Novel vero cell line that can be cultured in suspension in serum-free medium, method for preparing the same, and method for preparing virus for vaccine using the novel cell line

Abstract

The present invention relates to a sVERO 7C2 cell line, which is a Vero cell line derived from WHO distributed Vero cells (African green monkey kidney cell line) and capable of being cultured in suspension without serum components. In addition, the present invention relates to a culture method for culturing Vero cells and a method for preparing a virus for vaccine using the Vero cells.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2019-0154749, filed on October 27, 2019, and all contents disclosed in the specification and drawings of the corresponding application are incorporated herein by reference.

[0002] This invention relates to a novel cell line derived from Vero that can be cultured in suspension in serum-free medium, a method for preparing the same, and a method for preparing viruses using the cultured cells. Background Technology

[0003] With the commercialization of various types of vaccines, methods for manufacturing vaccines have been developed in various ways. A representative example of a traditional vaccine production method is the production of influenza vaccines using fertilized eggs. However, producing vaccines using fertilized eggs presents challenges in providing a stable supply, thus requiring adjustments to vaccine production. Furthermore, chickens suitable for product production must be raised in sterile facilities, increasing costs, and the difficulty in purifying egg protein-derived components limits vaccination to individuals with egg protein allergies.

[0004] As a method to overcome the problems of producing vaccines using fertilized eggs, there is a method for producing vaccines using cell culture. Because this vaccine is produced by inoculating the virus into animal cells that can grow indefinitely, it can be supplied through large-scale production in a short time and has the advantage of being suitable for people with egg protein allergies.

[0005] These animal cells are typically adherent cells and cultured using fetal bovine serum (FBS). However, this process can introduce unknown animal-derived factors into the product, leading to potential quality variations between products. Furthermore, FBS is expensive and carries the risk of contamination by infectious proteins such as prions, viruses, and mycoplasma, increasing manufacturing costs and compromising safety. Therefore, it is preferable to avoid using animal-derived additives and animal-derived serum in vaccine production.

[0006] On the other hand, cell culture in suspension, as an alternative method of cell culture, has many advantages compared to adherent cell culture, such as ease of large-scale culture, simplified passage process, and reduced manpower and space requirements. Therefore, extensive research is underway on the development of serum-free suspension cells and their use for viral propagation.

[0007] The Vero cell line is an established cell line capable of propagating various viruses due to its high sensitivity to these viruses, such as rotavirus, poliovirus, influenza virus, Japanese encephalitis virus, and dengue virus. However, because Vero cell lines have a very strong adhesion to surfaces, large-scale culture vessels or microcarriers are required, resulting in significant costs in vaccine manufacturing. Furthermore, a step is needed to remove cells attached to the carrier. In this case, animal-derived trypsin is used, and with the use of animal-derived components, there is a possibility of cell loss and damage.

[0008] Therefore, in order to produce vaccines through safe and effective animal cell culture, a Vero cell line that can be cultured in suspension in serum-free medium is needed, and the inventors have previously studied and obtained a patent for a Vero cell line that can be cultured in suspension in serum-free medium (Korean Patent No. 10-1831284).

[0009] However, the previously registered patent (Korean Patent No. 10-1831284) showed limited effectiveness in terms of cell proliferation and viral infectivity for the suspension Vero cell line Vero Sky7458. Summary of the Invention

[0010] Technical issues

[0011] In this study, during the development of new cells, the inventors have attempted to establish a new suspension Vero cell line with superior cell proliferation, cell morphology and viral proliferation compared to the previously registered patented cell line (Vero Sky 7458) by selecting cells with superior functions.

[0012] Therefore, the object of the present invention is to provide a Vero cell line that can be used for the propagation of vaccines and is capable of serum-free and suspension culture to solve the problems of contamination or low culture efficiency caused by the use of serum and adherent culture.

[0013] Another object of the present invention is to provide a more efficient method for viral proliferation and vaccine production by providing a cell line that has superior performance in terms of cell proliferation or viral proliferation capacity compared to the previously registered patented cell line (Vero Sky 7458).

