Non-tumorigenic MDCK suspension cells and their applications

Abstract

This disclosure provides an MDCK cell and its application. The MDCK cells screened in this disclosure were deposited on November 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO:46717. The screened non-tumorigenic MDCK cells can be used as a cell matrix for influenza vaccine production. The suspension process, compared to traditional adherent cell technology, offers advantages such as easier scale-up production, higher production efficiency and yield, better process control and consistency, reduced production costs, and no introduction of serum-derived animal components.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to a non-tumorigenic MDCK suspension cell and its application. Background Technology

[0002] Influenza vaccination is a primary and cost-effective strategy for reducing annual influenza-related morbidity and mortality. Traditional influenza vaccine production relies on chicken embryo culture, which is relatively inflexible in terms of meeting surging vaccine demand or responding to emergency pandemics. During continuous passage of influenza viruses in chicken embryos, the amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) often undergo mutations, a phenomenon known as "ovarian adaptation," which can affect antigenicity and thus alter vaccine efficacy. The WHO recommends using mammalian cells (such as MDCK cells) for influenza vaccine preparation as an alternative strategy to chicken embryo culture.

[0003] MDCK cells are an adherent cell line established in 1958 from the kidneys of female Cocker Spaniels. In the vaccine field, they are mainly used in the preparation of swine, avian, and human influenza vaccines and are considered one of the suitable cell substrates for influenza vaccine production.

[0004] MDCK cells are used in the production of human influenza vaccines, but the main challenges include difficulties in scaling up adherent culture and the inherent tumorigenicity of the cells themselves. There are reports of solving these problems by domesticating MDCK cells into suspension cells, addressing issues such as adherence to carriers and the need for trypsin digestion. However, to address the tumorigenicity of MDCK cells, purification processes are typically used to remove intact cells and control host cell residues, but this does not fundamentally solve the problem of tumorigenicity inherent in the cells themselves.

[0005] Therefore, there is an urgent need to provide tumor-free MDCK suspension cells to meet the needs of industrial vaccine production. Summary of the Invention

[0006] This disclosure provides a non-tumorigenic MDCK suspension cell and its application.

[0007] According to one aspect of this disclosure, a canine kidney passage (MDCK) cell is provided, which was deposited on November 6, 2025, at the China General Microbiological Culture Collection Center with accession number CGMCC NO:46717.

[0008] In some embodiments, the cells are capable of surviving and / or stably passaged in serum-free culture medium; and / or the cells are capable of being cultured in suspension as single cells in the culture medium; and / or the cells are non-tumorigenic.

[0009] In some embodiments, after the cells are inoculated into an animal, the inoculated animal does not develop nodules, or the nodules that appear regress and can be maintained in a state of nodule regression.

[0010] In some embodiments, the cells are sensitive to viruses; and / or the cells can be used to culture viruses.

[0011] In some embodiments, the virus includes one or more of the following: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, Coxsackie virus, adenovirus, Lassa virus, and vaccinia virus.

[0012] In some implementations, the virus is an influenza virus.

[0013] In some embodiments, the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and / or influenza D virus.

[0014] In some embodiments, the influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2 and H10N8.

[0015] In some embodiments, the influenza B virus includes at least one of the BY and BV influenza viruses.

[0016] In some embodiments, the cell species is identified as canine.

[0017] According to another aspect of this disclosure, a method for preparing a virus or a vaccine thereof from the cells described in the first aspect is provided, the method comprising:

[0018] (a) Infect the cells with a virus;

[0019] (b) Culture infected cells under conditions that allow the virus to multiply;

[0020] (c) Isolation of viruses from cell culture mixtures.

[0021] In some embodiments, serum-free culture medium is used in step (b).

[0022] In some implementations, in step (a), the virus is in increments of 10. -4 ~10 -9 The cells are infected with an MOI, preferably an MOI of 10. -5 ~10 -8 .

[0023] In some embodiments, the culture temperature includes 30–37°C.

[0024] In some embodiments, the culture temperature includes 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, and 37°C.

[0025] In some embodiments, the culture rotation speed includes 70 to 140 rpm. In some embodiments, the culture rotation speed includes 70 rpm, 75 rpm, 80 rpm, 85 rpm, 90 rpm, 95 rpm, 100 rpm, 105 rpm, 110 rpm, 115 rpm, 120 rpm, 125 rpm, 130 rpm, 135 rpm, and 140 rpm.

