Lactobacillus gasseri strain, screening method and application thereof
By screening and identifying the Lactobacillus gasseri push strain, the problem of unclear dosage and mechanism in the biopharmaceutical field was solved, achieving highly efficient inhibition of HPV-infected cells and providing a new treatment approach.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEKING UNIVERSITY SHENZHEN HOSPITAL
- Filing Date
- 2026-03-11
- Publication Date
- 2026-06-30
AI Technical Summary
The mechanism of action of Lactobacillus gasseri in inhibiting HPV infection in existing technologies is unclear, and the optimal dosage has not yet been determined, which affects its application in the biopharmaceutical field.
A strain of Lactobacillus gasseri push (CGMCC No. 35609) was screened and identified. The strain was isolated from the lower genital tract of healthy women through specific culture and screening methods, and morphological and molecular biological identification confirmed that it is Lactobacillus gasseri species. It can be used to inhibit the growth of HPV-infected cells.
The *Lactobacillus gasseri* strain exhibited a 99.3% inhibition rate in suppressing the growth of HPV-infected cells, providing a new strategy for treating HPV infection in the female lower genital tract. Furthermore, as a GRAS strain, it is non-pathogenic and suitable for the biopharmaceutical field.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a screened strain of Lactobacillus gasseri with specific functions, as well as the screening method, culture conditions, and application of the strain in the biopharmaceutical field. Background Technology
[0002] In healthy women, lactobacilli are the most frequently detected probiotics in the vagina, accounting for 50%-80% and more than 95% of vaginal microbiota. *Lactobacillus gasseri* is one of the most common vaginal lactobacilli and can be used as a food ingredient, fermentation agent, or probiotic microorganism to promote oral health. Furthermore, *Lactobacillus gasseri* has functions such as defending against pathogens, regulating inflammation, managing the gut microbiota, and preventing bacterial infections. Researchers have also found that this strain can inhibit the growth of cervical cancer cells in people who test positive for human papillomavirus (HPV).
[0003] The mechanism of action of Lactobacillus gasseri in inhibiting HPV-infected cells is currently unclear. There are few studies on the clinical use of Lactobacillus gasseri to improve the health of women's lower genital tract, and further research is needed to confirm the optimal dosage of Lactobacillus gasseri. Summary of the Invention
[0004] Based on the aforementioned deficiencies in the prior art, this invention screens and obtains a new strain of Lactobacillus gasseri with excellent properties that can inhibit the growth of HPV-infected cells, thereby solving the problem of unclear dosage and mechanism in the prior art and meeting its practical application needs in the biopharmaceutical field.
[0005] To achieve the above objectives, this invention provides a strain of *Lactobacillus gasseri*, classified and named *Lactobacillus gasseri* push, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35609, on August 13, 2025. Its 16S rRNA gene sequence is shown in SEQ ID NO. 1. It has been identified as having 99.93% homology with strains of the genus *Lactobacillus*, species *Lactobacillus gasseri*.
[0006] The *Lactobacillus gasseri* described herein was isolated from the lower genital tract of healthy women. Morphological characteristics of *Lactobacillus gasseri*: Colonies are round, approximately 1-2 mm in diameter, with regular edges, a smooth and moist surface, and a soft texture that is easily picked up. Colonies are milky white, opaque, and slightly convex in the center. Cell morphology is diverse, mainly short rod-shaped or oval, with blunt, rounded ends. They usually exist singly or in pairs, occasionally forming short chains, but without long chains or branching. The bacteria do not form spores or have flagella, therefore they are not motile.
[0007] The physiological and biochemical characteristics of *Lactobacillus gasseri* include: efficient utilization of carbon sources such as glucose, fructose, galactose, lactose, and maltose; this strain is a heterotrophic microorganism, unable to utilize inorganic nitrogen, and can grow by relying on organic nitrogen sources. It does not produce catalase. *Lactobacillus gasseri* has strong acid resistance, tolerating acidic conditions of pH 2.5-3.5, and can survive and colonize the intestinal mucosa.
