Non-tumorigenic MDCK cell line and its application

Abstract

The present disclosure discloses a non-tumorigenic MDCK cell strain and its application. The MDCK cells screened by the present disclosure can be used to culture and amplify influenza viruses under multi-scale conditions such as well plates and reactors, and the virus sensitivity and toxin production ability of the monoclonal cell strains are tested to screen out non-tumorigenic MDCK cell strains that are sensitive to influenza viruses and have high yields to improve yield and production efficiency. The screened non-tumorigenic MDCK monoclonal cell strain can be used as a cell matrix for influenza virus vaccine production, which has many advantages over traditional chicken embryo matrices, can solve the potential safety risks of cell strains from the source, eliminate product safety hazards, and improve product quality.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a non-tumorigenic MDCK cell line and application thereof. Background Art

[0002] For most countries, safe and effective inactivated and live attenuated vaccines have been the cornerstone of influenza prevention.

[0003] The influenza vaccine produced using chicken embryos has a long production cycle, and there may be a shortage of chicken embryos. When a large-scale outbreak of viral infection occurs, the vaccine supply cannot be met in time. Therefore, using mammalian cells instead of chicken embryos to culture influenza viruses will alleviate these problems.

[0004] MDCK cells are a continuous cell line isolated and cultured from the kidney tissue of healthy adult Coxswain female dogs. MDCK cells have a wide range of sensitivity to different types and subtypes of influenza virus strains and can effectively amplify the virus. They are currently the main cell line used for influenza virus isolation, culture and infectious titer detection, and are an important cell matrix for the development of cell culture-based influenza vaccines. However, the MDCK cell strains currently used in China have obvious tumorigenic characteristics, which brings great regulatory issues when used to produce virus vaccines. Summary of the invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a non-tumorigenic MDCK cell line and its application.

[0006] According to one aspect of the present disclosure, a cell line derived from Martin Darby canine kidney cells (MDCK) is provided, wherein the cell line is non-tumorigenic.

[0007] In some embodiments, after the cell line is inoculated into an animal, the inoculated animal does not develop nodules, or the nodules that appear disappear and can continue to disappear.

[0008] In some embodiments, the cell line is spindle-shaped with regular edges and an epithelial-like morphology.

[0009] In some embodiments, the cell line is sensitive to a virus.

[0010] In some embodiments, the cell line can be used to grow viruses.

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

[0012] In some embodiments, the influenza virus comprises influenza A virus, influenza B virus, influenza C virus, and / or influenza D virus.

[0013] In some embodiments, the influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N8.

[0014] In some embodiments, the influenza B virus comprises at least one of a BY influenza virus and a BV influenza virus.

[0015] In some embodiments, the cell line was deposited in the China General Microbiological Culture Collection Center on March 7, 2024, with the deposit number CGMCC NO:45824, and was classified and named MDCK monoclonal cell line.

[0016] According to another aspect of the present disclosure, there is provided a method for preparing a virus or a vaccine from the cell line described in the first aspect, the method comprising:

[0017] (a) infecting the cell line with a virus;

[0018] (b) culturing the infected cell line under conditions that allow the virus to propagate;

[0019] (c) isolating the virus from the cell line culture mixture.

[0020] In some embodiments, the method further comprises adding fresh culture medium to the cell culture or replacing part of the culture medium with fresh culture medium in step (b).

[0021] In some embodiments, step a) is performed at a cell density of more than 2×10 6 When the cell line is 10 cells / mL, the cell line is infected at an MOI of 0.0005 to 0.05, preferably at an MOI of 0.001 to 0.01.

[0022] In some embodiments, the culture medium in step b) includes one or more of the following: Eagle's medium, M199 medium, DMEM / F12 medium, IMDM medium, DMEM medium, H-DMEM medium, high-glucose DMEM medium or Knockout-DMEM medium.

[0023] In some embodiments, the Eagle's medium includes one or more of the following components: Eagle's basal medium, sodium bicarbonate, and / or glutamine.

[0024] In some embodiments, the culture medium further comprises serum.

[0025] In some embodiments, the serum is selected from one or more of the following: fetal bovine serum, calf serum, or newborn calf serum.

[0026] In some embodiments, the culture medium further comprises 5% to 20% serum.

[0027] In some embodiments, the culture medium further comprises 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% serum.

[0028] In some embodiments, the culture temperature is 30-37°C.

[0029] In some embodiments, the culturing temperature comprises 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C.

[0030] In some embodiments, the culture rotation speed is 50-80 rpm.

[0031] In some embodiments, the culture speed comprises 50rpm, 51rpm, 52rpm, 53rpm, 54rpm, 55rpm, 56rpm, 57rpm, 58rpm, 59rpm, 60rpm, 61rpm, 62rpm, 63rpm, 64rpm, 65rpm, 66rpm, 67rpm, 68rpm, 69rpm, 70rpm, 71rpm, 72rpm, 73rpm, 74rpm, 75rpm, 76rpm, 77rpm, 78rpm, 79rpm or 80rpm.

