JP6609110B2 - Virus culture method using fiber material support - Google Patents

Description

  The present invention relates to a method of proliferating virus in the cells by maintaining the virus-infected adhesive cells for a long period of time without detaching them from the support, that is, a method of cultivating viruses that are sensitive to adhesive cells, particularly The present invention relates to a method for culturing hepatitis B virus.

  Hepatitis A virus is an icosahedral Picornaviridae RNA virus with a diameter of 27 nm. There are seven types of genotypes, and there are differences in epidemic types in each region of the world, but since there is one type of serotype, there is no difference in immunogenicity by genotype.

  Hepatitis A virus is proliferative in cultured cells but is slow and grows slowly over 2-3 weeks. Generally, it does not show a cytopathic effect and accumulates in the cell.

  When hepatitis A virus infects humans, serum transamylase rises after fever, malaise, etc. after an incubation period of 2 to 6 weeks. In addition to symptoms such as anorexia and vomiting, cases such as jaundice, hepatomegaly, dark urine, and grayish white stool are observed. In general, it does not become chronic and recovers after 1 to 2 months, but rarely develops fulminant hepatitis.

  In recent years, the risk of infection with the hepatitis A virus has increased due to an increase in the number of overseas travelers, and the demand for hepatitis A vaccine has increased. On the other hand, since supply that can sufficiently meet the increase in demand is difficult, efforts to mass production by reviewing production methods are required.

The production method of the current hepatitis A vaccine is as follows. (Patent Document 1)
[1] Inoculate and infect GL37 cells, which are cell lines derived from African green monkey kidney, with hepatitis A virus for vaccine production;
[2] Infected hepatitis A virus is propagated in GL37 cells by performing roller bottle culture for 3 weeks;
[3] After culturing, the cells are lysed with a surfactant to recover hepatitis A virus to obtain a cell lysate;
[4] The cell lysate is clarified by filtration, concentrated by salting out and centrifuging, and then treated with chloroform, an enzyme and an organic solvent to obtain a purified virus solution;
[5] The purified virus solution is inactivated with formalin to obtain a vaccine stock solution.

Japanese justice 1-279843

  Roller bottle culture in the current hepatitis A vaccine production method has low productivity and is not suitable for mass production. Therefore, production methods by floating culture using microcarriers such as beads as a support have been studied. However, when floating culture is performed by attaching GL37 cells to microcarriers, the cells cannot endure during the three-week culture period and are detached from the microcarriers, so the problem of improving productivity cannot be solved.

  Therefore, the present invention provides a method for proliferating a virus in the cell by maintaining the virus-infected adherent cell in culture for a long period of 20 days or longer without detaching from the support, that is, an adherent cell-sensitive virus. An object is to provide a culture method.

  As a result of repeated studies to achieve the above-mentioned object, the present inventors have made adherent cells infected with viruses adhere to a fiber-made support, and filled the support into a culture vessel. In culturing, it was found that by controlling the discharge and addition of the medium, the cells can be cultured while maintaining the adhesion rate of the cells at 90% or more, and the present invention has been completed.

Accordingly, the present invention provides a method for culturing viruses infected with adherent cells, and specifically includes the following inventions.
[1] The method includes adhering virus-infected adhesive cells to a fiber material support, filling the support in a culture vessel, and culturing cells, and controlling the discharge and addition of the medium. While maintaining the adhesion rate of the adherent cells at 90% or higher;
[2] The method according to [1] above, wherein the adhesion rate of the adherent cells on the 20th day after the start of the culture is 90% or more;
[3] The method according to [1] or [2] above, wherein the fiber material is polyethylene terephthalate;
[4] The method according to any one of [1] to [3], wherein the support is a single-use product;
[5] The method according to any one of [1] to [4] above, wherein the virus is a Picornaviridae virus;
[6] The method according to [5] above, wherein the Picornaviridae virus is hepatitis A virus;
[7] The method according to any one of [1] to [6] above, wherein the adherent cells are African green monkey kidney-derived cells;
[8] The method according to [7] above, wherein the African green monkey kidney-derived cells are GL37 cells;
[9] An immunogenic composition prepared using a virus cultured by the method according to any one of [1] to [8] above;
[10] A hepatitis A vaccine produced using the hepatitis A virus cultured by the method according to any one of [6] to [8] above.

  The method of the present invention is a high-density culture method in which three-dimensional culture is performed at a cell density 10 times or more that of roller bottle culture, and is a long-term cell culture necessary for culturing a virus infected with adherent cells. Maintenance is possible. Thereby, productivity of virus culture is improved, and mass production of vaccine production using the virus becomes possible.

It is the figure which showed the process for making virus-infected cell adhere to a fiber material support | carrier reliably, ie, taking in and out of the culture medium of a culture container (adding and removing of the culture medium for 10% of culture container capacity). It is the figure which showed the process for making virus-infected cell adhere to a fiber material support body reliably, ie, taking in and out of the culture medium of a culture container (it puts and draws out almost the whole quantity of culture medium). It is the figure which showed the cell number at the time of culturing hepatitis A virus for 35 days on a 500 mL scale. The result using two types of culture media (MEM and VP-SFM) is shown. It is the figure which showed the cell number at the time of mass-cultivating hepatitis A virus on a 2L scale, and the amount of antigens. The result using a VP-SFM medium is shown. It is the figure which compared the cell number of culture | cultivation using a fiber material support body, and suspension culture using a microcarrier as a support body.

