A highly efficient method for removing cell contamination

Abstract

The present invention discloses a method for efficiently removing cell contamination, comprising the following steps: washing contaminated cells with a cleaning solution; adding a polyhexamethylene biguanide salt solution to a culture medium, adding the culture medium to a culture vessel containing contaminated cells, and then culturing in a constant temperature incubator; after washing the cells, adding cell culture medium, culturing for 6 to 24 hours, observing whether the cell contamination is eliminated, and if not, repeating the above steps. This method has high antibacterial efficiency, low cost, and does not produce any negative effects on cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for efficiently removing cell contamination. Background Art

[0002] Cell culture technology has a history of nearly a century. Contamination in cell culture primarily includes the following types: bacterial and fungal contamination, viral contamination, contamination by other cells, and contamination by chemicals. To reduce contamination in cell culture, researchers and manufacturers can implement a range of preventive measures, including aseptic techniques, cell identification, frequent culture medium changes, the use of qualified reagents and culture media, and waste disposal. However, contamination in cell or tissue culture remains a major challenge for researchers.

[0003] "Black worm" contamination is a common and serious contamination problem in cell culture. "Black worm" can parasitize animal cells or survive in culture medium, relying on nutrients in cells and culture medium for survival and propagating with cell generation. "Black worm" contamination has the following characteristics: (1) "Black worm" shows obvious motility and clustering under high-power microscope. (2) It can usually pass through a 0.22 μm filter membrane and conventional fluid exchange is ineffective against it. (3) Compared with contamination by other organisms, it does not make the culture medium turbid or significantly change its pH value. (4) It can reduce tetrazolium salt (MTT) to form blue crystals, indicating that it has mitochondria or is a prokaryotic microorganism.

[0004] Mycoplasma contamination is another common and serious contamination problem in cell culture. Mycoplasmas are the smallest bacteria without cell walls and can pass through bacterial filters, making mycoplasma contamination difficult to prevent. They can parasitize in animal and human cell cultures, either on the cell surface or inside the cell, and can have serious impacts on cell growth, metabolism, and function. Mycoplasma contamination is typically introduced into cell culture through several pathways: source cell contamination, reagent and culture medium contamination, laboratory environment, and operators. The effects of mycoplasma contamination on cell culture include, but are not limited to, impeded cell growth, altered cell morphology, abnormal cell metabolism, and changes in gene expression.

[0005] Currently, common methods for addressing contamination in cell or tissue culture include the following: (1) Antibiotics. For mycoplasma contamination, a common solution is to use specific antibiotics, such as ciprofloxacin, erythromycin, and tetracycline, to inhibit and eliminate mycoplasmas. However, these antibiotics may require a long treatment period and are less effective against some drug-resistant mycoplasma strains. (2) Antifungals. For fungal or mold contamination, a common solution is to use antifungals, such as amphotericin B. However, these antifungals are often toxic to cultured animal and plant cells. (3) Cell freezing and screening. Contaminated cells are frozen and screened to identify uncontaminated cell lines. This is a relatively thorough treatment method, but it can be time-consuming and resource-intensive. (4) Cleaning and disinfection. During the cell culture process, maintaining a clean laboratory environment and regularly disinfecting experimental equipment can prevent and reduce "black glue bug" or mycoplasma contamination.

[0006] However, the above methods have some defects in solving the problem of "black mold bugs", mycoplasma or fungal contamination in cell and tissue culture: (1) Drug resistance problem. Mycoplasma DNA is prone to mutation. Under the screening effect of drugs, some mycoplasma strains may become resistant to antibiotics, resulting in poor antibiotic removal of contamination; (2) Impact on cell health: Certain antibiotics, anti-mycoplasma agents or antifungal agents may be toxic to the cells themselves, affecting cell health and growth; (3) Long processing time: For certain contaminations, especially mycoplasma contamination, a long time of processing and screening is required, affecting the progress and time arrangement of the experiment; (4) Repeated infection: If the root cause is not completely resolved, the cells may be contaminated by "black mold bugs", mycoplasma or fungi again.

[0007] Therefore, a new method or means needs to be explored to solve the problem of "black glue worm", mycoplasma or fungal contamination in cell and tissue culture. Summary of the Invention

[0008] In order to effectively solve the problem of contamination such as "black glue bugs", mycoplasma or fungi in cell and tissue culture, we have proposed a method for effectively removing cell contamination, which includes the following steps:

[0009] Wash contaminated cells with washing solution;

[0010] adding a polyhexamethylene biguanide salt solution to a culture medium, adding the culture medium to a culture vessel containing contaminated cells, and then placing the culture vessel in a constant temperature incubator for culture;

[0011] After washing the cells, add cell culture medium and culture for 6 to 24 hours

[0012] Observe whether the cell contamination is eliminated. If not, repeat the above steps.

[0013] Preferably, the polyhexamethylene biguanide salt is polyhexamethylene biguanide hydrochloride or polyhexamethylene biguanide phosphate.

[0014] Preferably, the working concentration of the polyhexamethylene biguanide salt solution is 2 to 5 ppm.

[0015] Preferably, the solvent of the polyhexamethylene biguanide salt storage solution is deionized water, physiological saline, buffer solution or cell culture medium.

[0016] Preferably, the polyhexamethylene biguanide salt solution is sterilized by filtration through a filter membrane or filter.

[0017] Preferably, the temperature of the constant temperature incubator is 25°C to 37°C.

[0018] Preferably, the incubation time in the constant temperature incubator is 1 to 24 hours.

[0019] Preferably, the cleaning solution is cell culture medium, physiological saline or buffer.

[0020] Preferably, the buffer is a cell isotonic phosphate buffer or HEPES buffer.

[0021] Preferably, the cleaning is performed 2 to 4 times.

