Non-tumorigenic MDCK cell strain, screening method and application

Abstract

The invention discloses a non-tumorigenic MDCK cell strain, a screening method and application. The non-tumorigenic MDCK cell strain is MDCK-CA027 (the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2023329). The tumor formation rate of the cell strain is 0% when the cell strain is observed for at least 16 weeks in a nude mouse in-vivo tumor formation test, and the cell strain has good sensitivity to various influenza viruses (such as H1N1, H3N2, BY and BV). The invention also provides a method for screening the cell strain. The method comprises the following steps: preparing a single-cell suspension, carrying out limited dilution to obtain monoclone, carrying out enlarged culture, carrying out virus sensitivity detection, carrying out in-vitro tumorigenicity related detection (clone formation test / soft agar test), carrying out nude mouse in-vivo tumorigenicity test and the like. The non-tumorigenic MDCK cell strain is high in safety, can efficiently support influenza virus replication, can be safely and effectively applied to influenza vaccine production and influenza virus amplification, overcomes the tumorigenic risk of traditional MDCK cells, and has important scientific significance and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a non-tumorigenic canine kidney (Madin-Darby Canine Kidney, MDCK) cell line, its screening method, and the application of this cell line in influenza virus amplification and influenza vaccine production. Background Technology

[0002] Influenza (Flu) is an acute respiratory infectious disease caused by the influenza virus (IV). It is highly contagious and variable, posing a serious and ongoing threat to global public health. It can lead to seasonal epidemics and periodic pandemics, causing a large number of cases and deaths, and imposing a heavy economic burden.

[0003] Vaccination is the most economical and effective measure to prevent influenza epidemics, reduce influenza-related morbidity and mortality, and control influenza outbreaks. Currently, global influenza vaccine production mainly relies on chicken embryo culture technology for virus amplification. However, the chicken embryo production process has many inherent drawbacks: First, the production cycle is long, making it difficult to respond quickly to sudden influenza outbreaks; second, the operation is cumbersome, making large-scale production difficult; third, chicken embryos are susceptible to contamination by exogenous factors (such as avian influenza viruses), affecting vaccine safety; in addition, some influenza virus strains do not grow well in chicken embryos, and long-term passage in chicken embryos may lead to changes in viral antigenicity, affecting the immunogenicity and protective efficacy of the vaccine; finally, the supply of chicken embryos is affected by factors such as seasons and avian influenza outbreaks, making stability difficult to guarantee.

[0004] To overcome the shortcomings of chicken embryo culture technology, using mammalian cells as a substrate for influenza vaccine production has become an important direction and hot topic in vaccine research and development. Mammalian cell culture technology has advantages such as short production cycles, ease of large-scale and automated production, high controllability of product quality, continuous passage capability, and no risk of contamination from exogenous factors associated with chicken embryos. Madin-Darby Canine Kidney (MDCK) cells, due to their broad sensitivity to various influenza viruses and excellent amplification capacity, have become one of the main cell lines used internationally for influenza virus amplification and vaccine production.

[0005] While MDCK cells have shown great potential in influenza vaccine production, traditional MDCK cell lines (such as some ATCC MDCK cells) may accumulate genetic mutations and acquire tumorigenic potential during long-term passage. Tumorigenicity refers to the ability of cells to proliferate abnormally and form tumors in both in vivo and in vitro environments. This is a key factor limiting the safety of cell-based vaccine products and a major concern for drug regulatory agencies during the review and approval process. Using cell lines with tumorigenic risk in vaccine production could introduce potentially tumorigenic components during vaccine preparation, posing a potential threat to the health of vaccine recipients. Therefore, establishing stable, tumorigenic MDCK cell lines is one of the core technologies for overcoming the bottlenecks in the development of MDCK cell-based influenza vaccines in my country and achieving the market launch of safe and effective vaccine products.

