MDCK cell strain and application thereof in vaccine preparation

Abstract

The invention relates to the technical field of biology, in particular to an MDCK cell strain and application of the MDCK cell strain in preparation of influenza vaccines. The invention provides an MDCK cell strain, the preservation number of which is CCTCC NO: C202504. The cell strain not only can maintain high sensitivity and high yield to influenza viruses and ensure the yield and quality of vaccines, but also can remarkably improve the biological safety of cell matrix influenza vaccines due to elimination of tumorigenicity risk, and has a good application prospect. And a key technical support is provided for promoting domestic research and development and marketing of influenza vaccines based on cell culture.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the MDCK cell line and its application in vaccine preparation. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus, seriously endangering public health. The influenza virus is antigenically variable, spreads rapidly, and can cause seasonal epidemics annually. Globally available influenza vaccines are categorized into inactivated influenza virus vaccines, live attenuated influenza virus vaccines, and recombinant influenza virus vaccines. Based on their components, they are classified as trivalent and quadrivalent influenza vaccines. According to their manufacturing process, influenza vaccines are classified as chicken embryo-based, cell culture-based, and recombinant influenza vaccines. Currently, all influenza vaccines marketed in China are chicken embryo-based. While dozens of companies have received clinical trial approvals for cell culture-based influenza vaccines, none have yet been marketed. Internationally approved cell-based influenza vaccines primarily utilize MDCK cells and Vero cell matrix. In 2001, Solvay's trivalent influenza virus subunit vaccine (Influvac), using serum-free microcarrier adherent MDCK cells, received marketing approval in the Netherlands, but failed to be officially launched due to supply delays. Seqirus (which acquired Novartis's influenza vaccine business) in Australia and SK in South Korea have launched their suspension MDCK cell influenza subunit vaccines in Europe, Australia, the United States, and South Korea, respectively. In late 2020, the U.S. FDA approved Seqirus’s MDCK cell matrix-based MF59 adjuvant H5N1 influenza subunit vaccine.

[0003] Chicken embryo influenza vaccines have been on the market for over 70 years since the 1940s. The production process is mature, and they remain the mainstream influenza vaccine. However, chicken embryo influenza vaccines have several inherent drawbacks. For example: 1. Because vaccine preparation requires a large number of chicken embryos as a production substrate, manufacturers need to purchase them from chicken farms in advance before the WHO publishes the strain composition, often limiting vaccine production. 2. Chicken embryo influenza vaccines require inoculating each chicken embryo with the virus. Due to individual differences in chicken embryos and batch variations between different manufacturers, the quality and yield of each batch of stock solution often vary significantly. Furthermore, inactivated vaccines use ordinary chicken embryos, which are highly susceptible to contamination by exogenous factors, making quality control during production difficult. 3. Continuous passage of the influenza virus in chicken embryos can easily lead to mutations, causing a mismatch between the vaccine strain and the circulating strain, resulting in decreased vaccine effectiveness. 4. Although the quality of chicken embryo influenza vaccines has improved significantly, with a substantial reduction in residual chicken embryo proteins, concerns about chicken embryo allergies remain. 5. Once an avian influenza outbreak occurs, causing a large number of chicken deaths and resulting in a lack of chicken embryos available, the normal supply of influenza vaccines will be further restricted.

[0004] To overcome the various problems associated with chicken embryo influenza vaccine production processes, using animal cells as a production substrate for influenza vaccines has become a development trend in recent years. In 1995, the World Health Organization recommended that influenza vaccine manufacturers use mammalian cells as the culture medium for influenza viruses to improve vaccine production efficiency and protective efficacy. The advantages of cell-based influenza vaccines are as follows: 1. Using cells as a substrate to culture influenza viruses is simpler than using chicken embryos; the culture scale can be easily scaled up using bioreactors, and the short cell culture cycle reduces vaccine production time and significantly increases influenza vaccine yield; 2. During influenza outbreaks, there is no need to wait for chicken embryo supplies, allowing for timely and flexible responses; 3. Cell culture uses a single raw material, resulting in fewer allergic reactions; 4. Cell-cultured influenza viruses are less prone to antigenic mutation, have a higher antigenic match with circulating influenza viruses, and provide higher protection for vaccinated populations; 5. The cells used for culturing influenza viruses require the establishment of a three-tiered cell bank and undergo comprehensive testing, further reducing the risk of exogenous factor contamination.

[0005] Mammalian cells commonly used to culture influenza viruses include MDCK, Vero, PER.C6, and FRhK-4 cells. Among them, MDCK cells are more susceptible to influenza viruses and have a yield 10 times that of Vero cells, making them the preferred cell for cell-based influenza vaccine development by various vaccine companies.