[0026] In some embodiments, the culture time includes 1 to 5 days. In some embodiments, the culture time is 1 day, 2 days, 3 days, 4 days, or 5 days.

[0027] In some embodiments, the pH value of the culture includes 6.5 to 8.5. In some embodiments, the pH value of the culture includes 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0028] In some embodiments, the dissolved oxygen content of the culture comprises 20% to 60%. In some embodiments, the dissolved oxygen content of the culture comprises 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%.

[0029] In some embodiments, the cells can be used to prepare viruses under various culture conditions such as plates, well plates, shake flasks, square flasks, bioreactors, and cell factories.

[0030] In some embodiments, the virus includes one or more of the following: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, Coxsackie virus, adenovirus, Lassa virus, and vaccinia virus.

[0031] In some implementations, the virus is an influenza virus.

[0032] In some embodiments, the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and / or influenza D virus.

[0033] In some embodiments, the influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2 and H10N8.

[0034] In some embodiments, the influenza B virus includes at least one of the BY and BV influenza viruses.

[0035] According to another aspect of this disclosure, the use of the said cells in the production of viruses and / or their vaccines is provided.

[0036] In some embodiments, the virus production includes virus propagation culture.

[0037] In some embodiments, the virus includes one or more of the following: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, Coxsackie virus, adenovirus, Lassa virus, and vaccinia virus.

[0038] In some implementations, the virus is an influenza virus.

[0039] In some embodiments, the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and / or influenza D virus.

[0040] In some embodiments, the influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2 and H10N8.

[0041] In some embodiments, the influenza B virus includes at least one of the BY and BV influenza viruses.

[0042] One less.

[0043] The beneficial effects of this disclosure are:

[0044] The tumor-free MDCK suspension cells disclosed herein address potential cell safety issues at the source, ensuring product quality. These tumor-free MDCK suspension cells can be used as a cell matrix for influenza vaccine production. The suspension process, compared to traditional adherent cell methods, offers advantages such as easier scale-up production, higher production efficiency and yield, better process control and consistency, reduced production costs (eliminating the use of high-cost materials like microcarriers and serum), and no introduction of serum-derived animal components. The obtained tumor-free MDCK suspension cells can also replace chicken embryo matrix. Compared to traditional chicken embryo matrix, they offer stricter aseptic control, avoidance of residual chicken embryo-derived allergens, and reduced adaptive mutations of viral strains in chicken embryos, providing greater flexibility in responding to pandemics or emergencies.

[0045] Preservation Information

[0046] MDCKs-1F3 cells were deposited on November 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The scientific description is canine kidney cell line monoclonal cells, and the accession number is CGMCC NO: 46717. Attached Figure Description

[0047] Figure 1 The time for nodule regression in nude mice inoculated with MDCKs-1F3 at different generations was determined.

[0048] Figure 2 Images of nude mice used for tumorigenicity testing of MDCKs-1F3.

[0049] Figure 3 Histopathological sections for tumorigenicity examination of MDCKs-1F3 cells.

[0050] Figure 4 To enhance the toxicity sensitivity of MDCKs-1F3 cells.

[0051] Figure 5 The process for establishing a library of MDCKs-1F3 cells.

[0052] Figure 6 The distribution of chromosome number in MDCKs-1F3.

[0053] Figure 7 This represents the morphology of cell growth.

[0054] Figure 8 This is a cell growth curve.

[0055] Figure 9 To determine the cell density and viability for virus culture in bioreactors.

[0056] Figure 10This refers to the hemagglutination titer of the virus harvest fluid from the bioreactor. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0058] definition

[0059] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0060] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0061] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0062] Example

[0063] Example 1: MDCK cell suspension domestication

[0064] (1) MDCK adherent cell resuscitation and expansion

[0065] Multiple MDCK adherent monoclonal cell lines were obtained from Beijing Sinovac Biotech Co., Ltd. MDCK adherent cells were resuscitated in Eagle's medium (10% newborn calf serum NBS) in T25 cell culture flasks and statically cultured in a PHCbi incubator at 36.5℃ and 5% CO2. Once cell confluence reached over 75%, the culture medium was discarded, and the bottom of the flask was washed three times with 0.01M PBS solution. 0.04 ml / cm³ of PBS was then added to the culture medium. 2 Digest with recombinant trypsin (Yuanpei, catalog number S340KJ) for 10-20 minutes. Stop digestion after the cells become rounded, blow away the cell suspension, and take samples for cell counting.