[0032] In some embodiments, the culturing time is 48 hours to 72 hours.

[0033] In some embodiments, the culturing time comprises 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h, 61h, 62h, 63h, 64h, 65h, 66h, 67h, 68h, 69h, 70h, 71h or 72h.

[0034] In some embodiments, the pH value of the culture is comprised between 6.5 and 8.5.

[0035] In some embodiments, the pH of the culture comprises 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0036] In some embodiments, the culture comprises 20% to 60% dissolved oxygen.

[0037] In some embodiments, the dissolved oxygen in 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%.

[0038] In some embodiments, the cell line can prepare viruses under various culture conditions such as flat plates, well plates, shake flasks, square bottles, microcarriers, bioreactors, cell factories, etc.

[0039] In some embodiments, the cell lines include cell lines within 200 generations.

[0040] In some embodiments, the cell lines include cell lines within 200 generations, within 190 generations, within 180 generations, within 170 generations, within 160 generations, within 150 generations, within 140 generations, within 130 generations, within 120 generations, within 110 generations, or within 100 generations.

[0041] According to another aspect of the present disclosure, there is provided a method for obtaining the cell line according to the first aspect, the method comprising:

[0042] (a) dividing N Martin Darby canine kidney cells into M culture chambers for culture, so that there is at most one cell in each culture chamber, wherein N≤M;

[0043] (b) One cell in each culture chamber is cultured as a monoclonal cell population;

[0044] (c) injecting the monoclonal cell populations into animals respectively, and screening out the monoclonal cell populations that can cause nodule regression in a shorter time and maintain nodule regression continuously as the cell lines.

[0045] In some embodiments, the animal comprises a mammal, such as a mouse, rat, horse, rabbit, sheep, pig, cow, or monkey.

[0046] According to yet another aspect of the present disclosure, provided is a virus or vaccine produced by the cell line of the present disclosure.

[0047] According to another aspect of the present disclosure, provided is the use of the cell strain in virus production.

[0048] In some embodiments, the virus production includes virus propagation culture and / or vaccine production.

[0049] In some embodiments, the cell line is non-tumorigenic.

[0050] In some embodiments, after the cell line is inoculated into an animal, the inoculated animal does not develop nodules, or the nodules that appear disappear and can continue to disappear.

[0051] In some embodiments, the virus comprises one or more of the following: influenza virus, measles virus, Japanese encephalitis virus, mumps virus, rubella virus, poliovirus, herpes simplex virus-1, herpes simplex virus-2, rabies virus, respiratory syncytial virus, respiratory enterovirus type 3, yellow fever virus, parvovirus, coxsackievirus, adenovirus types 1 to 47, Lassa virus, and vaccinia virus.

[0052] In some embodiments, the virus is an influenza virus.

[0053] In some embodiments, the influenza virus comprises influenza A virus, influenza B virus, influenza C virus, and / or influenza D virus.

[0054] In some embodiments, the influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N8.

[0055] In some embodiments, the influenza B virus comprises at least one of a BY influenza virus and a BV influenza virus.

[0056] Beneficial effects of the present disclosure:

[0057] The non-tumorigenic / monoclonal MDCK cells screened in the present disclosure are sensitive to viruses and can efficiently produce viruses or vaccines under multi-scale conditions such as well plates, square bottles, and reactors, which can solve the potential safety risks of cell lines from the source, eliminate product safety hazards, and improve product quality. It can also improve yield and production efficiency.

[0058] The present invention discloses the use of cell matrix to replace the traditional chicken embryo matrix to develop influenza vaccine. The non-tumorigenic MDCK monoclonal cell strain screened is used as the cell matrix for the production of influenza virus vaccine, which has many advantages over the traditional chicken embryo matrix: the cell matrix influenza adopts the reactor microcarrier suspension culture process, which has a more stable process technology and is easy to scale up compared with chicken embryo culture; the raw materials used have stricter exogenous control and higher safety. MDCK cells are mammalian cells, which are more suitable for replicating viruses in the human body, and the probability of adaptive mutation in the passage is low; at the same time, they have higher flexibility in responding to influenza pandemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The MDCK-4D10 monoclonal screening process is shown.

[0060] Figure 2 The monoclonal toxin production ability test process is shown.

[0061] Figure 3 The results of monoclonal toxigenic capacity are shown.

[0062] Figure 4 Shown are the nodule regression times of MDCK-4D10 cell tumorigenic animals.

[0063] Figure 5 Pathological sections of MDCK-4D10 cell tumorigenic animals are shown.

[0064] Figure 6 The results of karyotyping of MDCK-4D10 monoclonal cells are shown.

[0065] Figure 7 A photograph of the MDCK-4D10 cell line is shown.

[0066] Figure 8 MDCK-4D10 cell growth curve is shown.