[0006] MDCK (Madin-Darby Canine Kidney) is an epithelial-like cell line derived from canine kidneys. It is an adherent cell line, but the imported MDCK cell line in China exhibits significant tumorigenicity and is not the optimal cell matrix for vaccine production. The upstream production process for influenza virus culture using MDCK cells includes adherent and suspension culture processes. Currently, the cells used for research and production are mainly low-serum-content, low / non-tumorigenic adherent cells and serum-free, low-tumorigenic suspension cells. Both methods have been submitted for clinical trials. However, serum-free suspension cells, being low-tumorigenic, still carry a risk of tumorigenesis. Therefore, providing an MDCK cell line with extremely low tumorigenicity that can be cultured in serum-free medium and its culture method for influenza vaccine production can effectively reduce potential safety risks and has significant clinical significance and application value. Summary of the Invention

[0007] In view of this, the present invention provides the MDCK cell line and its application in vaccine preparation.

[0008] This invention utilizes the MDCK(NBL-2)CCL-34 cell line to obtain a serum-free MDCK cell line through screening in serum-free medium containing gradient concentrations of bovine serum. Experimental verification confirmed its non-tumorigenicity. This cell line was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2025, with accession number CCTCC NO:C202504.

[0009] The MDCK cell line provided by this invention has successfully eliminated its tumorigenic potential through a specific screening and acclimatization process. Its non-tumorigenic characteristics have been confirmed by tumorigenicity experiments; for example, when this cell line was inoculated into immunodeficient nude mice, no tumor formation occurred at the inoculation site or in any major organs throughout the body during the observation period (usually 16 weeks), demonstrating a significant improvement in the biosafety of this cell line as a vaccine production matrix. Simultaneously, this cell line maintains good growth and proliferation capacity under serum-free culture conditions and can be efficiently amplified in adherent culture systems such as microspheres or sheet carriers. In terms of virus culture, this cell line exhibits high susceptibility and high yield to various circulating strains of influenza A and B viruses. Viral titers (such as TCID50 or HA titer) can reach or exceed the levels of existing commercial MDCK cell lines, ensuring vaccine production yield and subsequent purification and concentration efficiency. In addition, the cell line has a stable genetic background. After multiple passages, its key characteristics such as morphological features, growth rate, viral sensitivity, and non-tumorigenicity have not changed significantly, providing a solid guarantee for the stability and reproducibility of the vaccine production process.

[0010] This invention also provides the application of the MDCK cell line in the preparation of viral vaccines.

[0011] Furthermore, the virus includes at least one of the following viruses: influenza virus, Coxsackie virus, reovirus, and adeno-associated virus.

[0012] In some implementations, the influenza virus includes at least one of influenza A, influenza B, influenza C, and influenza D viruses. Specifically, the influenza A virus includes at least one of H1N1, H3N2, H5N1, H5N6, H7N9, and H9N2. The influenza B virus includes BV and / or BY influenza viruses.

[0013] The present invention also provides a method for preparing an influenza vaccine, which includes: culturing influenza virus using the MDCK cell line with accession number CCTCC NO:C202504.

[0014] Furthermore, the method for preparing the influenza virus includes:

[0015] After culturing the MDCK cell line with accession number CCTCC NO:C202504, the influenza virus strain was inoculated to carry out virus replication culture. The cultured virus supernatant was collected, clarified, concentrated, inactivated, purified and lysed to obtain the influenza vaccine.

[0016] In some implementation schemes, the MDCK cell line is cultured using serum-free adherent culture.

[0017] In some implementations, the culture includes: static culture in a square bottle. The static culture time is 48h~72h, specifically 48h, 60h, or 72h; the carbon dioxide concentration during static culture is 4.5%~5.5%, specifically 4.5%, 5.0%, or 5.5%; and the static culture temperature is 36.5℃~37.5℃, specifically 36.5℃, 37℃, or 37.5℃.

[0018] In some embodiments, the culture includes: seeding the MDCK cell line into a culture medium containing sheet-like carriers or microsphere carriers, and culturing under shaking conditions in a shake flask. The shaking rotation speed is preferably 60 rpm to 100 rpm, specifically 60 rpm, 70 rpm, 80 rpm, 90 rpm, or 100 rpm; the shaking culture time is 48 h to 96 h, specifically 48 h, 72 h, or 96 h; the carbon dioxide concentration during the shaking and static culture is 4.5% to 5.5%, specifically 4.5%, 5.0%, or 5.5%; and the shaking culture temperature is 36.5℃ to 37.5℃, specifically 36.5℃, 37℃, or 37.5℃.

