Serum-free full-suspension MDCK (Madin Darby Canine Kidney) cell and application thereof in preparation of influenza vaccine

Abstract

The invention discloses a serum-free full-suspension MDCK (Madin-Darby Canine Kidney) cell and application thereof in preparation of influenza vaccines, and belongs to the field of virology and biological medicines. The serum-free full-suspension MDCK cell is preserved in the China Center for Type Culture Collection, is classified and named as a canine kidney cell MDCK-JHS, and has a preservation number of CCTCC NO: C2024373. The invention further provides a method for culturing the influenza virus by using the cell line, high-titer amplification of the influenza virus can be realized, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the fields of virology and biopharmaceuticals, and particularly relates to a serum-free fully suspended MDCK cell and its application in the preparation of influenza vaccines. Background Art

[0002] Influenza, commonly known as the flu, is a highly infectious respiratory disease caused by influenza virus infection. A series of complications may occur after infection, leading to death. The World Health Organization states that influenza can cause approximately 3 million to 5 million people to suffer from severe illness globally each year, and result in 290,000 to 650,000 deaths.

[0003] Currently, vaccination remains the most effective strategy for preventing influenza; and due to the high replication level of influenza A and B viruses in eggs, the availability of existing infrastructure, and economic considerations, most seasonal influenza vaccines are still produced in large quantities using chicken embryos. However, there are many drawbacks to using chicken embryos to produce influenza vaccines, such as a relatively long production cycle, difficulty in automation leading to a large workload and high pollution risk, possible adaptive mutations, difficulty in monitoring the production process, and production capacity being limited by the supply of chicken embryos. Therefore, the WHO proposed in 1995 to use mammalian passaged cells to replace chicken embryos as the production matrix for influenza vaccines. Compared with the chicken embryo process, the influenza vaccine production process based on mammalian passaged cell culture is easier to achieve standardized large-scale culture, the production capacity is not limited by the supply of chicken embryos, production is carried out using a relatively closed bioreactor, which can ensure the quality of the vaccine, and the probability of the vaccine virus strain mutating during passage in cells is low, the vaccine antigenicity is closer to the natural epidemic strain, and the produced vaccine does not contain ovalbumin, which can reduce the risk of allergic reactions after vaccination.

[0004] MDCK cells (Madin-Daby Canine Kidney Cells, MDCK Cells) are a type of epithelial cells isolated and cultured from the renal tubular tissue of the kidneys of Cocker Spaniel dogs in the United States by Madin and Darby in 1958; studies have found that MDCK cells are suitable for the culture of influenza viruses, and the ineffectiveness of its interferon system gives it strong virus replication ability. Therefore, it is widely used in the isolation and amplification of viruses, and the influenza vaccine produced using MDCK cell matrix has better immunogenicity and protective effect compared to the influenza vaccine produced using chicken embryo matrix; therefore, MDCK cells are often used in the research and development of influenza vaccines, and are one of the recognized ideal cells for culturing influenza viruses and one of the cells approved by the WHO for producing influenza vaccines.

[0005] MDCK cells are adherent culture cells, which have the advantages of fast proliferation, short passage time, and easy cultivation. Serum needs to be added during adherent culture. Serum is an essential component in cell culture, providing various nutrients and hormones for cells. However, the composition of serum is complex, some components are not yet clear, and it may introduce mycoplasma, viruses, etc., which have an adverse impact on cells. Some macromolecular proteins in serum seriously affect the downstream purification of biological agents. Moreover, it is difficult to standardize the preparation and use of serum, the resources are severely scarce, and there may be differences in different batches of serum. Therefore, it is very necessary to develop and apply serum-free culture; and with the increasing requirements of industrialization, cell adherent culture increasingly restricts its industrial use value.

[0006] Therefore, there is an urgent need in the art to provide a cell substrate that can achieve high-titer amplification of influenza virus and is suitable for industrial production of influenza vaccines. Summary of the Invention

[0007] To solve the above problems, the purpose of the present invention is to provide MDCK cells cultured in serum-free suspension, which can be used to culture influenza virus for application in the production of influenza vaccines.

[0008] In the first aspect of the present invention, there is provided MDCK cells cultured in serum-free suspension. The adherent MDCK cells (CCL-34, P60) introduced from ATCC are domesticated to obtain MDCK cells cultured in serum-free full suspension. The MDCK cells cultured in serum-free suspension are named MDCK-JHS, and the preservation number is: CCTCC NO: C2024373.