[0006] To address the vaccine safety concerns posed by the tumorigenicity of MDCK cells in existing technologies, this invention aims to provide a novel non-tumorigenic MDCK cell line. This cell line not only maintains good sensitivity to influenza virus but also possesses clearly defined non-tumorigenic characteristics, thus laying a solid foundation for the production of safe and effective influenza vaccines. Furthermore, this invention also provides an efficient screening method for this non-tumorigenic MDCK cell line and its applications. Summary of the Invention

[0007] The purpose of this invention is to provide a non-tumorigenic MDCK cell line that overcomes the tumorigenicity of existing MDCK cells and is suitable for influenza virus amplification and influenza vaccine production.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a non-tumorigenic MDCK cell line, namely MDCK-CA027, which was deposited on October 29, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C2023329, and classified as Madin Darby Canine Kidney Cells MDCK-CAO27.

[0009] The MDCK-CA027 cell line was identified as epithelioid cells with regular morphology, exhibiting a typical "paving stone" appearance when the monolayer is dense. This cell line showed reduced or negative tumorigenicity in in vitro clonogenic assays and soft agar assays, and in a rigorous in vivo tumorigenicity assay in nude mice (observation period of at least 16 weeks), the tumorigenicity rate was 0%, confirming it as a non-tumorigenic cell line. Furthermore, this cell line maintained good sensitivity to multiple influenza viruses, including influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Yamagata lineage (BY), and influenza B virus Victoria lineage (BV), effectively supporting the replication of these viruses, thus making it highly suitable as a cell matrix for influenza vaccine production.

[0010] This invention provides the application of the above-mentioned non-tumorigenic MDCK cell line in influenza vaccine production. Because the non-tumorigenic MDCK cell line of this invention is safe and non-tumorigenic, and can efficiently support influenza virus replication, it can replace traditional chicken embryos or MDCK cells with tumorigenic risk for the industrial production of influenza vaccines, improving vaccine safety and production efficiency.

[0011] This invention also provides the application of the above-mentioned non-tumor-forming MDCK cell line in influenza virus amplification. Using the non-tumor-forming MDCK cell line of this invention, influenza virus can be amplified in large quantities and efficiently in an in vitro culture system, providing a sufficient source of viral antigen for influenza vaccine production. It can also be used in virological research, diagnostic reagent development, and other fields.

[0012] This invention provides a method for screening non-tumorigenic MDCK cell lines, comprising the following steps: a. Prepare single-cell suspensions from the original MDCK cell line; b. Seed the single-cell suspensions into culture plates using a limiting dilution method, and screen to obtain monoclonal cell wells; c. Expand the cell lines in the monoclonal cell wells; d. Perform virus susceptibility testing on the expanded monoclonal cell lines, and screen for monoclonal cell lines sensitive to influenza virus; e. Perform tumorigenicity-related tests on the monoclonal cell lines obtained in step d, including colony formation assays and / or soft agar assays, and screen for monoclonal cell lines with reduced tumorigenicity; f. Perform in vivo tumorigenicity assays on the monoclonal cell lines obtained in step e in nude mice, and screen for non-tumorigenic MDCK cell lines with a tumorigenicity of 0%.

[0013] In step a, the original MDCK cell line is derived from the MDCK cell line of ATCC.

[0014] In step b, the cell concentration of the limiting dilution method is 5 cells / mL, and the seeding volume is 200 μL / well in a 96-well plate to increase the probability of obtaining monoclonal cells.

[0015] In step d, the influenza viruses include influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Yamagata lineage (BY), and influenza B virus Victoria lineage (BV). Preferably, the cell lines are sensitive to all four influenza viruses to ensure broad applicability. Viral susceptibility is typically assessed by measuring hemagglutinin (HA) titers after viral infection.

[0016] In step e, the colony formation assay is performed at an inoculation density of 100 cells / well. After 7 days of culture, the number of colonies formed and the colony formation rate are counted. Compared with the original cell line, lines with reduced colony formation rate are screened. The soft agar assay is performed at an inoculation density of 1000 cells / well. After 21 days of culture, the formation of clonal clusters is observed and counted, and lines that do not form clonal clusters or have significantly reduced clonal cluster formation ability are screened. These in vitro assays are important indicators for assessing the tumorigenic potential of cells.