[0019] In some embodiments, the culture includes: inoculating the MDCK cell line into a reactor containing culture medium and a sheet-like carrier, and culturing under fixed-bed culture conditions; or inoculating it into a reactor containing culture medium and a microsphere carrier, and culturing under suspension culture conditions. The dissolved oxygen content of the suspension culture is preferably 40%–60%, specifically 40%, 50%, or 60%; the pH value is 7.0–7.4, specifically 7.0, 7.1, 7.2, 7.3, or 7.4; the rotation speed is controlled at 30–120 rpm, specifically 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, or 120 rpm. The culture time is 48 h–120 h, specifically 48 h, 72 h, 96 h, or 120 h. The culture temperature is 36.5℃–37.5℃, specifically 36.5℃, 37℃, or 37.5℃.

[0020] In some embodiments, the carrier for adherent culture includes at least one of the following: square flasks, microspheres, sheet-like carriers, hollow fiber carriers, porous microcarriers, and fixed-bed / filled-bed carriers. Microspheres can be solid or porous / macroporous; this invention does not impose any particular limitation and the choice can be made according to actual conditions. In this invention, the diameter of the microspheres is preferably 60-350 μm, more preferably using cross-linked dextran as a substrate, with a specific surface area of ​​approximately 2700-4400 cm² / g for solid carriers. In this invention, the sheet-like carrier can be a circular, rhomboid, or polygonal sheet structure, preferably a sheet structure with a diameter of approximately 6 mm, more preferably a sheet structure with a polyester fiber or non-woven fabric matrix. The specific surface area is typically 1200-1500 cm² / g.

[0021] In some implementation plans, according to 10 -2 ~10 -5 The MOI is used to inoculate the influenza virus. In some specific embodiments, the MOI is 10. -2 10 -3 10 -4 10 -5 The trypsin concentration is 2~20 μg / ml, specifically 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, and 20 μg / ml; the culture temperature for virus replication culture is 33~35℃, specifically 33℃, 33.5℃, 34℃, 34.5℃, and 35℃, and other culture conditions are the same as those for the MDCK cell line culture described above; the harvest time for the virus supernatant is 48h~72h, specifically 48h, 52h, 56h, 60h, 64h, 68h, and 72h.

[0022] The present invention also provides an influenza virus vaccine prepared by the above-described preparation method.

[0023] This invention also provides a method for screening MDCK cell lines, comprising:

[0024] After multiple monoclonal screenings and large-scale culture, the MDCK(NBL-2)CCL-34 cell line was cultured in medium containing graded concentrations of bovine serum. The tumorigenicity was verified, yielding the MDCK cell line described in this invention. Tumorigenicity testing showed that this cell line is non-tumorigenic and has high safety.

[0025] In some embodiments, the gradient concentrations of bovine serum include 10% bovine serum, 7.5% bovine serum, 5% bovine serum, 2.5% bovine serum, 0.5% bovine serum, and 0% bovine serum. The culture medium containing 10% bovine serum is a mixture of 10% bovine serum and serum-free culture medium. In this invention, the serum-free culture medium includes at least one of DMEM, MEM, DMEM / F12, CDVM01, EX-CELL, and OPM-AM146. In a specific embodiment of this invention, the serum-free culture medium is OPM-AM146.

[0026] This invention provides an MDCK cell line with accession number CCTCC NO:C202504. This cell line not only maintains high sensitivity and high yield to influenza virus, ensuring vaccine production and quality, but also significantly improves the biosafety of cell matrix influenza vaccines by eliminating the risk of tumorigenesis. This provides key technical support for promoting the research and development and marketing of cell culture-based influenza vaccines in China.

[0027] Biological Preservation Instructions

[0028] Canine kidney cells MDCK.3.1.6-T Canis lupus familiaris were deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202504. Attached Figure Description

[0029] Figure 1 The results of the adherent culture of MDCK cells on microspheres in this invention are shown; where a~d are the results of cell culture on medium containing 1% serum for 0h, 6h, 24h and 48h respectively, and e~h are the results of cell culture on serum-free medium for 0h, 6h, 24h and 48h respectively.

[0030] Figure 2 The microscopic examination results of the culture medium after MDCK cells adhered to a three-layer scaffold sheet carrier in this invention; wherein, a~b are the microscopic examination results of the culture medium containing 1% serum; c~d are the microscopic examination results of the serum-free culture medium;

[0031] Figure 3 The results show the adherent culture of MDCK cells on a three-layer scaffold sheet carrier according to the present invention; among them, the results with 1% serum are from top to bottom the results of cells cultured on medium containing 1% serum for 0h, 24h and 72h, and the results without serum are from top to bottom the results of cells cultured on medium without serum for 0h, 24h and 72h.