[0009] Preferably, the inoculation density of the serum-free full suspension MDCK cells is 0.5 - 1.0×10 6 cells / mL.

[0010] In the second aspect of the present invention, there is provided the application of the above MDCK cells in the preparation of influenza vaccines.

[0011] Preferably, the influenza vaccines include seasonal influenza vaccines and pandemic influenza vaccines

[0012] Preferably, the above MDCK cells are used as a cell substrate to culture influenza virus.

[0013] Preferably, the influenza viruses include influenza A virus and influenza B virus.

[0014] Preferably, the influenza A virus includes but is not limited to H1N1, H3N2, H5N1, H7N9; the influenza B virus includes BV strain and BY strain.

[0015] In the third aspect of the present invention, a method for culturing influenza virus is provided, which includes the steps of:

[0016] (1) Inoculating the above-mentioned MDCK cells into a serum-free medium for culture;

[0017] (2) After virus inoculation of the MDCK cells, continue to culture to allow the virus to amplify in the cells.

[0018] Preferably, the virus amplification is carried out in a shake flask and / or a bioreactor.

[0019] Preferably, the culture conditions for virus amplification in a bioreactor are: culture temperature 33 - 35 °C, stirring speed 60 - 80 rpm, culture pH 7.2 - 7.4, dissolved oxygen 40 - 60%.

[0020] Preferably, virus inoculation is carried out when the MDCK cell density reaches 3.0 - 6.0×10 6 cells / mL.

[0021] Preferably, the multiplicity of infection of the virus inoculated into MDCK cells is 0.001 - 0.1.

[0022] In view of the shortcomings and deficiencies of the existing production process technology, the present invention makes improvements at the cell level. Through one-step serum-free continuous suspension domestication, a serum-free fully suspended MDCK cell line (classified name: MDCK-JHS, preservation number: CCTCC NO: C2024373) is obtained. This cell line not only breaks through the growth limitation of adherent cells, avoids the problems introduced by the use of serum, but also can achieve high-titer amplification of influenza virus, providing a basis for carrying out relevant research and production of influenza vaccines.

[0023] Canine kidney cells MDCK-JHS (Madin Darby Canine Kidney Cells) were preserved in the China Center for Type Culture Collection (CCTCC) on November 12, 2024. The address is Wuhan University, Wuhan, China, Zip Code: 4730072, and the preservation number is: CCTCC NO: C2024373. Description of the Drawings

[0024] Figure 1 is the preparation process of MDCK-JHS cells.

[0025] Figure 2 is the subculture curve during the domestication process of MDCK cells.

[0026] Figure 3 is the doubling time of each generation during the domestication process of MDCK cells.

[0027] Figure 4 Morphological photos of adherent - cultured MDCK cells and MDCK - JHS cells cultured in serum - free full - suspension according to the present invention.

[0028] Figure 5 Growth curve of MDCK - JHS cells.

[0029] Figure 6 Passage stability of MDCK - JHS cells.

[0030] Figure 7 Hemagglutination titer results of culturing seasonal influenza virus vaccine strains with MDCK - JHS cells.

[0031] Figure 8 Hemagglutination titer results of culturing seasonal influenza virus vaccine strains with MDCK - JHS cells in a bioreactor. Detailed implementation manners

[0032] The following examples are further descriptions of the present invention, rather than limitations thereof.

[0033] The materials used in the following examples are as follows:

[0034] 1. MDCK cell line (CCL - 34), introduced from ATCC, preservation number: 70040764, is an adherent - cultured cell line. After introduction, a master cell bank was established according to the requirements of the 2020 Edition of the Pharmacopoeia of the People's Republic of China.

[0035] 2. Influenza virus vaccine strains are all purchased from the National Institute for Biological Standards and Control (NIBSC), UK.

[0036] 3. Reagents: MEM medium is purchased from Gibco, product number: 11095072; fetal bovine serum is purchased from Gibco, product number: 10099141C; trypsin solution is purchased from Gibco, product number: 25200 - 072.

[0037] 4. CO2 incubator (ThermoFisher, USA, 3951), CO2 shaker (Yiheng, Shanghai, WSZ - 200A), cell counter (Guangzhou Newton Optics Research Institute, Cytocube Auto).