[0067] Fig. 9 The figure shows the process of establishing the master cell bank of MDCK-4D10 cell line.

[0068] Fig.10 The design process of reactor culture MDCK-4D10 cell line toxin production is shown.

[0069] Fig.11 The MDCK-4D10 cell reactor culture and inoculation growth curve are shown. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0071] As an example of a virus, influenza has a high incidence rate, spreads quickly, and causes serious harm. Pregnant women, infants, the elderly, patients with chronic underlying diseases, and immunosuppression are susceptible to influenza and have a higher risk of severe illness and death after contracting influenza. The increased use of medical resources and loss of productivity caused by influenza each year will lead to serious public health and socioeconomic problems.

[0072] At present, the traditional chicken embryo process is mainly used to produce influenza vaccines in China. Chicken embryo matrix influenza vaccines have the following problems: the demand for chicken embryos is large, which consumes manpower and material resources, the cost of handling the debris is high, and it pollutes the environment; the chicken embryo culture is semi-open, and it is impossible to achieve completely sterile production, which has potential contamination; the virus may have adaptive mutations during chicken embryo passage, reducing the effectiveness of the vaccine; the chicken embryo allantoic fluid has many impurities, which is not conducive to the subsequent purification process; in special periods, the source and quantity of chicken embryos cannot be guaranteed.

[0073] All the cell influenza cell lines that have been marketed overseas are MDCK cells, but MDCK cells themselves are tumorigenic. There are two solutions to the tumorigenicity of MDCK cells abroad: ① Screening MDCK non-tumorigenic cell lines for cell influenza vaccine process research ② Removing intact cells, host DNA, proteins, etc. in the downstream process. Although the probability of intact MDCK cells existing after the purification process is extremely low, it is impossible to eliminate the tumorigenicity of MDCK cells from the source. At present, no vaccine products using MDCK cell lines have been marketed in my country.

[0074] The present disclosure provides a cell line derived from Martin Darby canine kidney cells, characterized in that, compared with the Martin Darby canine kidney passage cell line, the cell line is non-tumorigenic and can be used to culture viruses, such as influenza viruses.

[0075] In some embodiments, after the cell line is inoculated into an animal, the inoculated animal does not develop nodules, or the nodules that develop disappear and can continue to disappear, for example, the nodules disappear within 4 to 16 weeks and can continue to disappear.

[0076] In some embodiments, the more times the cell line is passaged, the longer it takes for the nodules to disappear.

[0077] In some embodiments, the cell line is spindle-shaped with regular edges and an epithelial-like morphology, and can form a single layer of cells, multiple layers of cells, or floating cells.

[0078] In some embodiments, the cell line is susceptible to influenza virus.

[0079] In some embodiments, the cell line is species-identified as canine.

[0080] In some embodiments, the cell line is free of bacterial and fungal contamination.

[0081] In some embodiments, the cell line is free of mycobacterial growth.

[0082] In some embodiments, the cell line is free of mycoplasma contamination.

[0083] In some embodiments, the cell line tests negative for retrovirus, species-specific exogenous factors, bovine, porcine and other specific exogenous viruses.

[0084] As an example, the method for preparing influenza virus in the cell line includes:

[0085] (a) infecting the cell line with influenza virus at an MOI of 0.001 to 0.01;

[0086] (b) culturing the infected cell line under conditions that allow the virus to grow, such as 34°C, 50-80 rpm, 5% CO 2， About 48h~72h;

[0087] (c) isolating the virus from the cell line culture mixture.

[0088] As an example, a method for screening a non-tumorigenic MDCK cell line for amplifying influenza virus comprises the following steps:

[0089] (1) Monoclonal screening by limiting dilution method: After ATCC MDCK (NBL-2) cells are subcultured and stored, monoclonal screening is performed by limiting dilution method. The number of cells is set to 50 when plating each 96-well plate, that is, the number of cells per well is about 0.5. Single cell inspection after plating can avoid inaccurate observation after cell sedimentation. Mark the single well with a cell number of 1 and perform fluid replacement or refilling in time.

[0090] (2) Screening of non-tumorigenic cell lines and library construction: After observing the single-well cells obtained in step (1) for one week, it can be found that there are significant differences in cell growth. Some single cells undergo apoptosis, while others proliferate, but the speed varies and the morphology is also different. The surviving single-well cells are amplified step by step. When the cells are expanded to a certain scale, the cells are frozen. Inject into SPF nude mice aged 4-7 weeks to screen out cell lines with nodules disappearing.

[0091] (3) High-passage tumorigenicity test of non-tumorigenic cell lines: The cells whose nodules disappeared during the observation period were serially passaged, and the same dose was injected into 4-7 week-old nude mice every 5-10 generations for retesting. It was found that the nodule disappearance time of low-passage cells was prolonged, and some cells did not disappear during the observation period. Among them, MDCK-4D10 cells underwent multiple tumorigenicity tests, and the nodules disappeared during the observation period.