[0032] Figure 4The results are from the tumorigenicity assay on soft agar; where a represents the results for the MDCK(NBL-2) cell line; and b represents the results for the MDCK.3.1.6-T cell line.

[0033] Figure 5 The results are the pathological examination results of P4 passage MDCK.3.1.6-T adherent cells from batch 20241120; among them,

[0034] Figure 6 The results are the pathological examination results of P29 passage MDCK.3.1.6-T adherent cells from batch 20250310;

[0035] Figure 7 The results are pathological examination results of adherent cells from batch 20250425 of MDCK.3.1.6-T. Among them, a is the result of adherent cells from animal No. 204 at passage P29, b is the result of adherent cells from animal No. 222 at passage P19, and c is the result of adherent cells from animal No. 238 at passage P15.

[0036] Figure 8 This is a network diagram for enrichment analysis of differentially expressed genes using string software. Each circle represents a gene, and different colors indicate different gene clusters.

[0037] Figure 9 This is a graph showing the number of differentially regulated genes. The horizontal axis represents the control group, and the vertical axis represents the number of differentially regulated genes. Red indicates upregulated genes, and blue indicates downregulated genes.

[0038] Figure 10 This is a heatmap of differential gene clustering. The horizontal axis represents sample information, and the vertical axis represents genes. Red represents high expression, and blue represents low expression.

[0039] Figure 11 This is a volcano plot of differentially expressed genes. The X-axis represents the fold change after log2 transformation, and the Y-axis represents the significance value after -log10 transformation. Red represents upregulated DEGs, green represents downregulated DEGs, and gray represents non-DEGs.

[0040] Figure 12 This is a Venn diagram of gene expression. A circle represents a set of genes, and overlapping areas of different circles represent the intersection of these gene sets. Unoverlapping areas indicate genes unique to that gene set, and the numbers on the diagram represent the number of genes in the corresponding region. Detailed Implementation

[0041] This invention provides the MDCK cell line and its application in the preparation of influenza vaccines. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0042] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0043] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0044] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0045] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it means that the units for the left and right endpoints are the same. For example, 60~80rpm means that the units for the left endpoint "60" and the right endpoint "80" are both rpm.

[0046] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0047] The present invention will be further illustrated below with reference to the embodiments:

[0048] Example 1

[0049] Adherent cell digestion and passage: Taking a fully grown T75 flask as an example. Wash twice with 10ml PBS, then add 4ml 0.25% trypsin, spread evenly over the bottom of the flask, and digest at 37℃ for 6-10 minutes. After the cells become rounded and detach, dilute with 5ml serum-free culture medium, mix well by pipetting, transfer to a 10ml centrifuge tube, centrifuge at 900rpm / 4min, remove the supernatant, resuspend the pellet, and passage according to the cell volume.

[0050] Monoclonal selection (culture medium containing 10% serum): MDCK (NBL-2) CCL-34 purchased from ATCC, LOT: 70040764. Monoclonal cell selection was performed three times using a limiting serial dilution method. Cells were passaged to their optimal viability before digestion, counting, and plating. To ensure ≤1 cell count per well in each 96-well plate, the cell count was set to 75 at plating. Immediately after plating, each well was checked to identify wells with 1 cell count and marked accordingly. The medium was changed weekly, and each well was examined microscopically weekly; any wells with multiple cell clusters were discarded. After 10-15 days of growth in the 96-well plates, when each well reached approximately 30% confluence, the cells were digested and passaged to 6-well plates. After approximately one week of growth in the 6-well plates, when each well reached approximately 60% confluence, the cells were digested and passaged to T25 flasks. When cells reached over 80% confluence, the cells were passaged to T75 flasks for serum acclimation. The first generation yielded 57 cell lines from 2400 single clones; the second generation further expanded these 57 lines, yielding 200 lines from 4320 single clones; the third generation further expanded these 200 lines, yielding 80 lines from 3840 single clones. From these 80 lines, we screened 30 cells that did not form colonies using a soft agar colony formation assay. These 30 lines were then sent to Hunan Silek Jingda for tumorigenicity testing, resulting in the selection of 6 low / non-tumorigenic cell lines.