[0066] (2) Direct suspension acclimatization

[0067] Centrifuge the cell suspension obtained in (1) at 200g for 10min (Sigma), discard the supernatant, and resuspend the cells in serum-free CD MDCK medium in an SF125 shake flask. The initial cell seeding density is 1.5×10⁶ cells / 10⁻⁶. 6 Approximately 100 cells / mL were cultured in suspension in a CO2 shaking incubator (Zhichu), with parameters set at 36.5℃, 5% CO2, and 100-140 rpm. Cells were counted and passaged by centrifugation every 2-3 days. After adaptive acclimatization and passage, when the cultured cells showed significant proliferation (3 times or more of the initial density) and a viability of not less than 90%, they were prepared for cryopreservation.

[0068] (3) Gradual acclimatization (suspended state)

[0069] Centrifuge the digested cell suspension from (1), resuspend it in CD MDCK medium containing 10% newborn calf serum in an SF125 shake flask, and initially seed the cells at a density of 1.5 × 10⁶ cells / year. 6 Cells were cultured at approximately 100 cells / mL in a CO2 shaking incubator at a temperature of 36.5℃, 5% CO2, and a rotation speed of 100-140 rpm. Cells were counted and passaged by centrifugation every 2-3 days. After acclimatization and passage, when cells showed a proliferative trend, serum concentrations were reduced to 5%, 1%, and 0% for continued culture. Once the cell density was three times or more of the initial density and the viability was not less than 90%, the cells were prepared for cryopreservation.

[0070] (4) Gradual acclimatization (adhering to the wall state)

[0071] The digested cell suspension from (1) was transferred to T75 cell culture flasks at a seeding ratio of 1:3 to 1:6 using CDMDCK medium containing 10% newborn calf serum. The flasks were then placed in a CO2 incubator for static culture. Under these conditions, the cells were cultured continuously for 3 generations using CDMDCK medium containing 10% newborn calf serum. The serum concentration was then reduced to 5%, and the cells were seeded at a 1:3 ratio in CDMDCK medium containing 5% newborn calf serum. The flasks were then placed in a CO2 incubator for static culture. Subculture continued until the adherent cells at the bottom of the flask showed loose intercellular spaces. The cells at the bottom of the flask were washed three times with 0.01M PBS solution, and 0.04 ml / cm³ of PBS was added. 2 Digest with recombinant trypsin for 5-10 minutes until the cells become rounded, then disperse the cell suspension and take samples for cell counting.

[0072] Centrifuge the cell suspension at 200g for 10 minutes, discard the supernatant, and resuspend the cells in CDMDCK medium containing 5% newborn calf serum in an SF125 shake flask. The initial cell seeding density is 1.5 × 10⁶ cells / year. 6Cells were cultured at approximately 100 cells / mL in a CO2 shaking incubator at a temperature of 36.5℃, 5% CO2, and a rotation speed of 100-140 rpm. Cells were counted and passaged by centrifugation every 2-3 days. After acclimatization and passage, when cells showed a proliferative trend and serum levels decreased to zero, culture was continued. Once the cell density was three times or more of the initial density and the viability was not less than 90%, the cells were prepared for cryopreservation.

[0073] (5) Cryopreservation of suspension cells

[0074] The MDCK suspension cells successfully domesticated in steps (2)-(4) were centrifuged at 200g for 10min and resuspended in CD MDCK cryopreservation medium containing 10% dimethyl sulfoxide (Sigma), 1mL per vial, 4.0 × 10 7 Cells / vial were placed in a gradient cooling box at -80°C overnight, and then transferred to liquid nitrogen for long-term storage. Multiple MDCK suspension cell lines, including MDCKs-4D10, MDCKs-1C10, MDCKs-4G9, and MDCKs-1F3, were obtained.