[0017] In step f, the in vivo tumorigenicity test in nude mice is the gold standard for assessing cell tumorigenicity. Typically, a certain number of cells (e.g., 1×10^6 to 1×10^7 cells) are inoculated subcutaneously into nude mice, and the observation period is at least 16 weeks. A tumor formation rate of 0% is used as the criterion for non-tumorigenicity.

[0018] This invention also screened other non-tumorigenic MDCK cell lines, including MDCK-CA005, MDCK-CA078, MDCK-CB057, and MDCK-CB082. These cell lines also underwent rigorous screening and identification, and showed a tumorigenesis rate of 0% in nude mice (observation period of at least 16 weeks), exhibiting non-tumorigenic characteristics and susceptibility to influenza virus similar to MDCK-CA027.

[0019] The beneficial effects of this invention are as follows: 1. Significantly improved safety: The MDCK cell lines (such as MDCK-CA027) obtained by screening in this invention showed a tumorigenesis rate of 0% in a rigorous in vivo tumorigenesis test in nude mice (observation period of 16 weeks), which completely eliminated the tumorigenesis risk that may exist in traditional MDCK cells, greatly improved their biosafety as a vaccine production matrix, and met the strict requirements of drug regulatory agencies for cell matrices used in the production of biological products.

[0020] 2. Good viral sensitivity: The non-tumorigenic MDCK cell line of the present invention maintains high sensitivity to a variety of influenza viruses, including influenza A H1N1, H3N2 subtypes and influenza B BY and BV lines, and can efficiently support the replication of these viruses, ensuring the viral titer and antigen yield in vaccine production.

[0021] 3. Scientific and efficient screening method: The screening method established in this invention, through single-cell cloning, virus sensitivity detection, in vitro tumorigenesis-related indicators (clonal formation, soft agar) pre-screening, and finally in vivo tumorigenesis gold standard verification in nude mice, is scientific and rigorous, and can efficiently screen out safe and efficient non-tumorigenic MDCK cell lines from a large number of single-cell clonal cells.

[0022] 4. Broad Application Prospects: The non-tumorigenic MDCK cell line of this invention can be widely used in the production of influenza vaccines, overcoming the shortcomings of chicken embryo culture and realizing the modernization, large-scale production, and safety of vaccine manufacturing. Simultaneously, it also has significant application value in basic research on influenza viruses and the development of diagnostic reagents.

[0023] In summary, the non-tumorigenic MDCK cell line and its screening method provided by this invention offer key technical support and safety assurance for the research and development and production of influenza vaccines, and have significant scientific value and great application prospects. Attached Figure Description

[0024] Figure 1 This image shows the growth status of MDCK monoclonal cells at different culture times in a 96-well plate.

[0025] Figure 2 This is a morphological diagram of MDCK monoclonal cells.

[0026] Figure 3(a) shows the screening results of CA series monoclonal cell lines for susceptibility (HA titer) to influenza virus H1N1; Figure 3(b) shows the screening results of CA series monoclonal cell lines for susceptibility (HA titer) to influenza virus H3N2; Figure 3(c) shows the screening results of CA series monoclonal cell lines for susceptibility (HA titer) to influenza virus BY; Figure 3(d) shows the screening results of CA series monoclonal cell lines for susceptibility (HA titer) to influenza virus BV.

[0027] Figure 4 This figure shows the screening results of CB series monoclonal cell lines for the sensitivity (HA titer) of four influenza viruses (H1N1, H3N2, BY, BV).

[0028] Figure 5 Venn diagram showing the number of MDCK monoclonal cell lines in the CA and CB series that are sensitive to all four viruses.