[0051] Six cell lines were acclimatized to serum-free culture: The serum-containing medium was discarded, the cells were washed twice with PBS, digested with trypsin for 6-10 min, neutralized with OPM-AM146 serum-free medium containing 10% bovine serum, mixed thoroughly by pipetting, centrifuged at 900 rpm for 4 min, the supernatant was discarded, and the cells were resuspended in serum-free medium containing 10% bovine serum. The cells were then passaged, with each serum-containing medium passaged at least twice. Serum-reduced passages were performed only after the cells showed good growth. The passage sequence was as follows: 10% bovine serum + serum-free medium → 7.5% bovine serum + serum-free medium → 5% bovine serum + serum-free medium → 2.5% bovine serum + serum-free medium → 0.5% bovine serum + serum-free medium → serum-free medium.

[0052] Tumorigenicity verification: After six serum-free cultured cell lines were degraded to stability, tumorigenicity verification was performed. Ten 4-7 week old nude mice were selected per group, and each mouse was subcutaneously injected with 0.2 ml of the cells to be tested (i.e., each mouse was inoculated with 1×10⁻⁶ cells). 7 (10 live cells); the positive control group (HeLa cells) was injected with 0.2 ml of positive control cells per cell, containing 1×10⁻⁶ live cells. 6 Live cells. Regularly observe and palpate all animals at the injection site for nodule formation, for at least 16 weeks. Select cell lines where nodules have regressed and perform further pathological examination to confirm the absence of tumor growth.

[0053] Simultaneously, the six serum-free cell lines selected were subjected to viral adaptability verification, with the viruses classified according to MOI of 10. -2 ~10 -5 Trypsin concentrations of 2 μg / ml to 20 μg / ml were used to inoculate T25 and T75 flasks and microspheres (microspheres with MDCK cells) and sheet carriers (sheet carriers with MDCK cells) to screen for cell lines with higher titers.

[0054] Based on tumorigenicity and viral adaptability results, the MDCK.3.1.6-T cell line of this invention was obtained through comprehensive screening.

[0055] Test Example 1: Blood Coagulation Titration Detection

[0056] Viral susceptibility testing was performed on MDCK.3.1.6-T cells using T25 vials, T75 vials, microspheres, and sheet vectors. The following strains were used for validation, and the results were compared with the original MDCK (NBL-2).

[0057] Strain H1N1: 23 / 250: Influenza Virus Infectious IVR-238 (A / Victoria / 4897 / 2022); 16 / 270: Influenza virus infectious IVR-180;

[0058] Strain H3N2: 17 / 196: Influenza Virus Infectious A / Singapore / INFIMH-16-0019 / 2016;

[0059] 21 / 204: Influenza Virus Infectious A / Thailand / 8 / 2022;

[0060] Strain BV: 22 / 204: Influenza Virus Infectious B / Austria / 1359417 / 2021 (B-Victoria lineage) BVR-26;

[0061] 23 / 228: Influenza Virus Infectious B / Austria / 1359417 / 2021 (B / Victoria).

[0062] The results of blood coagulation titer verification using T25 square bottles are shown in Table 1.

[0063] Table 1

[0064]

[0065] The results of blood coagulation titer verification using T75 square bottles are shown in Table 2.

[0066] Table 2

[0067]

[0068] The hemagglutination titer results of the microsphere culture are shown in Table 3.

[0069] Table 3

[0070]

[0071] The hemagglutination titer results for the sheet-like carriers are shown in Table 4.

[0072] Table 4

[0073]

[0074] Hemagglutination titer results showed that, under the culture conditions of T25 flasks, T75 flasks, microsphere culture systems, and sheet-like carrier culture systems, the MDCK.3.1.6-T cell line of this invention maintained influenza virus culture and replication capabilities comparable to the original MDCK cell line in different culture systems. It can effectively support the proliferation of multiple circulating influenza virus strains, providing a good cell matrix basis for the large-scale production of subsequent vaccines.

[0075] Test Example 2: Tumorigenicity Test Experiment

[0076] (a) Soft agar assay for tumorigenesis

[0077] The soft agar colony formation assay is a key technique for assessing the proliferative capacity of cells, especially tumor cells, in suspension. It is based on the cell's anchorage-independent growth characteristics and its ability to form colonies in a semi-solid soft agar medium, and is commonly used to evaluate cell tumorigenicity or transformation status.

[0078] Experimental procedure: Prepare two layers of agarose gel, with the lower layer containing 1.2% as support and the upper layer containing 0.7% mixed with cells. Maintain a cell density of 5000-10000 cells per cell. Incubate at 37℃ in a 5% CO2 incubator for 2-3 weeks. After incubation, add 0.005% crystal violet to stain the clones and count them.

[0079] See results Figure 4 .