[0008] Another aspect of the present invention provides a method for screening Lactobacillus gasseri strains, comprising the following steps:
[0009] (1) Sample collection: Secretions from the lower genital tract of healthy women were collected using sterile cotton swabs;
[0010] (2) Pretreatment: Place the sterile cotton swab in sterile MRS broth medium and vortex, then dilute the bacterial solution to 10-2, 10-3, and 10-4 times respectively with sterile MRS broth medium;
[0011] (3) Initial screening: The diluted bacterial solution was spread on the screening medium and the culture conditions were: culture temperature 37℃, anaerobic culture for 48h;
[0012] (4) Purification culture: Select single colonies of the suspected strain obtained in step (3) and streak them on the purification medium. Culture them anaerobically at 37°C for 24 h to obtain pure culture strains.
[0013] (5) Secondary screening: The pure culture strain obtained in step (4) was inoculated into MRS broth medium and cultured under the following conditions: culture temperature 37℃, anaerobic culture for 24h;
[0014] (6) Perform morphological and molecular biological identification on the strains obtained from the second screening in step (5).
[0015] Preferably, the screening medium is MRS agar medium, the composition of which is: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, agar powder 15 g / L, and anhydrous calcium carbonate 20 g / L.
[0016] Preferably, the purification medium is MRS agar medium, and the components of the MRS agar medium are: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, and agar powder 15 g / L.
[0017] Preferably, the MRS broth culture medium comprises 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L yeast extract, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 2 g / L triammonium citrate, 0.03 g / L manganese sulfate, and 1 mL / L Tween 80.
[0018] Another aspect of the present invention provides a method for culturing a strain of Lactobacillus gasseri, wherein the seed culture of Lactobacillus gasseri is inoculated into a liquid culture medium for fermentation culture, and the fermentation culture conditions are: culture temperature 37°C, anaerobic culture for 48 hours.
[0019] Seed culture: 10% inoculum, anaerobic culture at 37°C for 24 h in liquid culture medium.
[0020] Fermentation culture: The inoculum size of Lactobacillus gasseri was 1×10⁷ CFU, which was inoculated into 10 mL of liquid culture medium and cultured anaerobically at 37℃ for 48 h.
[0021] Preferably, the liquid culture medium is MRS broth medium, and the components of the MRS broth medium are: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L yeast extract, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 2 g / L triammonium citrate, 0.03 g / L manganese sulfate, and 1 mL / L Tween 80.
[0022] The Lactobacillus gasseri of this invention produces a variety of lactic acids, which have a great advantage in inhibiting the growth of HPV-infected cervical epithelial cells and have great potential in the treatment of HPV infection in the female lower genital tract.
[0023] The present invention also provides the application of the Lactobacillus gasseri strain in inhibiting the growth of HPV-infected cells.
[0024] The application of the *Lactobacillus gasseri* strain in inhibiting the growth of HPV-infected cells includes the following steps:
[0025] (1) Preparation of fermentation broth of Lactobacillus gasseri: Dissolve Lactobacillus gasseri at room temperature, take 100-200 μL and inoculate it into MRS broth medium and anaerobic culture at 37℃ for 24 h, then dilute it to 1×107 CFU / mL, take 1 mL and inoculate it into MRS broth medium, anaerobic culture at 37℃ for 48 h, and finally centrifuge and sterile filter the culture medium to obtain Lactobacillus gasseri supernatant;
[0026] (2) Co-culture: The activated and passaged Ect1 / E6E7 cells were seeded into 24-well plates with a concentration of 1×105 / mL in DMEM medium without fetal bovine serum, and each well contained 1 mL of the medium. The cells were starved at 37°C and 5% CO2 for 24 h. The medium was then replaced with DMEM medium containing 10% fetal bovine serum, and the supernatant of Lactobacillus gasseri obtained in step (1) was added to the DMEM medium at a ratio of 20% (v / v). The cells were cultured at 37°C and 5% CO2 for 24 h.
[0027] (3) Result detection: Each well of the 24-well plate was washed, and then trypsin was added to digest and detach Ect1 / E6E7 cells. After digestion was terminated, the cells were placed in a cell counting plate to count the viable cells in each well.
[0028] Preferably, in step (3), 1 mL of PBS without calcium and magnesium ions is used to wash each well of the 24-well plate twice. 100 μL of 0.25% trypsin-0.53 mM EDTA is added and the plate is placed in a 37°C incubator for 5-10 min until most Ect1 / E6E7 cells are observed to have detached under a microscope. 400 μL of DMEM with 10% fetal bovine serum is added and mixed by pipetting to stop the digestion. 15-20 μL of each DMEM is placed in a cell counting plate, and the viable cells in each well are counted using a cell counter.