[0029] Figure 6 This image shows the screening results of the MDCK monoclonal line colony formation test (the lines with a lower colony formation rate compared to the control group). Figure 7 This is an observational image showing the formation of clonal clusters in some MDCK monoclonal cell lines during a soft agar assay. Figure 8Images of tumors in nude mice, where: control mice were inoculated with MDCK-A cells on the left and MDCK-B cells on the right. Figure 9 The graphs show the tumor volume and mass of nude mice, where: A: tumor volume growth curve, B: final tumor volume, and C: final tumor weight.

[0030] Figure 10 For cell morphology observation.

[0031] Figure 11 The growth curves are for 5 cell lines.

[0032] Figure 12 This represents the maximum proliferation concentration for 5 cell lines.

[0033] Figure 13 The assay results are for isoenzyme detection, where: 1:MDCK-A, 2:CA005, 3:CA027, 4:CA078, 5:MDCK-B, 6:CB057, 7:CB082.

[0034] Figure 14 Chromosome karyotype diagram. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0036] Example: Screening and establishment of the non-tumorigenic MDCK cell line MDCK-CA027 1. Selection of MDCK monoclonal cells Single-cell suspensions were prepared using the MDCK-A progenitor cell line and diluted to 5 cells / mL using the limiting dilution method. 200 μL of the cell suspension was then seeded into 96-well plates using a pipette, and a total of 20 96-well plates were plated. After the cells adhered for 6 hours, they were observed and selected well by well using an optical microscope, and a total of 238 single-cell wells were selected. Similarly, the MDCK-B progenitor cell line was diluted to the same cell concentration and plated into 15 96-well plates for observation and selection, and a total of 154 single-cell wells were selected.

[0037] Observe the growth status and density of selected monoclonal cells daily, and promptly discard wells with poor growth, slow growth, or multiple clonal clusters. At 6 hours of culture, most MDCK monoclonal cells have just spread or are still round. These cells mostly adhere to the edge of the 96-well cell culture plate, increasing the difficulty of selecting monoclonal cell lines. Similarly, some cells have already divided at 6 hours of culture; because these cells have already divided or are in the process of dividing, different operators may make different judgments, so these cells are not labeled. As the culture time increases, some wells with two or more clonal clusters appear; these cells are promptly removed. Figure 1 As shown, newly adherent cells proliferate slowly, only beginning to divide after 48 hours of culture. Once cell division begins, cell proliferation gradually accelerates with increasing cell number. The fastest-proliferating cells have covered 40-80% of the surface area by 240 hours of culture, exhibiting uniform and consistent morphology, homogeneous transparency, and clear edges, suitable for further subculturing. Slower-growing cells continue to be cultured. Observations revealed three morphologies in the clonal cells: epithelial-like, island-like, and fibroblast-like. Furthermore, CB series monoclonal cells generally grow slower than CA series monoclonal cell lines.

[0038] 2. Large-scale culture of monoclonal cells Rapidly growing and healthy monoclonal cells were progressively expanded into 96-well cell culture plates, then 24-well cell culture plates, and finally T25 cell culture flasks. Monoclonal cells from the CA and CB series 96-well cell culture plates were digested and passaged using standard digestion methods, then transferred to 24-well cell culture plates. Cells were named CA001 and CB001 respectively based on passage order and cultured at 37°C in a 5% CO2 incubator. A total of 130 cells from the CA series were passaged and numbered up to CA130, and 121 cells from the CB series were passaged and numbered up to CB121.