[0080] The results showed that obvious cell clumps were visible in the soft agar of MDCK (NBL-2) cells, while no colonies were observed in the MDCK.3.1.6-T cells of this invention, indicating that no clones could be formed on the soft agar, suggesting that they do not have tumorigenicity.

[0081] (II) Tumorigenic Pathological Examination

[0082] The Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd. was commissioned to conduct histopathological testing.

[0083] In this embodiment, three tumorigenicity experiments were conducted, and representative samples were selected for pathological verification: batch 20241120 of MDCK.3.1.6-T adherent P4 passage, batch 20250310 of MDCK.3.1.6-T adherent P29 passage, and batch 20250425 of MDCK.3.1.6-T adherent P15, P19, and P29 passages. Although the pathological reports showed cystic cell arrangements, no tumors were observed.

[0084] The specific results are as follows:

[0085] (1) In the 20241120 batch of validation MDCK.3.1.6-T adherent P4 generation, all nodules in 10 animals disappeared, except for nodule No. 29, which disappeared the latest. Therefore, this animal was selected for pathological examination. The heart, liver, kidney, spleen, lungs, and lymph nodes of animal No. 29 were sent for examination. The injection site (tumor test site could not be sampled because the nodule disappeared).

[0086] Pathological examination results are shown Figure 5 .

[0087] The results showed that extramedullary hematopoiesis of the red pulp was observed in the spleen of experimental animals 3.1.6-29 (see...). Figure 5 (d) No obvious pathological changes were observed in other organs and tissues.

[0088] (2) In batch 20250310, MDCK.3.1.6-T adherent P29 passage, 2 out of 10 animals showed complete ablation. Of the remaining 8 animals, 4 were randomly selected for pathological examination: animals 085, 092, 093, and 095. These 4 animals were sent for injection site pathological examination. The pathological results are shown below. Figure 6 .

[0089] The results showed that in experimental animals 3.1.6-85, the implantation site revealed a cystic structure with cellular arrangement, and basophilic protein-like material was visible within the cyst. Figure 6 (a). 3.1.6-92 Experimental animals showed cellular arrangements forming a cystic structure at the implantation site, with basophilic protein-like material visible within the cysts. Figure 6 (b). 3.1.6-93 experimental animals: At the implantation site, cells were arranged in a cystic structure, and basophilic protein-like material was visible within the cyst. Figure 6 (c). 3.1.6-95 Experimental animals showed cystic cell arrangement at the implantation site, with basophilic protein-like material and a small amount of inflammatory cell infiltration within the cysts. Figure 6(d).

[0090] (3) In the MDCK.3.1.6-T adherent P15 generation (batch 20250425), 4 out of 10 animals were completely ablated, and animal number 238 was randomly selected from the remaining 6 animals for pathological examination. In the MDCK.3.1.6-T adherent P19 generation, 4 out of 10 animals were completely ablated, and animal number 222 was randomly selected from the remaining 6 animals for pathological examination. In the MDCK.3.1.6-T adherent P29 generation, 2 out of 10 animals were completely ablated, and animal number 204 was randomly selected from the remaining 8 animals for pathological examination.

[0091] Pathological examination results are shown Figure 7 .

[0092] The results showed that at the implantation site of experimental animal No. 204, cells were arranged in a cystic structure, and basophilic protein-like material was visible within the cyst. Figure 7 (a). At the implantation site in experimental animal number 222, cells were arranged in a cystic structure, and eosinophilic protein-like material was visible within the cystic cavity. Figure 7 (b). At the implantation site in experimental animal No. 238, cells were arranged in a cystic structure, and eosinophilic protein-like material was visible within the cystic cavity. Figure 7 (c) The results showed no tumor characteristics, no loss of cellular atypia, no pathological mitotic figures, no crab-like or cord-like infiltration of tumor cells in the surrounding tissues, and no large areas of coagulative or liquefactive necrosis within the structures. This fully demonstrates that the inoculated MDCK cells did not form tumors and possess good safety as non-tumorigenic cells.

[0093] Test Example 3 Transcriptome Analysis

[0094] Transcriptome sequencing studies the sum of all RNA that a specific cell can transcribe under a given functional state, primarily including mRNA and non-coding RNA. Transcriptome research is the foundation and starting point for gene function and structure research. Next-generation high-throughput sequencing can comprehensively and rapidly obtain almost all transcript sequence information for a specific tissue or organ of a species under a given state, and has been widely applied in basic research, clinical diagnosis, and drug development. With the commercialization of next-generation sequencing platforms, RNA sequencing technology has become one of the important tools in transcriptomics research. This technology uses next-generation high-throughput sequencing platforms to sequence the genome, calculates the expression levels of different mRNAs by statistically counting relevant reads, analyzes the structure and expression levels of transcripts, discovers unknown and rare transcripts, accurately identifies alternative splicing sites and coding sequence single nucleotide polymorphisms, and provides the most comprehensive transcriptome information.