[0022] The present invention provides a highly efficient method for removing cell contamination, which has a highly effective killing and removing effect on microbial contamination in cell or tissue culture, especially "black mold" and mycoplasma contamination. The active ingredient, polyhexamethylene biguanide (PHMB), is a compound widely used for antibacterial and disinfection. It has excellent broad-spectrum antibacterial properties and has strong killing and inhibitory effects on a variety of bacteria, fungi, and enveloped viruses. PHMB is not likely to cause bacterial and fungal resistance. When treated with cell and tissue culture at an appropriate concentration, it will not be toxic to the cultured cells. PHMB has a high antibacterial efficiency and only requires a short cell treatment time to quickly remove microbial contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Flowchart of the method for efficiently removing cell contamination;

[0024] Figure 2 This is the effect picture of black glue insect treatment;

[0025] Figure 3 This is a diagram showing the effect of mycoplasma treatment. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. Example 1

[0027] This embodiment provides a method for efficiently removing cell contamination, such as Figure 1 As shown, the following steps are included:

[0028] S100. When "black gelatin bugs" are observed in the cell culture dish or culture flask, completely discard the culture medium and wash the cells three times with sterile PBS buffer (completely discard the PBS buffer each time).

[0029] S200. Prepare polyhexamethylene biguanide stock solution. Dilute 20% (w / w) PHMB hydrochloride solution into DMEM medium to a concentration of 1000 ppm. Aseptically filter the solution through a 0.22-μm sterile filter. Aliquot the sterile filtrate and store at 4°C until ready for use.

[0030] The polyhexamethylene biguanide stock solution was diluted 500-fold in DMEM complete medium supplemented with serum to obtain a polyhexamethylene biguanide salt solution with a final concentration of 2 ppm.

[0031] Add the polyhexamethylene biguanide salt solution with a final concentration of 2 ppm to the culture dish or culture bottle of the contaminated cells, and culture in a constant temperature incubator at 37° C. for 2 hours;

[0032] S300, discarding the above culture medium, washing the contaminated cells 2 to 4 times with sterile PBS buffer, adding serum-containing DMEM complete medium and continuing to culture;

[0033] S400, culture until the next day, observe the cell status and the removal of "black plastic worms". If the removal is not complete, repeat the above steps until the "black plastic worms" are completely removed (usually 2-3 times of treatment can completely remove the "black plastic worm" contamination). Figure 2 This is the effect picture of the black glue insect treatment. Example 2

[0034] This embodiment provides a method for efficiently removing cell contamination, comprising the following steps:

[0035] S100. When mycoplasma contamination is observed in the cell culture dish or culture flask, completely discard the culture medium and wash the cells three times with sterile PBS buffer (completely discard the PBS buffer each time).

[0036] S200. Prepare a polyhexamethylene biguanide stock solution. Weigh 1 g of PHMB powder and dissolve it in 1000 mL of deionized water to prepare a stock solution with a concentration of 1000 ppm. Under sterile conditions, filter the solution through a 0.22-μm sterile filter to sterilize. Aliquot the sterile filtrate and store at 4°C until use.

[0037] The polyhexamethylene biguanide stock solution was diluted 200 times in DMEM complete medium containing serum to a final concentration of 5 ppm of polyhexamethylene biguanide salt solution.

[0038] Add the polyhexamethylene biguanide salt solution with a concentration of 5 ppm to the culture dish or culture bottle of the contaminated cells, and culture in a constant temperature incubator at 37° C. for 3 hours;

[0039] S300, washing the contaminated cells three times with sterile PBS buffer, adding serum-containing DMEM complete medium and continuing to culture;

[0040] S400, culture until the next day, observe the cell status and the clearance of "mycoplasma". If the clearance is not complete, repeat the above steps until the "mycoplasma" is completely cleared (usually 2-3 times of treatment can completely eliminate "mycoplasma" contamination). Figure 3 This is a diagram showing the effect of mycoplasma treatment.

[0041] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently removing cell contamination, characterized in that: The steps include: Wash contaminated cells with washing solution; Adding a polyhexamethylene biguanide salt solution to a culture medium containing serum, adding the culture medium to a culture vessel containing contaminated cells, and then placing the culture vessel in a constant temperature incubator for culturing; the working concentration of the polyhexamethylene biguanide salt solution added to the cells is 2 to 5 ppm; After washing the cells, add cell culture medium and culture for 6 to 24 hours; Observe whether the cell contamination is eliminated. If not, repeat the above steps.

2. The method for efficiently removing cell contamination according to claim 1, wherein: The polyhexamethylene biguanide salt is polyhexamethylene biguanide hydrochloride or polyhexamethylene biguanide phosphate.

3. The method for efficiently removing cell contamination according to claim 1, wherein: The solvent of the polyhexamethylene biguanide salt storage solution is deionized water, physiological saline, buffer solution or cell culture medium.

4. The method for efficiently removing cell contamination according to claim 1, wherein: The polyhexamethylene biguanide salt solution is filtered through a filter membrane or a filter for sterilization.

5. The method for efficiently removing cell contamination according to claim 1, wherein: The temperature of the constant temperature incubator is 25°C to 37°C.

6. The method for efficiently removing cell contamination according to claim 1, wherein: The incubation time in the constant temperature incubator is 1 to 24 hours.

7. The method for removing cell contamination according to claim 1, wherein: The cleaning solution is cell culture medium, physiological saline or buffer.

8. The method for efficiently removing cell contamination according to claim 3 or 7, wherein: The buffer is a cell isotonic phosphate buffer or HEPES buffer.

9. The method for efficiently removing cell contamination according to claim 1, wherein: The contaminated cells were washed 2 to 4 times with the washing solution.

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