valuable and difficult to obtain again, the following methods can be used to remove them.

1. Use antibiotics

Antibiotics are more effective at killing bacteria in cell factories. Combination medication is more effective than medication alone. Preventive medication is more effective than medication after contamination. Preventive medication generally uses a double antibiotic (penicillin 100u/mL plus streptomycin 100μg/mL). After contamination, the cleaning method needs to be 5 to 10 times greater than the usual amount. The drug should be used for 24 to 48 hours after the addition, and then replaced with the usual routine. Culture fluid. This method may be effective in the early stages of contamination. In addition to penicillin and streptomycin, the antibiotics used can also include gentamicin, kanamycin, polymyxin, tetracycline, nystatin, etc. Commonly used are 400 to 800 μg/mL kanamycin or 200 μg/mL tetracycline. The medium is changed every 2 to 3 days and passed on for 1 to 2 generations for treatment. In recent years, it has been reported that 4-fluoro, 2-hydroxyquinoline (Ciprofloxacin, Cip), Pleu-romutilin derivative (Pleu-romutilin derivative, BM-Cyclin2: BM-1 and tetracycline derivative (BM-2)) Antibiotics are effective in killing mycoplasma when used alone or in combination. These three antibiotics are all prepared into 250X concentrated solutions in PBS and stored at -20°C for later use. The usage concentration Cip is 10 μg/mL, BM-1 is 10 μg/mL, and BM-2 is 5μg/mL. When using, first aspirate the contaminated culture medium, add RPMI1640 culture medium containing BM-1, then aspirate the culture medium after 3 days, add RPMI1640 culture medium containing BM-2, and culture for 4 days, and so on for 3 consecutive days. rounds, until it is proved by 33258 fluorescent staining microscopy that mycoplasma has been eliminated, then normal culture medium is added for culture and passage 3-4 times.

2. Heating treatment

Incubating contaminated tissue culture at 41°C for 18 hours can kill mycoplasma, but has adverse effects on cells. Therefore, a preliminary test should be conducted before treatment to explore the heating time that can kill mycoplasma to the maximum extent and have the least impact on cells. This method is sometimes unreliable. If treated with drugs first and then heated at 41°C, the effect will be better.

3. Use mycoplasma-specific serum

Mycoplasma contamination can be removed with 5% rabbit mycoplasma immune serum (hemagglutination titer 1:320 or above). Because the specific antibody can inhibit the growth of mycoplasma, it turns negative 11 days after antiserum treatment and remains negative 5 months later. is negative. However, this method is more troublesome and not as convenient and economical as using antibiotics.

4. Other methods

In addition to the above-mentioned methods of removing contamination, there are also inoculation and sterilization methods in animals, macrophage phagocytosis methods, methods of adding bromouracil to contaminated culture bottles and then irradiating them with light, and filtration methods, etc., but they are all more troublesome and ineffective. Therefore, once mycoplasma contamination occurs, unless it is of particularly important value, it is generally discarded and re-cultured.

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