[0039] Daily observation of the growth status and density of clonal cells in 24-well cell culture plates revealed uneven growth and poorer overall condition of CB series monoclonal cells compared to CA series cells. In the 24-well cell culture plates, some CB series monoclonal cells exhibited filamentous growth, poor cell condition, and significant black residue, and were discarded. Other cells gradually increased in size, becoming 2-3 times the size of normal cells, with blurred edges and a dull appearance, and were also discarded. Some cells grew extremely slowly, requiring medium changes every 2-3 days, reaching only 40% growth after 7 days. These cells were digested and expanded into 12-well cell culture plates, where further observation revealed worse cell growth, increasingly viscous culture medium, and generally larger cell volume, and were therefore discarded. A portion of CB series cells were uniform in size, with transparent cytoplasm and clear edges, and successfully covered the 24-well plates within one week of culture, allowing for further passage. In contrast, most CA series cells were in good condition, homogeneous and transparent, with only a small number exhibiting enlargement and poor condition. Once the cells have grown to 70% or more, they can be expanded into larger cell cultures. Finally, 107 cell lines were selected from the CA series and 61 cell lines were selected from the CB series, for a total of 168 monoclonal cell lines, for expansion culture in T25 cell culture flasks.

[0040] 3. Cell cryopreservation Observe the cell growth and state of T25 cells in the culture flasks daily, and promptly remove monoclonal cells in poor condition. Depending on the cell growth rate, the culture period is 2-7 days. Cryopreservation is carried out when the cell monolayer is dense. 95 cell lines were selected from the CA series and 50 from the CB series, for a total of 145 cell lines. Five vials of each cell line were cryopreserved, with a cryopreservation volume of 1 mL and a density of 50 × 10⁻⁶ cells / vial. 4 cells / mL -80×10 4 cells / mL. Among them, the passage number of CA series cells was P64, and the passage number of CB series cells was P65.

[0041] 4. Screening of MDCK monoclonal cell resuscitation efficacy Frozen MDCK monoclonal cell lines were revived using a medium-change method. Cell morphology and growth rate were observed daily, and clones in poor condition or with slow growth (≥5 days) were removed promptly. Results showed that, through revival screening, 79 monoclonal cell lines from the CA series and 29 monoclonal cell lines from the CB series exhibited good growth and condition. Further morphological classification of these 108 MDCK monoclonal cell lines revealed three different morphologies: 87 epithelial-like cells, 5 fibroblast-like cells, and 16 cells exhibiting island-like growth. Figure 2 ).

[0042] Example 2 5. Detection of viral susceptibility in MDCK monoclonal cells 5.1 Sensitivity screening of CA series monoclonal cell lines for four influenza viruses Influenza A virus (MOI=0.001) and influenza B virus (MOI=0.0001) were inoculated into MDCK monoclonal cell lines. After 48 hours of culture, the HA titer was measured. The results are shown in Figure 3. A total of 49 strains had H1N1 HA titers that were higher than or equal to the control group (MDCK-A), 46 strains had H3N2 HA titers that were higher than or equal to the control group (MDCK-A), 60 strains had BY HA titers that were higher than or equal to the control group (MDCK-A), and 49 strains had BV HA titers that were higher than or equal to the control group (MDCK-A).

[0043] 5.2 Screening of CB series monoclonal cell lines for influenza virus susceptibility The viral load was the same as under the CA series conditions, and the HA titer statistical results were as follows: Figure 4 The results showed that 23 plants had H1N1 HA titers that were higher than or equal to the control group (MDCK-B), 29 plants had H3N2 HA titers that were higher than or equal to the control group (MDCK-B), 17 plants had BYHA titers that were higher than or equal to the control group (MDCK-B), and 20 plants had BV HA titers that were higher than or equal to the control group (MDCK-B).

[0044] 5.3 Screening of MDCK monoclonal cells sensitive to four influenza viruses Veen plot statistics were performed on virus-sensitive cells of the CA and CB series. Figure 5 MDCK monoclonal cells sensitive to all four viruses were screened out. The results showed that 29 strains of the CA series and 15 strains of the CB series were sensitive to all four viruses.

[0045] 6. Cloning assay 6.1 Screening of inoculation density MDCK-A cells were diluted using the limiting dilution method and seeded into 6-well plates at seven densities: 30 cells / well, 50 cells / well, 100 cells / well, 200 cells / well, 500 cells / well, 1000 cells / well, and 2000 cells / well. Three wells were seeded at each density, with a culture volume of 3 mL. Cells were cultured for 7 days, and the experiment was repeated three times. The results (Table 1) showed that seeding at 100 cells / well resulted in a higher colony formation rate.