[0095] In a broad sense, the transcriptome refers to the collection of all transcription products within a cell under a given physiological condition, including messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA; in a narrow sense, it refers to the collection of all mRNAs. Proteins are the primary carriers of cellular function, and the proteome is the most direct description of cellular function and state. As a primary means of studying gene expression, the transcriptome is the essential link between genomic genetic information and the proteome of biological function. Transcriptional regulation is the most extensively studied and is also the most important regulatory mechanism in organisms. Transcriptome research can study gene function and structure at the holistic level, revealing specific biological processes, and has been widely applied in fields such as plant candidate gene discovery, functional identification, and genetic improvement.

[0096] The transcriptome sequencing data for this study are MDCK(NBL-2) and MDCK.3.1.6-T. In the report, Ctrl represents MDCK(NBL-2) and M06 represents MDCK.3.1.6-T. Due to the need to avoid excessively long names during sequencing, these abbreviations have been used.

[0097] This embodiment primarily compares intercellular differences and further analyzes differentially expressed genes between MDCK (NBL-2) and MDCK.3.1.6-T. Transcriptome sequencing was performed using the DNBSEQ platform from BGI Genomics to further explore the functions of genes related to phenotypic changes. The results were validated using string software for enrichment analysis of differentially expressed genes (https: / / cn.string-db.org / ). A False Discovery Rate ≤ 0.01 and a Strength value of 0.4–1.61, with higher values ​​indicating significant enrichment, were defined as significant enrichment in the differentially expressed gene. It was concluded that the protein played a synergistic role in the biological function or pathway. Related network diagrams are shown below. Figure 8 .

[0098] The genes located at the center of the functional network are mainly as follows: MK167 EX01 TOP2A CCNB1 CLSPN TROAFTPIP13 AURKB PRC1 CENPF CCNB1 ASF1B BUB1 NUF2 SPAG5 CCNA2 IQGAP3 UHRF1 KIF18BNDC80 TTK BUB1B ASPM KIF23 E2F8 IQGAP3 CCNB3 TPX2 ESCO2 CENPU KIF4A CDCA8ESPL1 DIAPH3 CDCA3 CDCA8 ESPL1 APH NEK2 CENPE CDCA2 HJURP CCNB2 DEPDC1 MND1CENPT NCAPG CEP55 CDCA5 MAD2L1 PSRC1 KIF22 PSRC1 GTSE1 MIS18A KNL1 CKAP2LELOVL7 CIT BUB1, etc. Among them: CENPU: A complex that plays a central role in kinetochore assembly, mitotic progression, and chromosome segregation. TTK: Involved in mitotic spindle assembly checkpoint signaling, a process that delays anaphase until chromosomes are biologically oriented on the spindle, and participates in repairing incorrect kinetochore microtubule attachment in mitosis. CDCA8: A component of the chromosome complex (CPC), a key regulator of mitosis. NUF2: A component of the essential kinetochore-associated NDC80 complex, necessary for chromosome segregation and spindle checkpoint activity. CENPE: Microtubule-termined kinetochore motors play an important role in chromosome aggregation, microtubule-kinetochore binding, and spindle assembly checkpoint activation. ASPM: Involved in the regulation of the mitotic spindle and coordination of the mitotic process. SPAG5: A fundamental component of the mitotic spindle required for normal chromosome segregation and anaphase. KIF23: Microtubule-dependent and Rho-mediated signaling required for myosin contractile loop formation during cell cycle cytokinesis. PRC1: A key regulator of cytokinesis. As an oncogene, it promotes bladder cancer cell proliferation, apoptosis inhibition, and oncogenic progression. CCNB2: Essential for the control of the cell cycle during the G2 / M (mitotic) transition. PRC1: A key regulator of cytokinesis, it crosslinks antiparallel microtubules at an average distance of 35 nM. It is crucial for controlling the spatiotemporal formation of the intermediate zone and successful cytokinesis. It requires the localization of KIF14 to the central axis and centrosome. It requires the recruitment of PLK1 to the main axis. Stimulation of PLK1 phosphorylation of RACGAP1 allows ECT2 to be recruited to the central spindle. As an oncogene, it promotes bladder cancer cell proliferation, apoptosis inhibition, and oncogenic progression. AURKB: A serine / threonine protein kinase component of the chromosome complex (CPC).