[0092] In some embodiments, the present disclosure provides the use of a non-tumorigenic MDCK cell line for amplifying influenza virus.

[0093] In a specific embodiment, the non-tumorigenic MDCK cell line disclosed herein can be used to amplify type A and type B cell matrix influenza viruses, including H1N1 and BY influenza viruses.

[0094] The present disclosure screens for non-tumorigenic MDCK cell lines derived from ATCC MDCK (NBL-2). MDCK (NBL-2) cells can first be passaged a limited number of times in serum-containing medium (Eagle's + 10% newborn calf serum (NBS) + 2% of 7.5% sodium bicarbonate + 1% glutamine).

[0095] The cell strain screened by the present disclosure can solve the potential safety risks of the cell strain from the source, eliminate the potential safety hazards of the product, and improve the product quality. It can be used in the preparation of influenza virus vaccines as needed. Specifically, it can be used in small-scale culture of cell square bottles or in bioreactor microcarrier culture.

[0096] definition

[0097] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0098] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0099] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0100] In the present disclosure, the term "cell monoclone" refers to the process of culturing a cell and causing it to divide continuously to form a cell group. This group of cells originates from a common ancestral cell. Cell monoclonal screening is a technology that separates single cells from mixed cell samples based on the characteristics of the cells. It is a very important part of cell line optimization. The cell monoclonal screening technology can be used to establish MDCK monoclonal cell lines with low tumorigenicity, which can be used efficiently and stably for the proliferation of influenza viruses.

[0101] In the present disclosure, the term "influenza virus" belongs to the family of Orthomyxoviridae and is an enveloped virus. Its genome consists of segmented negative-sense single-stranded RNA. The influenza virus envelope consists of a lipid bilayer, in which influenza A virus contains three viral envelope proteins: HA, NA and M2. The glycoprotein structures of HA and NA protrude from the lipid membrane derived from the host cell. A smaller number of matrix (M2) ion channels pass through the lipid envelope. The envelope and its three integral membrane proteins HA, NA and M2 cover the M1 protein matrix, which surrounds the nuclear export protein (also known as non-structural protein 2, NS2) and ribonucleoprotein (RNP) complex in the virus particle. The ribonucleoprotein complex consists of nucleoprotein (NP) encapsulating viral RNA fragments and a heterotrimeric RNA-dependent RNA polymerase, which consists of two "polymerase basic" and one "polymerase acidic" subunits (PB1, PB2 and PA).

[0102] The influenza viruses discovered so far are divided into four types: A, B, C, and D. Among them, influenza A and B can cause more serious infections in humans. At present, people name and distinguish different strains in pandemics by the types of surface antigens. Influenza A viruses are divided into multiple subtypes based on the combination of hemagglutinin (H) and neuraminidase (N) proteins expressed. There are currently 18 different hemagglutinin (H) subtypes and 11 different neuraminidase (N) subtypes (H1-18 and N1-11). More than 130 combinations of influenza A subtypes have been identified in nature (mainly from wild birds), but given the tendency of viruses to "reassort", there may be more combinations of influenza A subtypes. Reassortment is the process by which influenza viruses exchange gene fragments. Reassortment occurs when two influenza viruses infect a host at the same time and exchange genetic information. In some embodiments, the influenza A virus comprises at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8. In some embodiments, the influenza B virus comprises at least one of BY influenza virus and BV influenza virus.

[0103] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0104] Example

[0105] Example 1. Screening, identification and virus sensitivity testing of non-tumorigenic MDCK cell lines

[0106] Operation process see Figure 1 , specifically including the following steps:

[0107] (1) Cell digestion and counting

[0108] ATCC MDCK (NBL-2) P55 was passaged a limited number of times in Eagle's medium containing 10% newborn calf serum (NBS) (Minhai) to a frozen cell bank of P63, revived in T-75 (P64), and grown for two days until the cells covered the bottom of the square flask.

[0109] Discard the cell culture medium in the T-75 flask, wash the cell surface thoroughly 3 times with 0.01M PBS solution, add 3mL 0.25% trypsin (Gibco), incubate in a CO2 incubator, take out after 10-15min and observe the cells under a microscope to see if they have become round, add 9mL culture medium to stop digestion, mix the cell suspension, and take 100uL for cell counting.

[0110] (2) Adjustment of cell density and plating

[0111] In order to ensure that there are as many monoclonal cells as possible in the 96-well cell plate, the total number of cells should be reduced when plating. The total number of cells in each 96-well plate is 50, and 100uL of Eagle's medium containing 15% fetal bovine serum FBS (Gibco) is added to each well, that is, the cell density is 50 cells / (100uL*96 wells)≈5 cells / mL. The plated cell plate is observed for single cells under a microscope to avoid the inability to observe the number of cells in the cell plate wells after cell sedimentation, which makes it impossible to determine whether the cells amplified later are from monoclonal. For single cell wells, mark them and place them in a CO2 incubator for culture. On the second day, add 100μL of liquid to a total volume of 200μL / well, and change 1 / 2 of the liquid every 2 to 3 days. When the monoclonal clone forms obvious clumps, the liquid change time can be appropriately shortened to prevent the volatilization of the culture medium and ensure the normal growth of the cells. The specific judgment needs to be made according to the cell growth situation.