[0014] More specifically, one object of the present invention is to provide the following embodiments.

[0015] Implementation method 1, Vero cell line sVERO 7C2 (accession number KCLRF-BP-00470).

[0016] In Embodiment 2, the Vero cell line of Embodiment 1 is derived from Vero cells distributed by the WHO. It does not require serum for cell growth and does not require an attachment carrier, and can be cultured in suspension.

[0017] In embodiment 3, the virus is propagated in any of the Vero cell lines described in the preceding embodiments.

[0018] Embodiment 4: A method for preparing a vaccine virus using any of the cell lines in the foregoing embodiments; or the use of any of the cell lines in the foregoing embodiments for preparing a vaccine virus.

[0019] In embodiment 5, in any of the methods or uses described in the preceding embodiments, the virus is selected from the group consisting of yellow fever virus, Zika virus, rotavirus, dengue virus, influenza virus, measles virus, Japanese encephalitis virus, mumps virus, rubella virus, poliovirus, HSV-1, HSV-2, rabies virus, RS virus, reovirus type 3, parvovirus, Coxsackie virus, adenovirus types 1-47, Lassa virus, bullous stomatitis virus, and vaccinia virus.

[0020] In embodiment 6, in any of the methods or uses described in the foregoing embodiments, the virus is yellow fever virus or Zika virus.

[0021] Embodiment 7: A method for preparing a cell line according to any one of the foregoing embodiments, wherein the cell line does not require serum for cell growth and does not require an attachment carrier, and can be cultured in suspension, the method comprising:

[0022] (a) Preparing Vero cells obtained from WHO;

[0023] (b) Adapting the Vero cells to grow in a serum-free culture medium; and

[0024] (c) Adapt the adherent Vero cells selected in step (b) to grow in a suspension state without an attachment carrier.

[0025] Implementation method 8, a method for producing a virus for a vaccine, comprising:

[0026] (a) Vero cells from any of the foregoing embodiments are used at a concentration of 1 × 10⁻⁶. 5 -9×10 5 The cells were seeded at a concentration of 10 cells / mL into serum-free cell culture medium.

[0027] (b) The step of culturing the cells under culture conditions of maintaining a stirring speed of 40-90 rpm and a pH of 6.5-7.5, and proliferating the Vero cells to 5.0 × 10⁶ cells in a roller flask. 5 -4.7×10 6 Cell density per cell / mL;

[0028] (c) Infecting proliferating Vero cells with yellow fever virus or Zika virus;

[0029] (d) Culture infected, proliferating Vero cells; and

[0030] (e) Isolation of yellow fever virus or Zika virus from cell culture compositions.

[0031] In embodiment 9, during step (b) of any of the preceding embodiments, fresh culture medium is added to the cell culture, or a portion of the culture medium is removed and replaced with fresh culture medium.

[0032] Other objects and advantages of the present invention will become more apparent from the following description, claims and drawings.

[0033] Technical solution

[0034] To achieve the above objectives, this invention provides a novel suspension Vero cell line (Korean Foundation for Cell Research Accession No.: KCLRF-BP-00470) which exhibits excellent viral infectivity and a 70-100% higher growth rate compared to the previously registered patented cell line Vero Sky 7458. This cell line is a novel cell line that can be suspended in serum-free culture medium and is derived from Vero cells (African green monkey kidney cell line) distributed by the WHO.

[0035] Furthermore, to achieve the above objectives, the present invention provides a method for preparing the novel suspended Vero cell line, the method comprising:

[0036] 1) Adapt the adherent Vero cell line to serum-free culture medium;

[0037] 2) Select cells that exhibit excellent growth;

[0038] 3) Suspend the selected cells in serum-free culture medium; and

[0039] 4) Select cells with low cell aggregation and excellent proliferation ability from suspension cells and perform continuous passage culture.