[0075] (6) Tumorigenicity test of suspension cells

[0076] The obtained multiple MDCK suspension cells were processed at a ratio of 1×10⁻⁶. 7 0.2 mL / cell per 4-7 week old nude mice (n=10 per group) was injected for initial tumorigenicity testing. Cells that did not initially show tumorigenicity were re-injected at a dose of 1×10⁻⁶ cells / mL every 10 passages. 7 0.2 mL / cell / mouse was injected into 4-7 week old nude mice for continued tumorigenicity testing. The injection site was observed and palpated weekly for nodule formation, and the nodules were measured. Notably, MDCKs-1F3 was passaged to the P50 generation without tumorigenesis. Nodules at the injection site in nude mice regressed within the 16-week observation period; nodules in the P19 generation regressed within 2 weeks, and in the P50 generation within 10 weeks. The nodule regression time and images of the nude mice injected with MDCKs-1F3 are shown below. Figure 1 and Figure 2 At the end of the observation period, nude mice injected with MDCKs-1F3 were dissected, and skin, heart, liver, spleen, lungs, kidneys, and lymph nodes from the injection site were collected for pathological examination. After fixation, slide preparation, staining, and microscopic observation, no neoplastic lesions were found in any of the samples. Histopathological sections are shown below. Figure 3 .

[0077] (7) Suspension cell toxicity test

[0078] MDCKs-1F3 suspension cells were cultured at approximately (1.2±0.3)×10⁻⁶. 6Cells were initially seeded at a density of 100 cells / mL in serum-free CD MDCK medium and cultured in a CO2 shaking incubator at 36.5℃, 5.0% CO2 concentration, and 100 rpm. The cells were then passaged and expanded to a certain volume. The cell density was adjusted to 4.00 × 10⁶ cells / mL. 6 Cells / mL were inoculated with recombinant trypsin at 3 mg / L, and the virus multiplicity of infection (MOI) was set at 0.0001. Influenza viruses of types H1N1 (IVR-238, CNIC China), H3N2 (IVR-237, CNIC China), and BV (B / Michigan / 01 / 2021, CNIC China) were added. The virus-infected cells were then cultured in a CO2 shaking incubator at 34.0℃, 5.0% CO2 concentration, and 100 rpm. Hemagglutination titers were measured at different time points. Results are as follows: Figure 4 As shown in the figure. The results showed that MDCKs-1F3 suspension cells were sensitive to H1N1, H3N2 and BV strains, and were feasible for use in the preparation of influenza virus.

[0079] Blood coagulation titer assay procedure: Take a clean 96-well U-shaped blood coagulation plate. Add 50 μL of 0.9% sodium chloride injection to each well except the first column. Add 100 μL of the sample to be tested to the wells in the first column. Add 50 μL to the second well for serial dilution. Dilute to the last well and discard 50 μL. Add 50 μL of 1% chicken red blood cells to each well of the blood coagulation plate. After incubating at room temperature for 30 minutes, observe the results.

[0080] (8) Preservation of suspension cells

[0081] MDCKs-1F3 cells were deposited on November 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO: 46717.

[0082] Example 2: Establishment and testing of a cell bank

[0083] The master cell bank and working cell bank for MDCKs-1F3 cells were established. The bank establishment process is described in [link to documentation]. Figure 5 Cell bank testing was conducted in accordance with the requirements of the 2025 edition of the Chinese Pharmacopoeia, Part III, including identification tests, sterility, mycobacteria, mycoplasma, intracellular and extracellular viral factors, retroviruses, bovine, porcine and canine viruses, tumorigenicity, tumorigenicity, chromosome karyotype (number) analysis, pathogen metagenomics (DNA+RNA), and growth curve detection.

[0084] Identification test: Cell species identification was performed using PCR, and the results showed that the cells were of canine origin.

[0085] Bacterial and fungal sterility tests: Bacterial and fungal sterility tests were performed in accordance with the requirements of the sterility test method in General Chapter 1101 of the Chinese Pharmacopoeia. 20 mL × 2 supernatants of MDCKs-1F3 cell culture were prepared (without antibiotics) and the membrane filtration method was used. The results showed that the cells were free of bacterial and fungal contamination.

[0086] Mycobacterial examination: Mycobacterial examination was performed according to the requirements of General Chapter 1101, Sterility Examination Method, of the Chinese Pharmacopoeia. Take 10... 7 Cell lysates were prepared from the culture supernatant of live cells and inoculated into three suitable solid culture media, with three replicates for each medium. The cells were incubated at 37°C for 56 days for mycobacterial examination, and the results showed no mycobacterial growth in the cells.