[0038] Example 1 Serum - free suspension domestication of MDCK cells

[0039] Resuscitate 1 vial of cells from the master cell bank of adherent - cultured MDCK cells, resuspend with serum - free medium and transfer to a 125 mL Erlenmeyer flask, and control the inoculation density at 1.0 - 1.5×106 The cells were cultured at 37°C, 5% CO2, on a shaker at 80-140 rpm, and samples were taken and counted every 24 hours.

[0040] If the viable cell density increases, the culture can be continued until 48h treatment.

[0041] If the cell density is higher than 2.0×10 6 Cells were diluted to 1.0-1.5×10 cells / mL using serum-free medium. 6 Pieces / mL.

[0042] If the cell density is lower than 2.0×10 6 Depending on the condition of the cells under microscopic examination, the whole or a portion of the volume can be centrifuged and resuspended in serum-free medium to control the density to 1.0-2.0×10 6 / mL. Samples are taken and counted every 24 hours until the viable cell density doubles for 48 hours and the viability is higher than 90%, indicating initial success of acclimatization. Usually, as the number of passages increases, the cells become more and more adapted to the culture medium, and the specific growth rate continues to increase. If there are many cell clusters visible to the naked eye during the culture process, centrifuge at 50-100g for 1 minute, discard the precipitate, retain the supernatant, centrifuge at 200g for 5 minutes to collect the cells, and resuspend them in serum-free culture medium.

[0043] MDCK-JHS cell preparation process Figure 1 shown.

[0044] The subculture curve during the acclimation of MDCK cells is as follows Figure 2 shown.

[0045] The doubling time of each generation during the acclimation of MDCK cells is as follows: Figure 3 shown.

[0046] The results showed that the MDCK cells that were originally adapted to adherent culture were domesticated to achieve full suspension culture in serum-free medium. The results of continuous subculture showed that the cell size of each generation was basically uniform (diameter about 20μm), the cell growth was stable (doubling time was 42.22±14.87h), and the cell state was good (cell viability was 92.51±3.53%).

[0047] According to the above method, continuous subculture and acclimatization were carried out, and a serum-free suspension-adapted MDCK cell strain was obtained after screening, named "MDCK-JHS", which was preserved in the China Center for Type Culture Collection (CCTCC) on November 12, 2024, with the address of Wuhan University, Wuhan, China, Postal Code: 4730072, and the preservation number is: CCTCC NO: C2024373.

[0048] The morphology of adherent - cultured MDCK cells and MDCK - JHS cells cultured in serum - free full - suspension according to the present invention is shown in Figure 4 .

[0049] The passage stability of MDCK - JHS cells is as shown in Figure 6 .

[0050] Study on the culture characteristics of MDCK cells domesticated in serum - free suspension in Example 2

[0051] After resuscitating and culturing MDCK - JHS cells in serum - free full - suspension for three generations, inoculate the cells into 125 - mL Erlenmeyer flasks at initial densities of 0.25×10 6 cells / mL, 0.5×10 6 cells / mL, 1.0×10 6 cells / mL, and 2.0×10 6 cells / mL. Set 3 replicates for each group of culture flasks. After inoculation, place them on a shaker at 37°C, 5% CO₂, and a rotation speed of 110 rpm for culture. Sample and detect every 24 h until the cell density begins to decline. Record the cell density and cell viability, and plot the cell growth curve. At the same time, monitor the passage stability of the cells through continuous passage of the cells.

[0052] The results show that after resuscitating and culturing MDCK - JHS cells in serum - free full - suspension for three generations, inoculating and culturing at four densities of 0.25×10 6 cells / mL, 0.5×10 6 cells / mL, 1.0×10 6 cells / mL, and 2.0×10 6 cells / mL, the cell growth curves basically show an "S" shape, as shown in Figure 5 . The maximum proliferation densities are 10.44×10 6 cells / mL, 12.28×10 6 cells / mL, 10.49×10 6 cells / mL, and 12.70×10 6 cells / mL respectively. The times when the maximum proliferation densities appear are 120 h, 120 h, 96 h, and 96 h respectively. The results of counting the cells at different sampling times are shown in Table 1.