[0112] (3) Cryopreservation of monoclonal cells

[0113] Observe the laid cell plates regularly. After about a week, the cell growth status between different single wells will be different, some will undergo apoptosis, and some will grow more. When the confluence rate of most surviving monoclonal cells reaches more than 50%, they are digested and transferred to 24-well cell plates, and 1mL of Eagle's medium containing 10% newborn bovine serum (NBS) is added and cultured in a CO2 incubator. The remaining slower growing cells continue to be cultured in 96-well cell plates. When the confluence of monoclonal cells reaches more than 80%, they are transferred to 6-well cell plates, T-25 square bottles and T-75 square bottles in turn. When the cells in the T-75 square bottle are fully spread, they are frozen for seed preservation. The formula of cell freezing solution is DMSO (Sigma): FBS: Eagle's medium = 1:3:6.

[0114] (4) Screening of monoclonal cell toxin production ability and virus sensitivity testing

[0115] according to Figure 2 The process shown was used to examine the toxin-producing abilities of four monoclonal cell lines: 1B1, 1C10, 4D10, and 4G9.

[0116] Simply put, use hemostatic forceps to take out the cryopreserved cell tube from the liquid nitrogen tank, confirm that there are no cracks on the surface and the outer packaging is intact, then quickly put it in a 37°C water bath and shake the cryopreserved tube until the ice crystals melt. Spray alcohol to disinfect the outer surface of the cryopreserved tube and move it to a safety cabinet for operation. Transfer the cloned cells in the tube to a centrifuge tube containing 9.0mL complete culture medium and centrifuge at 125×g for 5 minutes. Discard the supernatant, resuspend the cells in a T25 bottle with culture medium, and place it in a CO2 incubator at 36.5°C to culture until a single layer.

[0117] Pour out the cell growth medium in the T25 flask, take 10mL PBS to wash the cell surface 2 to 3 times, and then 2 0.25% trypsin (Gibco) was added and the cells were placed in a 36.5°C incubator for digestion. 3 mL of Eagle's medium containing 10% newborn calf serum was added to terminate the digestion and samples were taken for counting. Each cloned cell was plated in 3 wells of a 6-well plate, approximately 2×10 5 Cells / well, 2 strains of cells per 6-well plate. The remaining cells of each strain on the 6-well plate were transferred to T25 for the next inoculation. The initial ATCC MDCK (NBL-2) cells were also plated on a 6-well plate as a positive control. Each strain of cells was cultured to a monolayer at a temperature of 36.5°C ± 0.5°C and 5% CO2.

[0118] Form a monolayer of 3-well cells, wash the cells twice with PBS, add 4 mL of Eagle's medium without serum, inoculate two wells with virus (the amount of virus inoculated is 1 / 10000 of the culture system, such as 4 mL in a single well of a 6-well plate, that is, 0.4 μL of virus inoculation), and the remaining well is used as a control (no virus inoculation but an equal amount of TPCK trypsin). The culture conditions are 34°C and 5% CO2. After the cells are basically completely diseased, the virus is harvested, packaged, stored in an ultra-low temperature refrigerator, and the hemagglutination titer is measured. The results are as follows Figure 3 shown.

[0119] The results showed that the four MDCK monoclonal cell strains 1B1, 1C10, 4D10, and 4G9 were all sensitive to BY, H1N1, and BV influenza virus strains, among which the MDCK-4D10 monoclonal cell strain had the highest hemagglutination titer and was the most sensitive to influenza virus strains.

[0120] (4) Monoclonal cell tumorigenicity assay

[0121] The injection test was performed using SPF nude mice aged 4-7 weeks. The digested MDCK cells were counted and injected into the nude mice within half an hour. The injection dose was 1×10 7 cells / 200uL / mouse, and regularly observe the nude mice injected with MDCK monoclonal cells to observe the nodules at the injection site.

[0122] The observation period was set at 16 weeks, and the size of the nodules was measured regularly with a vernier caliper according to the requirements of the pharmacopoeia. Some nodules formed by cells can disappear in a short period of time, some nodules formed by cells need a longer time to disappear, and some nodules formed by cells have not disappeared at the end of the observation period. It was found that the nodules at the injection site of SPF-grade 4-7-week-old nude mice injected with MDCK-4D10 cells disappeared in a shorter time during the observation period and the nodules could continue to disappear.