[0040] In addition, to achieve the above objectives, another aspect of the present invention provides a method for preparing a vaccine, the method comprising:

[0041] 1) Infect the novel suspension Vero cell line of this invention with a virus;

[0042] 2) Culture virus-infected cells in suspension; and

[0043] 3) Isolate viruses from cell cultures.

[0044] Furthermore, to achieve the above objectives, the present invention provides characterization results of the novel cell line of the present invention, including:

[0045] 1) Evaluate the tumorigenicity of the novel suspension Vero cell line;

[0046] 2) Determine the origin of the novel suspension Vero cell line; and

[0047] 3) Evaluate the long-term stability of the novel suspension Vero cell line.

[0048] Technical effect

[0049] This invention provides a novel sVERO 7C2 cell line that can be cultured and suspended in culture in a serum-free environment, exhibits a faster growth rate compared to the Vero Sky 7458 cell line, and can be effectively used for viral proliferation. This invention can also be used to produce viruses or vaccines using these cell lines. Attached Figure Description

[0050] The accompanying drawings illustrate preferred embodiments of the invention and are used to further understand the technical concept of the invention and the above-described content of the invention. Therefore, the invention should not be construed as limited to the content described in these drawings.

[0051] Figure 1 This is a diagram showing the cell proliferation of Vero Sky 7458 and the novel sVERO 7C2 cell line of the present invention in a 125 mL roller bottle;

[0052] Figure 2 This is a diagram showing the cell proliferation of Vero Sky 7458 and the novel sVERO 7C2 cell line of the present invention in a 5L bioreactor;

[0053] Figure 3 Images of the shapes of cells in Vero Sky 7458 and the novel sVERO 7C2 cell line of this application;

[0054] Figure 4 and 5 This is a diagram showing the cell proliferation patterns and cell metabolite concentrations used to evaluate the long-term stability of the novel sVERO 7C2 cell line of the present invention.

[0055] Figure 6 and 7 This is a graph showing the viral titer results after viral inoculation in Vero Sky 7458 and the novel sVERO 7C2 cell line of the present invention. Detailed Implementation

[0056] The novel sVERO 7C2 cell line of this invention is derived from Vero cells (African green monkey kidney cell line) distributed by the WHO. It does not require serum for cell growth and can be cultured in suspension without an attachment carrier.

[0057] In this invention, the term "serum-free culture medium" refers to a culture medium in which the cell lines established in this invention can be cultured, wherein no serum is substantially added, and the term "substantially not added" means containing less than 0.5% (v / v) of serum, preferably less than 0.1% (v / v), more preferably less than 0.01% (v / v), and most preferably completely free of serum.

[0058] The serum-free culture medium may be at least one selected from the group consisting of Sky FM03 (Lonza), SFM4CHO (Hyclone), ProVero1 (Lonza), EX-CELL VERO (Sigma), and VP-SFM (Gibco), but is not limited thereto, and any serum-free culture medium that can be used to culture animal cells may be used in this invention without limitation.

[0059] Compared to the Vero Sky 7458 cell line, the novel sVERO 7C2 cell line of this invention preferably exhibits a 70% or 100% or greater increase in cell growth. It can be prepared according to a method comprising a series of steps, thereby selectively selecting cells with relatively rapid cell proliferation and low cell aggregation. With the improved cell growth, the number of virus-infected cells increases, which is beneficial for the large-scale production of viruses or vaccines.

[0060] Furthermore, the sVERO 7C2 cell line of the present invention can be suspended in serum-free medium and exhibits a higher viral titer compared to Vero Sky 7458, thus demonstrating superior viral production capacity. Additionally, species validation assays using tumorigenicity tests, karyotype analysis, and PCR showed that the novel cell line of the present invention exhibited no tumorigenicity even at high passage numbers (220 passages) and showed that it was derived from monkey cells, thus making it advantageous for the production of vaccines.

[0061] Furthermore, the inventors named the newly established cell line capable of serum-free and suspension culture from Vero cells “sVERO 7C2”, and deposited it at the Korean Cell Line Research Foundation (KCLRF) on September 9, 2019, with accession number KCLRF-BP-00470.