[0046] 6.2 Screening by MDCK monoclonal strain colony formation assay Based on viral susceptibility testing, a total of 44 MDCK monoclonal cell lines were screened and cultured at a density of 100 cells / well. The results were statistically analyzed after 7 days of culture. Figure 6 As shown, the results indicate that 13 CA series clones had a lower clone formation rate than the control group (MDCK-A), namely CA005, CA021, CA027, CA028, CA043, CA044, CA056, CA057, CA064, CA066, CA078, CA085, and CA103. Four CB series clones had a lower clone formation rate than the control group (MDCK-B), namely CB009, CB037, CB057, and CB082.

[0047] 7. Soft agar test 7.1 Screening for inoculation density HeLa cells, MDCK-A cells, MDCK-B cells, and KMB17 cells were diluted using the limiting dilution method and seeded into the upper gel at six densities: 500 cells / well, 1000 cells / well, 2000 cells / well, 5000 cells / well, 10000 cells / well, and 20000 cells / well. Each density was replicated in triplicate, and the cells were cultured for 21 days. The experiment was repeated three times. As shown in Table 2, the results indicate that seeding at 1000 cells / well resulted in a higher colony formation rate.

[0048] 7.2 Screening of MDCK monoclonal strains using soft agar test Thirteen CA series and four CB series monoclonal cell lines were seeded at 1000 cells / well and cultured for 21 days. The results were then analyzed. The results showed that the 13 selected CA series cell lines and CB009 and CB037 cells from the CB series all formed clonal clusters, and the soft agar results were positive. CB057 and CB082 cells from the CB series did not form clonal clusters, and the soft agar results were negative. Among them, CA043, CA066, and CA085 cell lines penetrated the lower gel layer, adhered to the bottom of the 6-well plate, and formed obvious cell proliferation colonies. The results are as follows: Figure 7 As shown.

[0049] 8. Tumorigenicity assay in nude mice Seventeen selected monoclonal cell lines, HeLa cells, MRC-5 cells, MDCK-A cells, and MDCK-B cells were expanded into larger cultures. During the expansion process, the MDCK CA056 monoclonal cells, which exhibited island-like growth, gradually slowed down and showed poor cell morphology. When passaged at a ratio of 1:4, the cells failed to achieve monolayer density and were in extremely poor condition within one week when expanded to two single-layer factories; therefore, they were not inoculated into nude mice. The remaining cell lines showed good growth and remained stable during continuous passage.

[0050] After cell inoculation, the activity of nude mice in each group was observed. The mice survived well for 16 weeks. Tumor formation was statistically analyzed. No tumors formed in the negative control group inoculated with MRC-5 cells, while 100% of the nude mice in the positive control group inoculated with HeLa cells developed tumors. This indicates that the nude mouse model was established successfully. The results are shown in Table 3. After injection, bulges appeared at the injection sites in all nude mice, which were absorbed within 2 weeks. During the observation period, some tumors in the cell control groups (MDCK-A and MDCK-B) regressed. At the end of the observation period, 8 and 6 mice, respectively, developed tumors that did not regress. In contrast, in the experimental groups, some nodules gradually decreased in size within one month of inoculation. By the end of the observation period, a varying number of mice in each group had nodules smaller than 5 mm. These nodules were smooth, uniform, and spherical. Figure 8 During the observation period, the tumorigenesis rate of the selected MDCK cell monoclonal cells was not higher than that of their respective original cell lines. Among them, the tumorigenesis rate of the five MDCK cell monoclonal cells CA005, CA027, CA078, CB057, and CB082 was 0%. At the end of the observation period, the weight and tumor volume of the nude mice were recorded, and they were euthanized by dislocation. After dissection, it was found that no metastatic tumors had formed in the organs (heart, liver, spleen, lung, and kidney) of the nude mice with tumors.