[0029] The beneficial effects of this invention are as follows: Lactobacillus gasseri is a beneficial strain isolated from the lower genital tract of healthy women, and it has been designated as a Generally Recognized As Safe (GRAS) strain by the U.S. Food and Drug Administration. It is non-pathogenic and does not produce harmful metabolites. The Lactobacillus gasseri strain provided by this invention can achieve an inhibition rate of 99.3% in inhibiting the growth of HPV-infected cells, providing a new strategy for the treatment of HPV infection in the lower genital tract of women. Attached Figure Description
[0030] Figure 1 This is a morphological diagram of the Lactobacillus gasseri push strain of the present invention;
[0031] Figure 2 This is the experimental result of Lactobacillus gasseri push inhibiting the growth of HPV-infected cells according to the present invention. Detailed Implementation
[0032] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0033] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0034] A strain of *Lactobacillus gasseri*, classified and named *Lactobacillus gasseri* push, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35609, dated August 13, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. Its 16S rRNA gene sequence is shown in SEQ ID NO. 1. It exhibits 99.93% homology with strains of the genus *Lactobacillus*, species *Lactobacillus gasseri*.
[0035] The 16S rRNA gene sequence (SEQ ID NO.1) of Lactobacillus gasseri push is as follows:
[0036]
[0037] Example 1: Isolation, Screening and Identification of Strains
[0038] (a) Screening of strain 23-6
[0039] 1. Sample Collection: In December 2024, lower genital tract secretion samples were collected from healthy women at Peking University Shenzhen Hospital in Shenzhen, Guangdong Province. The collection method involved using sterile vaginal swabs to collect secretions from the deep vaginal wall, posterior fornix, and cervix. The swabs were gently rolled for approximately 10 seconds until saturated, avoiding contact with the vulva and vaginal opening to prevent contamination. The samples were typically clear or slightly yellow viscous liquids. The vaginal swabs containing secretions were placed in sterile centrifuge tubes, refrigerated at 4°C, and brought back to the laboratory for later use.
[0040] 2. Sample pretreatment: Add 1 mL of sterile MRS broth to a centrifuge tube containing a vaginal swab, vortex vigorously to prepare a 10⁻¹ dilution, and then serially dilute with sterile MRS broth to 10⁻², 10⁻³, and 10⁻⁴.
[0041] 3. Initial Screening: Spread 0.1 mL of serially diluted samples onto screening medium and incubate at 37℃ for 48 h. Select strains with a clear zone on the screening plates as suspected strains. Select several single colonies producing clear zones and number them 1-1, 1-2, 1-3, etc. Screening medium (selective medium): MRS agar medium (formulation: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, agar powder 15 g / L, anhydrous calcium carbonate 20 g / L), pH: 6.2±0.2, sterilization conditions: sterilize at 121℃ for 15 min.
[0042] 4. Purification Culture: Single colonies of suspected strains were picked and streaked multiple times on purification medium until pure cultures were obtained. The suspected strains were then purified by streaking on purification medium and incubated at 37℃ for 24 h to obtain pure cultures. Purification medium: MRS agar medium (formulation: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, agar powder 15 g / L), pH: 6.2±0.2, sterilization conditions: 121℃ for 15 min.
[0043] 5. Secondary Screening: The pure cultured strains were inoculated into MRS broth medium and cultured anaerobicly at 37°C for 24 h. The obtained strains were then stored at -80°C in preparation for 16S rRNA sequencing. MRS broth medium (formulation: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L), pH 6.2±0.2.
[0044] (II) Identification of strain 23-6
[0045] 1. Morphological identification: Please refer to Figure 1 After being cultured on MRS agar at 37°C for 24 h, strain 23-6 produced round colonies, 1-2 mm in diameter, milky white in color, with a smooth, moist surface, regular edges, opaque, and slightly convex in the center. The cells exhibited diverse morphologies, primarily short rod-shaped or oval, with blunt ends, and lacked spores and flagella.