[0062] One aspect of the present invention provides a novel sVERO7C2 cell line capable of serum-free suspension culture. For example, it can be prepared according to a method comprising the following steps: (a) thawing adherent Vero cells (WHO) and culturing them in a serum-containing medium; (b) culturing the cells obtained in step a) while reducing the serum content, and finally culturing them in a serum-free medium; (c) selecting individuals with rapid cell proliferation from the cells obtained in step b); (d) adapting the cells obtained in step c) to suspension culture with stirring at 40-90 rpm; and (e) selecting individuals with low cell aggregation and excellent proliferation capacity from the cells adapted for suspension culture obtained in step d).

[0063] The preparation method of the sVERO 7C2 cell line of the present invention will be described in detail below.

[0064] Step (a)

[0065] In step (a), Vero cells (WHO) were thawed and then cultured in a candidate medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0066] Step (b)

[0067] In step (b), the cells cultured in step (a) were obtained by suspending them at the bottom of the flask with 0.25% trypsin EDTA after 3–4 days, and then passaged in an attached state while reducing the serum content. When the cell doubling time was less than 48 hours, the serum ratio was reduced and eventually replaced with serum-free medium.

[0068] Step (c)

[0069] In step (c), cells are selected by single-cell cloning of the cells obtained in step (b). Cells are seeded into 96-well plates at a concentration of 1 cell / well, and cell proliferation patterns are observed. Specifically, by selecting a single cell with superior proliferative capacity from cells capable of adapting to serum-free culture, homogeneous cell lines from a single cell are ensured, thereby ensuring the consistency of future cell proliferation or viral infectivity. In this invention, single-cell cloning is performed separately in the serum-free adaptation step (c) and the suspension culture adaptation step (e) of Vero cells (WHO), and the homogeneity of newly developed cells is ensured through a total of two rounds of cell selection. This differs from previous studies on suspension-cultureable Vero cells.

[0070] For example, seeding and culturing single cells in a 96-well plate is not easy. Even if a single cell attaches to the culture vessel, cell proliferation can take four weeks or longer, or the cells may not grow well and may degenerate. Therefore, to overcome this problem, suspension culture medium is used to prepare a medium suitable for single-cell cloning and to culture single cells. Through this process, individual cells with relatively rapid cell proliferation are selected.

[0071] Step (d)

[0072] In step (d), the cells obtained in step (c) are suspended in a roller bottle with stirring at 40-90 rpm. Specifically, the cells from step (c) are seeded into serum-free suspension medium at a seeding concentration of 1.0 × 10⁻⁶. 5 -9.0×10 5 Cells / mL, preferably 5.0 × 10⁻⁶. 5 Cells / mL. While maintaining culture conditions of 40-90 rpm, preferably 60 rpm, and pH of approximately 6.5-7.5, passage the cells at 3-4 day intervals to adapt them to suspension culture.

[0073] Step (e)

[0074] In step (e), the cells obtained in step (d) are subjected to single-cell cloning as in step (c) to select individuals with low cell aggregation and excellent proliferation capacity. At this time, ultra-low adhesion 96-well plates are used instead of standard adhesion culture containers to induce cell proliferation in suspension. Through this double single-cell selection process, a suspension Vero cell line exhibiting superior performance compared to existing suspension Vero cell lines in the literature is established and named "sVERO 7C2". Furthermore, sVERO 7C2 is continuously passaged in roller bottles to examine the long-term stability and cell proliferation pattern of the cell line, and to perform characterization such as tumorigenicity assays.

[0075] Another aspect of the present invention provides a method for proliferating a vaccine virus using the sVERO 7C2 cell line according to the present invention.