[0051] By comparing the growth curves of tumors in nude mice ( Figure 9 We found that the tumors formed by the selected cell lines after inoculation into nude mice were all relatively small during the 16-week culture period. At the end of the observation period, the tumor volumes of each experimental group differed from their respective original cell lines and were all smaller than those of the HeLa group. The final tumor volumes also showed significant differences among the selected cell lines. Among them, the final tumor volumes of 8 monoclonal cell lines (CA021, CA028, CA043, CA044, CA057, CA064, CA066, and CA103) were significantly different from those of the control group MDCK-A. p <0.05%, except for the group without tumor emergence, the remaining single-clonal cell line (CA085) showed no significant difference from the control group MDCK-A cells. p>0.05), CB009 and CB037 tumor cells from the CB series showed significant differences compared to the control group MDCK-B cells. p <0.05. Observation of the final tumor weight in nude mice revealed no significant difference between CA085 monoclonal cells of the CA series and the control group MDCK-A cells. p >0.05), and the tumor weight of the remaining tumor-bearing nude mice was significantly different from that of the control group. p <0.05), while CB009 and CB037 cells from the CB series, when injected into nude mice, formed tumors, and the final tumor weight was significantly different from that of the control group MDCK-B cells. p <0.05).

[0052] 9. Cell cryopreservation Five low-tumorigenic MDCK monoclonal cell lines (CA005, CA027, CA078, CB057, and CB082) were revived from their original liquid nitrogen cryopreservation tanks using a medium-change method. After 6-8 hours of cell adhesion, the medium was changed again, and cell status was continuously monitored. Once a dense monolayer of cells was formed, the cells were passaged at a 1:6 ratio for expansion. The cells were then transferred to six T225 cell culture flasks. After the monolayers became dense, they were digested using standard digestion methods, counted, and centrifuged. The cells were then resuspended in cryopreservation medium and the density was adjusted to 100 × 10⁶ cells / mL. 4 Cells / mL, cell viability 95%-98%, 1mL per tube, 50 tubes of each type frozen. CA series frozen passage number P67, CB series frozen passage number P68.

[0053] 10. Cell morphology and resuscitation viability detection The cryopreserved cells were thawed using a medium-change method, and the cell viability was as high as 92%, indicating that the cells maintained high viability before and after cryopreservation and were in good condition after thawing. Close observation of cell morphology revealed that all five cell lines were epithelial-like cells with regular morphology, exhibiting a "paving stone" appearance when the monolayer was dense. Figure 10 CA005 cells showed a dense monolayer after 48 hours of growth, with good cell condition, relatively uniform size, clear edges, and homogeneous transparency.

[0054] 11. Cell growth curve Growth curves were plotted for the five cell lines CA005, CA027, CA078, CB057, and CB082, as follows: Figure 11 As shown, all cells exhibit an "S" shape, and their cell growth density is lower than that of the original cells. By comparing their maximum proliferation density ( Figure 12 The study found that the five low-tumorigenicity MDCK monoclonal cell lines were all lower than their original cell lines, and the differences were significant.p <0.05). By comparing their specific growth rates (Table 4), a significant difference was found between the CA series and the original cell line (MDCK-A). p <0.05), there was no significant difference between CB057 and the original CB cell line (MDCK-B). p >0.05), CB082 was significantly different from the original CB cell line (MDCK-B). p <0.05. The PDT of the CA series low-tumorigenic MDCK monoclonal cell lines was significantly different from that of the original cell line (MDCK-A). p <0.05), while there was no significant difference among the CB series ( p >0.05 (Table 4).

[0055] 12. Detection of bacteria, fungi, mycoplasma, and exogenous contaminants. 12.1 Detection of bacteria and fungi During routine passage, no contamination was observed with the naked eye, and microscopic examination also showed no defects. The supernatant of the examined cells was added to THIO and TSB media for culture; after 14 days, both media remained clear and showed no contamination.

