Removal and prevention of contamination in cell culture
1. Removal of pollution
Once the cultured cells are contaminated, most are more difficult to handle. If the contaminated cells are of little value, they should be discarded; if there are cell lines remaining or can be purchased, the operation room can be completely disinfected after the cause is found, the cells can be recovered or repurchased, and then cultured. If the value of the contaminated cells is large and it is difficult to retrieve them, the following methods can be used to remove them.
(1) Use of antibiotics
Antibiotics are more effective in killing bacteria. Combination medication is better than medication alone. Preventing medication is better than contaminating it. Preventive medication generally uses dual antibiotics (penicillin 100u / mL plus streptomycin 100μg / mL). After the pollution is removed, the medication needs to be washed 5 to 10 times larger than the usual amount. After adding the drug, it will act for 24 to 48 hours, and then change to the routine Culture medium. This method may be effective in the early stages of pollution. In addition to penicillin and streptomycin, antibiotics used can also use gentamicin, kanamycin, polymyxin, tetracycline, nystatin and so on. Commonly used 400-800μg / mL kanamycin or 200μg / mL tetracycline treatment, fluid replacement every 2 to 3 days, pass 1 to 2 generations for treatment. In recent years, it has been reported that three antibiotics such as 4-fluoro, 2-hydroxyquinoline (Ciprofloxacin, Cip), leucoromin derivative (Pleu-romutilinderivative, BM-Cyclin2: BM-1 and tetracycline derivative (BM-2), etc. Single or combined use is effective for killing mycoplasma. These three antibiotics are formulated with PBS into 250X concentrated solution, and stored at -20 ℃ for use. The concentration of Cip is 10 μg / mL, BM-1 is 10 μg / mL, and BM-2 is 5 μg. / mL. Aspirate the contaminated culture solution first, add RPMI1640 culture solution containing BM-1, and then aspirate the culture solution 3 days later, add RPMI1640 culture solution containing BM-2, culture for 4 days, so 3 consecutive rounds Times, until 33258 fluorescence staining microscopic examination proves that Mycoplasma has been removed, and then add normal culture medium to culture and passage 3-4 times.
(2) Heating treatment
Incubating the contaminated tissue culture at 41 ° C for 18 hours can kill mycoplasma, but has an adverse effect on the cells. Therefore, pre-testing should be carried out before treatment, to explore the heating time that can maximize the killing of mycoplasma and minimize the impact on cells. This method is sometimes unreliable. If it is treated with medicine first, then the effect of heating treatment at 41 ℃ is better.
(3) Use mycoplasma-specific serum
Use 5% of rabbit mycoplasma immune serum (hemagglutination potency 1: 320 or more) to remove mycoplasma contamination, because specific antibodies can inhibit the growth of mycoplasma, so after 11 days after treatment with antiserum turned negative, and after 5 months Is negative. However, this method is more cumbersome and not as convenient and economical as antibiotics.
(4) Other methods
In addition to the above decontamination methods, there are also inoculation methods for animals, macrophage phagocytosis, bromouracil in contaminated culture bottles and light irradiation, and filtration methods, but they are all cumbersome and have no effect determine. Therefore, once mycoplasma is contaminated, unless it has particularly important value, it is generally discarded and re-cultivated.
2. Prevention of pollution
Prevention is the best way to prevent contamination during cell culture. Only when prevention work is done before can the possibility of pollution be minimized. General prevention can start from the following aspects:
(1) Start with disinfection and sterilization of articles and supplies
The items used for cell culture should be thoroughly cleaned and disinfected. Various solutions should be sterilized and sterilized carefully, and only used after the sterility test is negative. The operating room and the remaining sterile equipment should be regularly cleaned, disinfected and sterilized.
(2) Start with the operator
1. The operator should have a strong sense of responsibility, be careful and steady, and be skilled in operation techniques. Before entering the sterile room, wash your hands with soap or soak for 5 minutes with 5% chlorpheniramine and wear a gown as required. Close the door after entering, sit down and move as little as possible. To start work, use 75% alcohol cotton balls to wipe hands, wipe bottle mouth and burn bottle mouth. Strictly check the equipment, solution and culture in advance, and do not bring contaminated or unsterilized items into the sterile room, or use it casually, so as not to cause a large amount of pollution.
2. The action of the operator should be light. The bottle mouth must be opened in the sterile area around the flame, and the bottle mouth should be turned and burned. Try not to talk during operation. If you sneeze or cough, you should turn to the back.
3. Always replace the straws during operation, do not make one straw to the end. Once it is found that the mouth of the straw has touched the hand and other contaminated objects, it should be discarded. After the experiment, pack it up in time, keep the laboratory clean and tidy, and finally wipe the table with gauze soaked in disinfection water.
(3) Prevent cross-contamination of cells
When performing a variety of cell culture operations, the equipment used must be strictly distinguished, and it is best to mark them for easy identification. Operate in order to avoid confusion when proceeding together.
Do not touch the mouth of the cell culture bottle with the syringe or dropper during the medium exchange or passage operation, so as not to bring the cells into the culture medium and contaminate other cells.
Once all the cells are purchased, introduced from elsewhere, or established by themselves, the seeds should be kept frozen early, and once contaminated, they can be discarded for recovery and cultured again.