[0087] Mycoplasma testing: Mycoplasma testing was performed according to General Chapter 3301 of the Chinese Pharmacopoeia. MDCKs-1F3 cell supernatant was prepared using two 10 mL culture methods (antibiotic-free) and one 10 mL indicator cell culture method. Results showed no mycoplasma growth in the direct inoculation method and the indicator cell culture method was negative.

[0088] Intracellular and extracellular viral factor detection: MDCKs-1F3 live cells, lysates, and supernatants were prepared according to the requirements of the Pharmacopoeia "Preparation and Quality Control of Animal Cell Matrix for the Production and Testing of Biological Products" for the detection of intracellular and extracellular viral factors. Results showed that in vitro inoculation culture methods (erythrocyte adsorption assay and erythrocyte agglutination assay) were negative; in vivo inoculation methods (suckling mice, adult mice, guinea pigs, chicken embryos) showed over 80% survival and no exogenous viral contamination; retroviruses were negative using PERT and transmission electron microscopy; bovine, porcine, and canine viruses were all negative using PCR.

[0089] Tumorigenicity test: Prepare 5×10 live cells 7 Cells / mL, viability not less than 90%, and a minimum of 2 mL. Inoculated into 4-7 week old nude mice, results showed no tumor growth at the injection site or in other sites such as the heart, liver, spleen, lungs, and kidneys.

[0090] Tumorigenicity test: Cell lysates and DNA were prepared and inoculated into newborn nude mice, hamsters, and rats. The results showed no tumor growth at the injection site or in other sites such as the heart, liver, spleen, lungs, and kidneys.

[0091] Chromosome karyotype (number) analysis: 0.5 × 10⁻⁶ live cells were prepared. 6Cells / mL, at least 5 mL per flask, with viability not less than 90% were used for the experiment. Colchicine was used to induce metaphase in the cells. Metaphase cells were harvested, fixed under hypotonic conditions, prepared, stained, and mounted. Cells with clear and concentrated chromosome distribution were selected for microscopic scanning slides. Chromosome counts were performed using Cytovision software, with 100 cells counted per sample. Results showed that 28% of the 100 cells contained 78 chromosomes, and 75% contained 78±2 chromosomes. Karyotype analysis results are shown below. Figure 6 .

[0092] Next-generation sequencing was used to detect more than 25,000 pathogenic microorganisms, including bacteria, fungi, mycobacteria, viruses, parasites, mycoplasma / chlamydia, and rickettsia, using the new pathogen metagenomic (DNA+RNA) technology. No cell contamination was found.

[0093] Cell morphology and growth curve: MDCKs-1F3 cells were cultured at (1.2±0.3)×10⁻⁶ cells. 6 Initially seeded at a density of [number] cells / mL, cells were inoculated into serum-free CD MDCK medium and cultured in a CO2 shaking incubator at 36.5℃, 5.0% CO2 concentration, and 100 rpm. Photos and samples were taken daily for cell count until viability decreased. Results showed that MDCKs-1F3 cells exhibited a uniformly dispersed spherical morphology. Figure 7 From day 0 to 6 post-inoculation, cell density gradually increased; from day 6 to the plateau phase, cell viability dropped below 90%. Figure 8 .

[0094] Example 3: Feasibility of Scale-up of Suspension Cell Reactor

[0095] (1) Shake flask culture

[0096] Resuscitate MDCK working cell bank cells into SF500 shake flasks using serum-free CD MDCK medium, 120 mL in volume, and culture at the set conditions of 36.5℃, 5.0% CO2, and 100 rpm. Inoculate at a density of (1.2±0.3)×10⁻⁶ cells / mL. 6 The cells / mL were calculated, and the cells were passaged into secondary, tertiary, and quaternary SF2L shake flasks every 3 days, with a final culture volume of approximately 1500 mL.

[0097] (2) Bioreactor culture

[0098] The cells in the grade IV shake flasks were seeded at a density of (1.2±0.3)×10⁶ cells / mL. 6Cells were passaged at a rate of 100 cells / mL and cultured in a 12 L primary bioreactor for 3 days. After 3 days, the culture was scaled up to a 60 L secondary bioreactor. The culture temperature was set at 36.5℃, pH 7.20, and dissolved oxygen at 50.0%. The primary fermentation scale was 6.5L-7.0L at 110 rpm, and the secondary fermentation scale was 26.0kg-30.0kg at 70 rpm. During the reactor culture phase, the cells were in good condition and, after 3 days of culture, expanded to 3 × 10⁶ cells / mL. 6 Cells with a density of at least 100 cells / mL and a viability of at least 90% are provided for virus inoculation, ensuring they are in good condition and at an appropriate density.