[0076] Viruses that can be propagated using the sVERO 7C2 cell line of this invention include yellow fever virus, Zika virus, rotavirus, dengue virus, influenza virus, measles virus, Japanese encephalitis virus, mumps virus, rubella virus, poliovirus, HSV-1, HSV-2, rabies virus, RS virus, reovirus type 3, parvovirus, Coxsackie virus, adenovirus types 1-47, Lassa virus, bullous stomatitis virus, and vaccinia virus. Among these viruses, the cell line of this invention is particularly suitable for the production of yellow fever virus and Zika virus.

[0077] The present invention will be described in more detail below by way of examples. However, the following examples are for illustrative purposes only, and the scope of the invention is not limited to the examples. The examples of the present invention are provided to explain the invention more completely to those skilled in the art.

[0078] <Example 1> Preparation of Vero cell line by serum-free suspension culture

[0079] 1.1 Thawing adherent Vero cells

[0080] The cryopreserved adherent Vero cells (WHO) were thawed and cultured in EMEM medium containing 10% serum in a T-75 flask at 37°C and 5% CO2.

[0081] 1.2 Selection of serum-free cell lines

[0082] Cells obtained in step 1.1 were passaged at 3-4 day intervals. Cell counts were checked at each passage; if the cell doubling time was 48 hours or less, the serum content in the medium was reduced and the cells were cultured again. The serum content in the medium was reduced to 10%, 5%, 2%, and 1%, and passaged repeatedly until serum-free culture was achieved. Single-cell cloning of serum-free adapted cells was performed in 96-well plates. The medium was changed weekly for approximately 3-4 weeks, and groups with relatively rapid cell proliferation were selected by microscopic observation. Cell groups exhibiting rapid proliferation were expanded and cultured in T-flasks.

[0083] 1.3 Selection of cell lines suitable for suspension culture

[0084] After sufficient expansion, the cells obtained in step 1.2 were transferred to roller bottles for suspension culture. The cells were cultured at 37°C and 5% CO2 with a stirring speed of 60 rpm. When the pH of the medium decreased or the cells grew above a certain level, the medium was replaced or the cells were passaged. After acclimatization to suspension culture for 2 months, the cell concentration reached approximately 1.5 × 10⁻⁶ cells / year. 6 Cells / mL. Cell viability was confirmed to be greater than 90%. Single-cell cloning was performed in ultra-low attachment 96-well plates to ensure rapid cell proliferation and low cell aggregation in cells adapted for suspension culture. After approximately 3-4 weeks of culture, the group with relatively rapid cell proliferation was selected by microscopic observation. Cells were expanded using ultra-low attachment T-flasks to select the group with the fastest proliferation rate, which was named "sVERO 7C2".

[0085] <Example 2> Cell line proliferation capacity and characterization

[0086] 2.1 Evaluation of the proliferation capacity of the novel cell line

[0087] The sVERO 7C2 cell line was cultured in suspension in roller bottles or a 5L bioreactor to evaluate its proliferation capacity. The VeroSky 7458 cell line was used as a control group.

[0088] The initial cell concentration for suspension culture was 5.0 × 10⁻⁶. 5 Cells / mL, in the case of roller flasks, cells are harvested after 3-4 days, centrifuged at 1200 rpm for 5 minutes, and then centrifuged at 5.0 × 10⁻⁶ cells / mL. 5 Cells / mL were passaged.

[0089] Compared to the Vero Sky 7458 cell line, the sVERO 7C2 cell line prepared in this invention showed approximately 1.7 times the cell proliferation rate in roller bottles and approximately 2 times the cell proliferation rate in bioreactors.

[0090] 2.2 Long-term passage stability of novel cell lines

[0091] The sVERO 7C2 cell line was passaged in roller bottles for 4 months, and the stability of long-term passage was evaluated by analyzing cell proliferation, cell viability, and cell metabolites. The cell line of the present invention showed the same level of cell proliferation during the 4-month culture period, and a cell viability of 90% or higher could be determined. Furthermore, it was confirmed that the concentrations of cell metabolites were in a similar pattern.