[0123] (5) Tumorigenicity test of MDCK-4D10 monoclonal cell line in serial culture

[0124] The cells whose nodules disappeared during the observation period were serially passaged to P111, and the number of cells per 10-cell cycle was calculated every 5-10 generations. 7 The injection volume of 10 cells / 200uL / mouse was re-injected into 10 4-7 week old nude mice for re-testing. It was found that after the low-generation cells were inoculated into nude mice, the time required for the nodules at the injection site to disappear was prolonged, and some cells failed to disappear during the observation period. The MDCK-4D10 cell line was tested for tumorigenicity at multiple generations, and the nodules at the injection site of SPF-grade 4-7 week old nude mice all disappeared during the observation period. The disappearance time of the nodules at the injection site after different generations of MDCK-4D10 monoclonal cell lines were inoculated into SPF-grade 4-7 week old nude mice was shown in Table 1. Figure 4 .

[0125] Ten SPF nude mice aged 4-7 weeks were inoculated with 111th generation MDCK-4D10 cells. After 16 weeks, the nude mice were dissected and the brain, heart, liver, kidney, spleen, lung and axillary lymph nodes were taken for pathological examination. The samples were placed in fixative for 7 days and then trimmed. After dehydration, transparency, wax immersion, embedding, sectioning and HE staining, tissue slides were prepared. Microscopic observation and analysis confirmed that all samples had no tumor lesions. Pathological examination sections showed Figure 5 .

[0126] (6) Karyotype analysis of MDCK-4D10 monoclonal cells

[0127] The MDCK-4D10 monoclonal cell line was passaged normally to three T25 cell bottles, and the experiment was carried out when the confluence was about 80%-90%. The cells were placed in metaphase by the action of colchicine, and the metaphase cells were harvested and fixed by hypotonic fixation, slide preparation, staining, and sealing. The cells with clear chromosomes and concentrated distribution were selected for oil microscope observation, counting, and karyotype analysis. MDCK (NBL-2) cells were used as controls, and a total of 100 cells were counted. The results showed that the proportion of 78 chromosomes in the control cell MDCK (NBL-2) was 5%, and the distribution proportion of chromosomes was 78±2 was 28%. The proportion of 78 chromosomes in MDCK-4D10 was 28%, and the distribution proportion of chromosomes within 78±2 was 89%. The results of karyotype analysis are shown in Table 1. Figure 6 .

[0128] (7) Storage of MDCK-4D10 monoclonal cell line

[0129] The cell was named MDCK-4D10, and the recommended classification name was: MDCK monoclonal cell line. It was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on March 7, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is CGMCC NO: 45824.

[0130] Example 2: Observation and culture of MDCK-4D10 monoclonal cells

[0131] (1) MDCK-4D10 cells have an epithelial-like morphology

[0132] Observe the cultured MDCK-4D10 cells under a MDCK-4D10 microscope: the cells are spindle-shaped with regular edges, adherent cells, and have an epithelial-like morphology, such as Figure 7 Observation of MDCK cells at different times revealed that MDCK cells grew faster after attaching to the culture flask. At 24h-48h, the cells were spindle-shaped with clear and translucent boundaries. At 72h, a compact cell layer was formed with small cells and clear boundaries. At 96h, multiple layers of cells could be observed, with a small number of floating cells.

[0133] (2) MDCK-4D10 has a higher growth density

[0134] After the MDCK-4D10 cells were completely digested, they were dispersed with Eagle's medium containing 10% newborn calf serum at a dispersion ratio of 1:6. The cell suspension was divided into 6 bottles with 10mL / T25 square bottles, and each cell was divided into 6 bottles. The cells were cultured at 36.5℃ with aeration, and samples were taken for counting. Observation: Microscopic observation was performed at 24h, 48h, 72h, 96h, and 120h after culture. After the sample culture medium was poured out after microscopic observation, it was washed twice with 0.01mol / LPBS solution, and the residual liquid was sucked dry with a pipette, and 1mL of preheated 0.25% trypsin solution was added. The cells were digested at 36.5℃ for 15min, and the cells were dispersed with 4mL 0.01mol / LPBS for counting.

[0135] The cell numbers of MDCK-4D10 cells inoculated with the same starting density at 0h, 24h, 48h, 72h, 96h, and 120h were 6×10 5 cells, 11.8×10 5 cells, 35×10 5 cells, 42.8×10 5 cells, 45.3×10 5 cells, 40.6×10 5 Cells. The growth curve of MDCK-4D10 cells is shown in Figure 8 .

[0136] Example 3: Establishment and verification of MDCK-4D10 master cell bank

[0137] Establish a master cell bank for adherent MDCK-4D10 cell line. The operation procedure is shown in Fig. 9 , the cell bank is tested in accordance with the requirements of the third volume of the 2020 edition of the "Pharmacopoeia of the People's Republic of China", including cell identification tests, bacterial and fungal sterility tests, mycobacteria tests, mycoplasma tests, intracellular and exogenous viral factor tests, tumorigenicity tests, and tumorigenicity tests.