[0099] Biological processes of differentially expressed genes were enriched and arranged in ascending order of false discovery rate (see Table 5). Expression analysis graphs of differentially expressed genes are shown below. Figures 9-12 Differentially expressed genes are involved in biological functions such as chromosome segregation, cell cycle, sister chromosome segregation, cell mitosis, nuclear division, cell division, spindle formation, microfilament formation, and cell proliferation.

[0100] Table 5. Significantly Differential Genes Between MDCK (NBL-2) and MDCK.3.1.6-T

[0101]

[0102] It is evident that differentially expressed genes primarily regulate biological processes related to cell proliferation and division cycles, thereby causing uncontrolled cell proliferation.

[0103] The transcriptome results above indicate that MDCK.3.1.6-T cells exhibit significant differences in gene expression at the transcriptome level compared to MDCK (NBL-2) cells. These differentially expressed genes are significantly enriched in biological processes closely related to cell proliferation, such as chromosome segregation, cell cycle, sister chromatid separation, mitosis, nuclear division, cell division, spindle formation, and microfilament formation. This suggests that MDCK.3.1.6-T cells may alter their proliferative capacity by regulating the expression of these genes related to cell proliferation and the cell cycle, potentially leading to their non-tumorigenic nature.

[0104] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A MDCK cell strain, characterized in that, Its accession number is CCTCC NO:C202504.

2. The use of the MDCK cell line of claim 1 in the preparation of a viral vaccine, wherein the virus includes at least one of influenza virus, Coxsackie virus, reovirus, and adeno-associated virus; and the vaccine includes at least one of inactivated split vaccine, live attenuated vaccine, and subunit vaccine.

3. Use according to claim 2, characterized in that, The influenza virus includes at least one of influenza A, influenza B, influenza C, and influenza D viruses.

4. Use according to claim 3, characterized in that, The influenza A virus includes at least one of the following types: H1N1, H3N2, H5N1, H5N6, H7N9, and H9N2; the influenza B virus includes BV and / or BY influenza viruses.

5. A method of preparing a vaccine, characterized in that, It includes: The virus was cultured using the MDCK cell line with accession number CCTCC NO:C202504.

6. The production method according to claim 5, characterized by, It includes: After culturing the MDCK cell line with accession number CCTCC NO:C202504, the virus strain was inoculated and trypsin was added for virus replication culture. The cultured virus supernatant was collected, clarified, concentrated, inactivated, purified, and lysed to obtain the vaccine.

7. The production method according to claim 6, characterized by, The culture was a serum-free culture. And / or, the culture includes: seeding the MDCK cell line into a square bottle and incubating it statically; the conditions for static culture include: time of 48h to 96h, carbon dioxide concentration of 4.5% to 5.5%, and temperature of 36.5℃ to 37.5℃; And / or, the culture includes: seeding the MDCK cell line into a culture medium containing sheet-like carriers or microsphere carriers, and culturing under shaking conditions in a shake flask; the shaking conditions include: a rotation speed of 60~100 rpm, a time of 48h~96h, a carbon dioxide concentration of 4.5%~5.5%, and a temperature of 36.5℃~37.5℃; And / or, the culture includes: inoculating the MDCK cell line into a bioreactor containing culture medium and a sheet-like carrier, and culturing under fixed-bed culture conditions; or inoculating it into a bioreactor containing culture medium and a microsphere carrier, and culturing under suspension culture conditions; the culture conditions include: dissolved oxygen of 40%~60%, pH of 7.0~7.4, rotation speed of 30rpm~120rpm, culture time of 48h~120h, and culture temperature of 36.5℃~37.5℃; Preferably, the carrier includes at least one of the following: square bottle, microsphere, sheet carrier, hollow fiber carrier, porous microcarrier, and fixed bed / filled bed carrier.

8. The preparation method according to claim 6, characterized in that, The virus is calculated at 1×10 -5~ 1×10 -2 The MOI was inoculated; the concentration of the trypsin in the culture system was 2~20 μg / ml; the temperature for virus replication culture was 33℃~35℃; and the harvest time of the virus supernatant was 48h~72h of virus replication culture.

9. A vaccine prepared by the method according to any one of claims 5 to 8.

10. A screening method for MDCK cell strains, characterized by, include: After multiple monoclonal screenings and expansion culture of the MDCK(NBL-2)CCL-34 cell line, it was acclimatized and cultured in a medium containing gradient concentrations of bovine serum. Obtain the MDCK cell line as described in claim 1; The gradient concentrations include 10%, 7.5%, 5%, 2.5%, 0.5%, and 0%. The culture medium includes at least one of DMEM, MEM, DMEM / F12, CDVM01, EX-CELL, and OPM-AM146.

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