[0053] Table 1 Results of inoculating and culturing MDCK - JHS cells in serum - free full - suspension at different densities

[0054]

[0055]

[0056] Study on the amplification conditions of influenza virus in suspension cells in Example 3

[0057] After culturing MDCK-JHS cells in serum-free suspension culture for three generations and centrifuging, resuspend them in serum-free medium to a density of 3.0 - 6.0×10 6 cells / mL. Inoculate influenza virus at a multiplicity of infection (MOI) of 0.1, 0.01, and 0.001, and supplement trypsin. Set up 3 replicates of culture flasks for each group. After inoculation, adjust the culture temperature to 33 - 35°C. At 48 h and 72 h after virus inoculation, take 1 mL of sample from each flask, and detect the hemagglutination titer of the virus in the sample through hemagglutination test to judge the proliferation of the virus in the cells, and draw a column chart of hemagglutination titer.

[0058] The results of inoculating influenza virus into serum-free suspension MDCK-JHS cells showed that: when H1N1 was inoculated at MOI = 0.01, the hemagglutination titer was the highest, reaching 1:320 (2 8.32 ); when H3N2 was inoculated at MOI = 0.1, the hemagglutination titer was the highest, reaching 1:320 (2 8.32 ). When BV was inoculated at MOI = 0.1, the hemagglutination titer was the highest, reaching 1:1120 (2 10.13 ).

[0059] The results of sampling and detecting the hemagglutination titer are shown in Table 2, and the virus proliferation rule is as Figure 7 shown.

[0060] Table 2 Detection results of hemagglutination titer

[0061]

[0062]

[0063]

[0064] Example 4 Amplification of influenza virus in a bioreactor

[0065] Culture serum-free suspension MDCK-JHS cells in a 15L bioreactor. The culture conditions are 37°C, the stirring speed is controlled at 80 - 140 rpm, the culture pH is 7.0 - 7.2, and the dissolved oxygen is 40 - 60%. When the cells are cultured to the target cell amount, inoculate the virus, and inoculate the virus according to the optimal virus inoculation parameters determined above. The culture conditions after virus inoculation are 33 - 35°C, the stirring speed is 60 - 80 rpm, the culture pH is 7.2 - 7.4, and the dissolved oxygen is 40 - 60%. The culture period is 72 h, and samples are taken every 24 h during this period to measure the hemagglutination titer of the samples.

[0066] As Figure 8 shown, cell culture and virus amplification are carried out in the bioreactor: the highest hemagglutination titer of H1N1 can reach 1:240 (2 7.91 ); the highest hemagglutination titer of H3N2 can reach 1:160 (27.32 ); The highest BV hemagglutination titer can reach 1:800 (2 9.64 ); The highest BY hemagglutination titer can reach 1:280 (2 8.13 ).

[0067] The above results show that the serum-free fully suspended MDCK cells provided by the present invention can effectively amplify influenza virus. Compared with the adherent culture system, this suspension culture system is easier to monitor data, the virus inoculation method is simple, and it is more conducive to subsequent process scale-up. It should be noted that the culture mode in this example has not been further optimized. In the future, precise control of relevant parameters by means of a bioreactor can be considered to further optimize the culture conditions to achieve the purpose of further improving the virus amplification efficiency.

[0068] The above is only the preferred embodiment of the present invention. It should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art in this technical field, without departing from the spirit and scope of the present invention, several improvements and refinements can also be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A serum-free suspension culture of MDCK cells, whose deposit number is CCTCC NO: C2024373.

2. Use of the MDCK cells according to claim 1 in preparing influenza vaccines.

3. The use according to claim 2, characterized in that: The influenza vaccines include seasonal influenza vaccines and pandemic influenza vaccines.

4. The use according to claim 2, characterized in that: The MDCK cells are used as cell matrix to culture influenza virus.

5. The use according to claim 4, characterized in that: The influenza virus includes influenza A virus and influenza B virus.

6. A method for culturing influenza virus, characterized in that: Includes steps: (1) inoculating the MDCK cells according to claim 1 and culturing them in a serum-free medium; (2) After the virus is inoculated into MDCK cells, the cells are continuously cultured to allow the virus to amplify in the cells.

7. The culture method according to claim 6, characterized in that: The virus amplification is performed in shake flasks and / or bioreactors.

8. The culture method according to claim 7, characterized in that: The culture conditions for virus amplification in the bioreactor are: culture temperature 33-35°C, stirring speed 60-80 rpm, culture pH 7.2-7.4, and dissolved oxygen 40-60%.

9. The culture method according to claim 6, characterized in that When the MDCK cell density reaches 3.0-6.0×10 6 Virus inoculation was performed at 1.54 μg / mL.

10. The culture method according to claim 6, characterized in that: The virus was inoculated into MDCK cells at a multiplicity of infection of 0.001-0.1.

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