[0138] Cell identification test: MDCK-4D10 cells and supernatant were prepared and the species and exclusive characteristics of MDCK-4D10 cells were identified by cell morphology observation and species identification (isoenzyme method). The results showed that the cells grew in an epithelial manner and the species was identified as canine.

[0139] Sterility test of bacteria / fungi: According to the requirements of General Chapter 1101 Sterility Test Method of the Chinese Pharmacopoeia, the sterility test of bacteria and fungi was carried out. 10 mL of mixed cell supernatant of MDCK-4D10 cell culture was prepared, and the cells were subjected to sterility test of bacteria / fungi by membrane filtration. The results showed that the cells were free of bacterial and fungal contamination.

[0140] Mycobacterium test: Mycobacterium test was carried out according to the requirements of General Chapter 1101 Sterility Test Method of Chinese Pharmacopoeia. 7 The cell lysate was prepared from the culture supernatant of MDCK-4D10 cells and inoculated into a suitable solid culture medium in triplicate. The cells were cultured at 37°C for 56 days and then mycobacteria were examined. The results showed that no mycobacteria grew in the cells.

[0141] Mycoplasma test: MDCK-4D10 cell supernatant was prepared and tested for mycoplasma using culture method and indicator cell culture method according to General Chapter 3301 of the Chinese Pharmacopoeia. The results showed that there was no mycoplasma contamination in the direct inoculation method and the indicator cell culture method was negative.

[0142] Method for testing exogenous viral factors in cells: Prepare MDCK-4D10 cell-related samples (square bottles, live cells, cell supernatants, cell lysates) according to the requirements of the pharmacopoeia for testing exogenous viral factors in cells. The detection of exogenous factors in cells includes two detection methods: in vitro and in vivo. The in vitro method includes cell morphology observation and red blood cell hemagglutination test, and in vitro different indicator cell methods (Vero, human diploid, MDCK). The results show that the morphology of MDCK-4D10 cells is normal, the red blood cell hemagglutination test is negative, and the different indicator cell passage culture inspection (red blood cell adsorption test) and direct cell culture direct observation method (red blood cell agglutination test) are all negative; the in vivo method includes animal inoculation method (suckling mice, adult mice) and chicken embryo (chicken embryos of different days of age) inoculation method. The results show that all suckling mice and adult mice survived, and all survived after inoculation of chicken embryos of different days of age, and the allantoic fluid red blood cell agglutination test was negative. At the same time, the detection of retroviruses, species-specific exogenous factors, bovine, porcine and other specific exogenous viruses was tested, and the test results were all negative.

[0143] Tumorigenicity test: MDCK-4D10 cell lysate and DNA were prepared and inoculated into newborn nude mice, hamsters and rats by in vivo inoculation method for tumorigenicity test. The results showed that no nodules appeared after MDCK-4D10 monoclonal cells were inoculated into newborn nude mice, hamsters and rats, and the tumorigenicity results were negative.

[0144] Tumorigenicity test: prepare no less than 2 mL of 5×10 7 MDCK-4D10 cells / mL were inoculated into 4-7 week old SPF nude mice for cell tumorigenicity test. The results showed that after nude mice were inoculated with MDCK-4D10 monoclonal cells, the nodules at the inoculation site disappeared within 4 weeks, and the tumorigenicity results were negative.

[0145] Example 4: Process feasibility test of MDCK-4D10 monoclonal cell reactor

[0146] (1) Cell recovery and subculture

[0147] MDCK-4D10 cells were revived and cultured in T75 cell culture flasks at 36.5°C ± 0.5°C and 5% CO2. The required cell amount was expanded to one CF5 and one CF10 cell factory for 2L bioreactor microcarrier cell culture after three consecutive passages.

[0148] (2) Cell culture using bioreactors

[0149] Digested cell factory cells, 3 × 10 5The cells were inoculated into a 2L bioreactor at a cell density of 100 / mL and a 4g / L Cytodex-1 microcarrier, with a culture volume of 1.5L. The conditions of the bioreactor cell culture phase were 37°C, a rotation speed of 50-80rpm, a pH value of 7.2, and a dissolved oxygen setting of 40%. Samples were taken and counted daily, and biochemical indicators were tested. When the glucose content was lower than 1 / 2 of the initial sugar content, half of the Eagle's medium containing 10% newborn calf serum (NBS) was replaced.