[0046] 2. Molecular biological identification: Genomic DNA was extracted from strain 23-6 and amplified by PCR using universal primers for the 16S rRNA gene (forward primer 27F: 5'-AGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 2), reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3)). Sequencing of the amplified product yielded a 1500 bp gene sequence (SEQ ID NO: 1). This sequence was submitted to the GenBank database for BLAST alignment, which showed 99.93% homology with strain *Lactobacillus gasseri* (accession number: CP049762.1). Based on morphological and physiological / biochemical characteristics, strain 23-6 was identified as *Lactobacillus gasseri* and named *Lactobacillus gasseri push*.
[0047] Example 2
[0048] Application of strain 23-6 in inhibiting the growth of HPV-infected cells
[0049] 1. Preparation of fermentation broth for strain 23-6: Lactobacillus gasseri, stored at -80℃, was dissolved at room temperature. 100-200 μL was inoculated into 5 mL of MRS broth (Solarbio, Beijing, China) and anaerobically cultured at 37℃ for 24 h. The concentration of the Lactobacillus gasseri suspension was measured using a McFarland turbidimeter (Yuanhengtong, China), and then diluted to 1×10⁷ CFU / mL. 1 mL of this solution was inoculated into 10 mL of MRS broth and anaerobically cultured at 37℃ for 48 h. The culture medium after 48 h of culture was centrifuged at 10,000 rpm for 10 min and then aseptically filtered through a 0.22 μm filter membrane (Merck Millipore, USA) to obtain the Lactobacillus gasseri supernatant. MRS broth medium (formulation: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L), pH: 6.2 ± 0.2.
[0050] 2. Co-culture experiment: Ect1 / E6E7 cells (Jinyuan Biotechnology, Shanghai, China) were activated and passaged in complete culture medium (Jinyuan Biotechnology, Shanghai, China) according to the manufacturer's instructions. After culture, the cells were removed, the culture medium was discarded, and the cells were rinsed twice with calcium- and magnesium-free PBS (Biosharp, Beijing, China). Then, 2 mL of 0.25% trypsin-0.53 mM EDTA (Gibco, USA) was added, and the cells were incubated at 37°C for 3-5 min. Cell detachment was observed under a microscope. If most cells became rounded and detached, the cells were quickly returned to the worktable, and digestion was terminated by adding at least 5 mL of complete culture medium containing 10% fetal bovine serum (Procell, Wuhan, China). 15-20 μL of the cultured Ect1 / E6E7 cells were placed in a cell counting chamber and counted using a cell counter (Thermo Fisher Scientific, USA). The concentration of DMEM medium (Gibco, USA) without fetal bovine serum was adjusted to 1×10⁵ / mL and inoculated into 24-well plates, with 1 mL per well. The plates were starved at 37°C and 5% CO₂ for 24 h. The medium was then replaced with DMEM medium containing 10% fetal bovine serum, and the fermentation broth of strain 26-3 was added to the medium at a ratio of 20% (v / v). Each strain culture was prepared in triplicate. The control group was recultured in DMEM medium without the addition of lactobacillus supernatant and cultured at 37°C and 5% CO₂ for 24 h.
[0051] 3. Result Detection: Wash each well of the 24-well plate twice with 1 mL of calcium- and magnesium-free PBS (Biosharp, Beijing, China). Add 100 μL of 0.25% trypsin-0.53 mM EDTA (Gibco, USA) and incubate at 37°C for 5-10 min until most cells detach under a microscope. Take 400 μL of DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Procell, Wuhan, China) and mix thoroughly by pipetting to stop digestion. Place 15-20 μL of each solution into a cell counting plate and count the viable cells in each well using a cell counter (Thermo Fisher Scientific, USA). Please refer to [link to relevant documentation]. Figure 2 The results showed that the inhibition rate of Ect1 / E6E7 cells in the experimental group was 99.3% compared with that in the control group, indicating that strain 23-6 has a significant inhibitory effect on the growth of Ect1 / E6E7 cells.
[0052] Those skilled in the art can implement the present invention in various modified forms without departing from the essence and spirit of the present invention. The above description is only a preferred and feasible embodiment of the present invention and is not intended to limit the scope of the present invention. All equivalent structural changes made in accordance with the present invention specification are included within the scope of the present invention.
Claims
1. A strain of Lactobacillus gasseri, characterized in that, The strain of *Lactobacillus gasseri* was classified and named *Lactobacillus gasseri* push, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35609 on August 13, 2025.