[0056] 12.2 Mycoplasma test Cell supernatant was collected after 48 hours of culture without medium change and poured onto mycoplasma liquid and solid culture media for culture. No mycoplasma growth was observed. After freeze-thaw treatment, the cells were centrifuged to collect the supernatant, which was then seeded onto Vero cells for culture and staining. Blue fluorescence was observed in the cell nuclei, but no fluorescence was observed in other parts.

[0057] 12.3 Detection of exogenous pollutants Direct observation under a microscope showed no change in cell morphology, and the cells examined before and after cryopreservation also exhibited normal morphology.

[0058] B. Blood Adsorption Virus Factor Test: The supernatant after freezing and thawing and centrifugation was inoculated into Vero and KMB17 cells. The cells were examined at 7, 14 and 21 days. No blood adsorption was observed, and the cell morphology was normal in the blood adsorption test.

[0059] C. Cytopathic Factor Test: The supernatant after centrifugation following freeze-thaw cycles was inoculated into Vero, MDCK, and KMB17 cells. After 21 days, no cytopathic effect was observed in the cells.

[0060] D. Direct fluorescent antibody assay for specific virus showed no fluorescence when the supernatant after freeze-thaw centrifugation was inoculated into primary bovine kidney cells, Vero cells, and MDCK cells.

[0061] 13. Isoenzyme assay Isoenzyme samples were collected from five low-tumorigenic MDCK monoclonal cell lines, and species contamination was detected by SDS-PAGE gel electrophoresis. Results are as follows: Figure 13 As shown, the five low-tumorigenic MDCK monoclonal cells migrated at the same distance as the original cell line, and the bands were relatively clear, indicating that there was no cross-contamination.

[0062] 14. Chromosome testing Add 30 μL of colchicine to well-grown, low-tumorigenic MDCK monoclonal cells in the logarithmic growth phase in a T75 flask and continue culturing for 6 hours. Prepare a cell suspension, slide, stain, and photograph the cells according to the above procedures. Figure 14 The figure shown is a chromosome observation diagram of CA005 cells. The chromosome results of the five cell lines are statistically summarized in Table 5.

Claims

1. A non-tumorigenic MDCK cell line, characterized in that: The non-tumorigenic MDCK cell strain is MDCK-CA027, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2023329.

2. The non-tumorigenic MDCK cell strain of claim 1 for use in production of influenza vaccine.

3. The non-tumorigenic MDCK cell strain of claim 1 for use in amplification of influenza virus.

4. A method for screening a non-tumorigenic MDCK cell line, characterized by, The method comprises the following steps: a. preparing a single cell suspension from a MDCK original cell strain; b. inoculating the single cell suspension into culture plates by limiting dilution method to obtain single clone cell wells; c. expanding the cells in the single clone cell wells; d. detecting the virus sensitivity of the expanded single clone cell strain to screen out a single clone cell strain sensitive to influenza virus; e. detecting the in vitro tumorigenicity of the single clone cell strain screened in step d, including clone formation test and / or soft agar test, to screen out a single clone cell strain with reduced tumorigenicity; f. performing a nude mouse in vivo tumorigenicity test on the single clone cell strain screened in step e to screen out a non-tumorigenic MDCK cell strain with a tumorigenic rate of 0%.

5. The method of claim 4, wherein: In step b, the cell concentration of the limiting dilution method is 5 cells / mL, and the inoculation volume is 200 μL / well in a 96-well plate; in step d, the influenza virus includes influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Yamagata lineage, and influenza B virus Victoria lineage.

6. The method of claim 4 or 5, wherein: In step e, the inoculation density of the clone formation test is 100 cells / well; the inoculation density of the soft agar test is 1000 cells / well; in step f, the observation period of the nude mouse in vivo tumorigenicity test is at least 16 weeks, and the tumor formation rate of 0% is used as the determination standard of non-tumorigenicity.

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