[0150] (3) Bioreactor inoculation and harvesting (BY and H1N1)

[0151] When the cell density is not less than 2×10 6 / mL, wash the cells twice with PBS, wash once with serum-free cell maintenance medium, replace the culture medium with serum-free Eagle's medium, count again and keep 100mL of sample in a stirring bottle as a cell control. Add 1mg / mL of TPCK trypsin stock solution to make the final concentration of TPCK trypsin in the culture system to 2mg / L. After stirring and mixing, take out 100mL of the suspension and culture it in a stirring bottle as a control for uninfected cells. The cells in the reactor are inoculated at MOI=0.001~0.01, and the culture conditions are 34℃, 50~80rpm, and 5% CO2. Take samples and count them every day, keep samples, and harvest according to the degree of cell pathology, about 48h~72h. For specific operations and experimental design, see the process Fig.10 In addition, during the second passage stage, a T75 was amplified and used for BY virus culture (the cells used for BY infection can be used as backup cells for H1N1 infection. Before BY infection, another batch of cells was amplified and used as backup cells for BY infection).

[0152] (4) Cell growth curve and toxicity results

[0153] According to the cell density at different times of the reactor cell culture stage and the cell density at the inoculation stage when the adherent MDCK-4D10 cells were inoculated with H1N1 (A / Delaware / 55 / 2019, CDC USA) and BY strain (B / Singapore / INFTT-16-0610 / 2016, VIDRL Australia), the cell growth curve was drawn. For details, see Fig.11 According to the cell growth curve, after the adherent MDCK-4D10 cells were inoculated into the bioreactor, the cells gradually grew to a cell density higher than 2×10 6 The cells were inoculated with H1N1 and BY strains at an MOI of 0.001 to 0.01. The cell number increased with time after inoculation, remained stable at first and then gradually decreased. The cells were harvested at 48 to 72 hours.

[0154] The results of virus production in the MDCK-4D10 reactor: After the adherent MDCK-4D10 cell line was inoculated with the 2022-2023 WHO-recommended northern hemisphere cell matrix influenza H1N1 and BY strains, the virus fluid samples were harvested and sent for testing of hemagglutination titer, hemagglutinin content, and virus titer. The results showed that MDCK-4D10 cells were sensitive to H1N1 and BY strains, and the feasibility of the pilot process of using the adherent MDCK-4D10 cell line for Cytodex-1 microcarriers to amplify influenza viruses in a 2L reactor was preliminarily verified. The test results are shown in Table 1.

[0155] Table 1. Test results of virus fluid samples harvested from inoculation of H1N1 and BY strains

[0156]

[0157] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An MDCK cell line, characterized in that The cell line is non-tumorigenic; the cell line was deposited in the China General Microbiological Culture Collection Center on March 7, 2024, with the deposit number CGMCC NO:45824.

2. The cell line according to claim 1, characterized in that After the cell line is inoculated into an animal, no nodules appear in the inoculated animal, or nodules appear and then disappear, and the state of nodule disappearance can be maintained continuously.

3. The cell line according to claim 1, characterized in that The cell line is sensitive to influenza virus.

4. The cell line according to claim 1, characterized in that The cell line can be used to culture influenza virus.

5. The cell line according to claim 3 or 4, characterized in that The influenza virus includes influenza A virus, influenza B virus, influenza C virus and / or influenza D virus.

6. The cell line according to claim 5, characterized in that The influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8.

7. The cell line according to claim 5, characterized in that The influenza B virus includes at least one of a BY influenza virus and a BV influenza virus.

8. A method for preparing influenza virus using the cell line according to any one of claims 1 to 7.

9. The method according to claim 8, characterized in that The method comprises: (a) infecting the cell line with influenza virus; (b) culturing the infected cell line under conditions that allow virus propagation; (c) isolating the virus from the cell line culture mixture.

10. The method according to claim 9, characterized in that In the step b), one or more culture media selected from the group consisting of Eagle's culture medium, M199 culture medium, DMEM / F12 culture medium, IMDM culture medium, DMEM culture medium, H-DMEM culture medium, high-glucose DMEM culture medium or Knockout-DMEM culture medium are used.

11. The method according to claim 10, characterized in that The culture medium also includes serum.

12. The method according to claim 10, characterized in that The culture medium also includes 5% to 20% serum.

13. The method according to claim 9, characterized in that The culture temperature is 30-37°C.

14. The method according to claim 9, characterized in that The culture temperature was 34°C.

15. The method according to claim 9, characterized in that The pH value of the culture is 6.5 to 8.

5.

16. The method according to claim 9, characterized in that The pH of the culture was 7.

2.

17. The method according to claim 9, characterized in that The dissolved oxygen in the culture is 20% to 60%.

18. The method according to claim 9, characterized in that The dissolved oxygen in the culture was 40%.

19. Use of the cell line according to any one of claims 1 to 7 in the production of influenza virus.

20. The use according to claim 19, characterized in that The influenza virus production includes virus proliferation culture and / or vaccine production.

21. The use according to claim 19, characterized in that The influenza virus includes influenza A virus, influenza B virus, influenza C virus and / or influenza D virus.

22. The use according to claim 21, characterized in that The influenza A virus includes at least one of H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8.

23. The use according to claim 21, characterized in that The influenza B virus includes at least one of a BY influenza virus and a BV influenza virus.

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