[0099] (3) Receiving and Harvesting the Poison

[0100] The cell density in the secondary reactor was adjusted to (3.00-5.00)×10⁻⁶ cells by adding culture medium. 6 Cells / mL, recombinant trypsin added at 3.0 mg / L, MOI at 10 -5 10 -8 10 -5 Influenza viruses of type H1N1, H3N2, and BV were inoculated. The culture temperature was set at 34.0℃, pH at 7.4, dissolved oxygen at 40.0%, and rotation speed at 70 rpm. During the virus culture phase, samples were taken daily to detect cell density and viability. Virus fluid samples were harvested for hemagglutination titer, sterility test, and mycoplasma test (according to the method in Example 2).

[0101] Cell density and viability after infection are shown in the figure. Figure 9 The results of the blood coagulation titer are shown below. Figure 10 Based on cell growth curves, MDCKs-1F3 suspension cells were cultured in a bioreactor to detect H1N1, H3N2, and BV influenza virus strains. After inoculation, the cell count initially increased and then decreased over time, with harvesting occurring after 3 days. Results showed that the virus harvested from all three batches of the reactor was sterile and free of mycoplasma growth. MDCKs-1F3 cells were sensitive to H1N1, H3N2, and BV influenza virus strains, indicating the feasibility of culturing influenza virus in a 60L bioreactor using MDCKs-1F3 suspension cells.

[0102] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An MDCK cell, characterized in that, The cell is preserved in the China General Microbiological Culture Collection Center on November 6, 2025, and the preservation number is CGMCC NO: 46717.

2. The cell of claim 1, wherein, The cell can survive and / or stably passaged in serum-free medium; and / or The cell can be cultured in a single cell form in the medium; and / or After the cell is inoculated into an animal, the inoculated animal does not appear nodules, or the nodules appear and then subside, and can continuously maintain the state of nodule subsidence.

3. The cell according to claim 1 or 2, characterized in that, The cell is sensitive to viruses; and / or The cell can be used for culturing viruses; and / or The virus includes one or more of the following viruses: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, coxsackie virus, adenovirus, lassa virus and vaccinia virus.

4. The cell according to claim 3, characterized in that, The influenza virus includes at least one of the following: influenza A virus, influenza B virus, influenza C virus and / or influenza D virus; and / or The influenza A virus includes at least one of the following: H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2 and H10N8; and / or The influenza B virus includes at least one of the following: BY-type influenza virus and BV-type influenza virus.

5. A method for preparing a virus or a vaccine thereof from the cell of any one of claims 1-4, the method comprising: (a) infecting the cell with a virus; (b) culturing the infected cell under conditions that allow the virus to propagate; and (c) isolating the virus from the cell culture. The virus includes one or more of the following viruses: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, coxsackie virus, adenovirus, lassa virus and vaccinia virus.

6. The method of claim 5, wherein, The influenza virus includes at least one of the following: influenza A virus, influenza B virus, influenza C virus and / or influenza D virus, and / or 7. The method of claim 6, wherein, The influenza A virus includes at least one of the following: H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2 and H10N8, and / or The influenza B virus includes at least one of the following: BY-type influenza virus and BV-type influenza virus.

8. Use of the cell of any one of claims 1-4 in the production of a virus or a vaccine thereof. The virus includes one or more of the following viruses: influenza virus, measles virus, encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus, rabies virus, respiratory syncytial virus, respiratory enterovirus, yellow fever virus, parvovirus, coxsackie virus, adenovirus, lassa virus and vaccinia virus.

9. Use according to claim 8, characterized in that, The influenza virus includes at least one of the following: influenza A virus, influenza B virus, influenza C virus and / or influenza D virus; and / or 10. Use according to claim 9, characterized in that, ​ The influenza A virus comprises at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H3N2, H7N9, H9N2, and H10N8, and / or The influenza B virus comprises at least one of a BY-type influenza virus and a BV-type influenza virus.

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