2. The method for screening Lactobacillus gasseri strains as described in claim 1, characterized in that, Includes the following steps: (1) Sample collection: Secretions from the lower genital tract of healthy women were collected using sterile cotton swabs; (2) Sample pretreatment: The sterile cotton swabs were placed in sterile MRS broth medium and vortexed. The bacterial solution was then diluted to 10⁻², 10⁻³, and 10⁻⁴ times with sterile MRS broth medium. (3) Initial screening: The diluted bacterial solution was spread on the screening medium and the culture conditions were: culture temperature 37℃, anaerobic culture for 48h; (4) Purification culture: Select single colonies of the suspected strain obtained in step (3) and streak them on the purification medium. Culture them anaerobically at 37°C for 24 h to obtain pure culture strains. (5) Secondary screening: The pure culture strain obtained in step (4) was inoculated into MRS broth medium and cultured under the following conditions: culture temperature 37℃, anaerobic culture for 24h; (6) Identification: Morphological and molecular biological identification of the strains obtained in step (5) re-screening.
3. The method for screening Lactobacillus gasseri strains as described in claim 2, characterized in that, The screening medium is MRS agar medium, and the composition of MRS agar medium is as follows: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, agar powder 15 g / L, and anhydrous calcium carbonate 20 g / L.
4. The method for screening Lactobacillus gasseri strains as described in claim 2, characterized in that, The purification medium is MRS agar medium, and the components of the MRS agar medium are: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, Tween 80 1 mL / L, and agar powder 15 g / L.
5. The method for screening Lactobacillus strains as described in claim 2, characterized in that, The MRS broth culture medium consisted of 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L yeast extract, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 2 g / L triammonium citrate, 0.03 g / L manganese sulfate, and 1 mL / L Tween 80.
6. The method for culturing the *Lactobacillus gasseri* strain according to claim 1, characterized in that, The seed culture of Lactobacillus gasseri was inoculated into a liquid culture medium for fermentation culture. The fermentation culture conditions were: culture temperature 37℃, anaerobic culture for 48h.
7. The method for culturing the *Lactobacillus gasseri* strain as described in claim 6, characterized in that, The liquid culture medium is MRS broth medium, and the components of the MRS broth medium are: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, yeast extract 5 g / L, sodium acetate 3.02 g / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, triammonium citrate 2 g / L, manganese sulfate 0.03 g / L, and Tween 80 1 mL / L.
8. The use of the Lactobacillus gasseri strain according to claim 1 in inhibiting the growth of HPV-infected cells.
9. The application as described in claim 8, characterized in that, Includes the following steps: (1) Preparation of fermentation broth of Lactobacillus gasseri: Dissolve Lactobacillus gasseri at room temperature, take 100-200 μL and inoculate it into MRS broth medium and anaerobic culture at 37℃ for 24 h, then dilute it to 1×107 CFU / mL, take 1 mL and inoculate it into MRS broth medium, anaerobic culture at 37℃ for 48 h, and finally centrifuge and sterile filter the culture medium to obtain Lactobacillus gasseri supernatant; (2) Co-culture: The activated and passaged Ect1 / E6E7 cells were seeded into 24-well plates with a concentration of 1×105 / mL in DMEM medium without fetal bovine serum, and each well contained 1 mL of the medium. The cells were starved at 37°C and 5% CO2 for 24 h. The medium was then replaced with DMEM medium containing 10% fetal bovine serum, and the supernatant of Lactobacillus gasseri obtained in step (1) was added to the DMEM medium at a ratio of 20% (v / v). The cells were cultured at 37°C and 5% CO2 for 24 h. (3) Result detection: Each well of the 24-well plate was washed, and then trypsin was added to digest and detach Ect1 / E6E7 cells. After digestion was terminated, the cells were placed in a cell counting plate to count the viable cells in each well.
10. The application as described in claim 9, characterized in that, In step (3), 1 mL of PBS without calcium and magnesium ions was used to wash each well of the 24-well plate twice. 100 μL of 0.25% trypsin-0.53 mM EDTA was added and the plate was placed in a 37°C incubator for 5-10 min until most Ect1 / E6E7 cells were observed to have detached under a microscope. 400 μL of DMEM with 10% fetal bovine serum was added and mixed by pipetting to stop the digestion. 15-20 μL of each DMEM was placed in a cell counting plate and the viable cells in each well were counted using a cell counter.