[0092] 2.3 Characterization of novel cell lines

[0093] The novel sVERO 7C2 cell line of the present invention was subjected to tumorigenicity testing. The test was conducted at QuBEST BIO Co., Ltd., a non-clinical evaluation center, in accordance with the European Pharmacopoeia guidelines for tumorigenicity evaluation, and clinicopathological results confirmed that the cell line of the present invention did not exhibit tumorigenicity even at high passage numbers (220 passages).

[0094] Furthermore, karyotype analysis and species identification tests (PCR) confirmed that the cell line of the present invention is derived from monkeys.

[0095] Furthermore, sterility and mycoplasma-negative tests proved that it was not contaminated with bacteria.

[0096] <Example 3> Comparison of Viral Proliferation Capacity

[0097] Viruses were propagated using the novel sVERO 7C2 cell line of this invention under suspension culture conditions. VeroSky7458 was used as a control group to compare viral propagation capacity. The viruses used in this experiment were yellow fever virus and Zika virus, and the culture conditions are as follows.

[0098] Cell concentration: 5.0 × 10⁻⁶ 5 cells / mL

[0099] Culture scale: 125mL roller bottle

[0100] Mixing speed: 60 rpm

[0101] Culture conditions: 37℃, 5% CO2, humidified

[0102] Incubation period: 4 days (yellow fever virus), 6 days (Zika virus)

[0103] Viral infectivity assays were performed at the same starting cell and virus inoculation concentrations to compare the viral proliferation capacity of the two cell lines. Virus was cultured after inoculation, and cytopathic effect (CPE) and cell viability were assessed. Yellow fever virus was cultured for 4 days, and Zika virus for 6 days. The culture supernatant was recovered, and the viral titer was determined. The titers of both viruses were determined using a plaque assay. The results showed that the viral titer proliferated in the novel cell line of this invention was superior to that of Vero Sky 7458, which was used as the control group. Furthermore, sVERO 7C2 was found to have a higher cell proliferation capacity than Vero Sky 7458, thus allowing for a larger number of cells to be infected during viral infection, thereby enabling more efficient production of large-volume viruses.

[0104] [Collection Number]

[0105] Name of the depositary institution: Korea Cell Line Research Foundation (KCLRF)

[0106] Accession number: KCLRF-BP-00470

[0107] Date of preservation: September 9, 2019

[0108]

Claims

1. A Vero cell line sVERO 7C2, with accession number KCLRF-BP-00470.

2. The Vero cell line according to claim 1, characterized in that, The cell line is derived from Vero cells distributed by the WHO. The cell line does not require serum for cell growth and does not require an attachment carrier, and can be cultured in suspension.

3. The Vero cell line according to claim 1, characterized in that, The cell line propagates the virus.

4. A method for preparing a vaccine virus using the cell line of claim 1.

5. The method according to claim 4, characterized in that, The viruses are selected from the group consisting of yellow fever virus, Zika virus, rotavirus, dengue virus, influenza virus, measles virus, Japanese encephalitis virus, mumps virus, rubella virus, poliovirus, HSV-1, HSV-2, rabies virus, RS virus, reovirus type 3, parvovirus, Coxsackie virus, adenovirus types 1 to 47, Lassa virus, bullous stomatitis virus, and vaccinia virus.

6. The method according to claim 4, characterized in that, The virus in question is either yellow fever virus or Zika virus.

7. A method for producing a vaccine virus, characterized in that, The method includes: (a) The Vero cells of claim 1 are used at a concentration of 1 × 10⁻⁶. 5 -9×10 5 The cells were seeded at a concentration of 10 cells / mL into serum-free cell culture medium. (b) Proliferate Vero cells to 5.0 × 10⁻⁶ in roller bottles. 5 -4.7×10 6 A cell density of cells / mL, including the step of culturing the cells under culture conditions of maintaining a stirring speed of 40-90 rpm and a pH of 6.5-7.5; (c) Infecting proliferating Vero cells with yellow fever virus or Zika virus; (d) Culture infected, proliferating Vero cells; and (e) Isolation of yellow fever virus or Zika virus from cell culture composition.

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