  The present invention is used for viruses that infect adherent cells. Specifically, it is characterized by a method for culturing hepatitis A virus comprising the steps of inoculating hepatitis A virus-sensitive cells with hepatitis A virus and culturing the cells.

  In cell culture, a medium containing virus-infected cells is put into a culture container filled with a fiber material support, and then the cell is adhered to the support by taking in and out the medium for 10% of the culture container capacity. (FIG. 1A). The culture in which the medium corresponding to 10% of the culture container capacity is taken in and out may be performed for about a day and night. Furthermore, adhesion is ensured by putting in and out almost all of the medium a plurality of times (FIG. 1B). During the culture period, by culturing while exchanging the medium with fresh one, the cells are placed in both gas phase and liquid phase environment, so that gas exchange and acquisition of growth components can be performed efficiently. Control. Thereby, the said cell is culture-maintained over a long period of time, and a virus can be cultured.

  The above-described method for exchanging the medium with a fresh one is performed by repeatedly discharging the old medium and charging the new medium. During the culture period, the culture vessel is placed in a temperature-controllable apparatus and cultured at a constant temperature.

  Examples of the adhesive cells used in the present invention include Vero cells and GL37 cells, which are cell lines derived from African green monkey kidney cells.

  The medium is a medium usually used for cell culture, for example, MEM (Thermo Fisher Scientific, special order), Eagle MEM (Nissui Pharmaceutical), Dulbecco's modified MEM (Nissui Pharmaceutical), VP-SFM (Thermo Fisher). Scientific company), etc., but any of them may be used. During virus culture, a medium supplemented with fetal calf serum can also be used.

  The support is not particularly limited as long as it is a fiber material, but a preferable material is polypropylene, and a more preferable material is polyethylene terephthalate. The shape of the support is not particularly limited, but a circular shape, a rectangular shape, or a unique geometric shape may be used to enhance functionality. Usually, it is a small and light piece of about 10 mm, but it is characterized by having a large surface area because it is a fiber material.

  By using a support made of such a fiber material, cells adhere not only to the surface of the support but also to the inside thereof, so that three-dimensional high-density culture is possible. In addition, since there is no share stress due to taking in and out of the medium, cells can be cultured for a long period of time. Furthermore, when a cell has a three-dimensional structure, it can take a form similar to in vivo, and the growth of the virus is improved.

Supports available on the market can be used. For example, the support that is provided under the trade name of "BioNOCII ^R" is available from CESCO Corporation. It may be a single-use product, or may be sterilized or surface-treated.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples at all.

(1) Hepatitis A virus culture (500 mL scale)
GL37 cells adherently cultured in VP-SFM medium were suspended in 500 mL of fetal bovine serum-added MEM medium so that the initial cell count was 7.6 × 10 ⁷ cells, and this was inoculated with hepatitis A virus (moi 0.1). .
Fibrous material support (trade name: BioNOCII ^R, CESCO Co.) high density culture vessel filled with (trade name: BelloCell500A ^R, CESCO Inc.) was charged with the MEM medium, overnight, in 10% fraction of said culture vessel Cells were allowed to adhere to the support by taking in and out of the volume of medium. Thereafter, almost all of the medium was taken in and out to ensure adhesion. Cultivation was carried out for 3 weeks in an environment of 37 ° C. and 5% CO ₂ , and during the culturing period, the discharge of the old medium in the entire volume and the introduction of a new medium were repeated every 2-3 days to maintain the culture.

(2) Cell adhesion rate On the 15th and 22nd days from the start of the culture, sampling from the culture supernatant and measuring the number of cells dropped by the trypan blue method using a hemocytometer, the number of cells measured on both days was 0.
Since the culture supernatant amount is 500 mL, the number of cells of 1 × 10 ⁷ cells can be detected as long as the cell number detection sensitivity is 1. Since the number of adherent cells on the 15th and 22nd days was 2.8 × 10 ⁹ cells and 2.9 × 10 ⁹ cells, it was confirmed that 99% of the cells were adhered.

(3) Measurement of viral antigen amount The viral antigen amount was measured by ELISA using an antibody against hepatitis A virus. The results are shown in Table 1.

(1) Long-term culture of hepatitis A virus (500 mL scale)
Hepatitis A virus was cultured for 35 days. The same procedure as in Example 1 was performed except that the initial cell number was 7.7 × 10 ⁷ cells. The virus antigen amount measurement results are shown in Table 2. Moreover, the measurement result of the number of cells is shown in FIG. In FIG. 2, the results of the same culture using VP-SFM medium are also plotted. From the results of FIG. 2, it was found that not only the MEM medium but also the VP-SFM medium can be used.

(1) Hepatitis A virus culture (2L scale)
GL37 cells adherently cultured in VP-SFM medium were suspended in 1600 mL of fetal bovine serum-added MEM medium so that the initial cell count was 1.2 × 10 ⁹ cells, and cultured using a 2 L high-density culture vessel. . During the 3-week culture period, the discharge of the old medium and the addition of the new medium were repeated in the same manner as in Example 1 except that the culture medium having a volume of 2 L was repeated at a rate of 5 L / day. The virus antigen amount measurement results are shown in Table 3 and FIG. The measurement result of the number of cells is as shown in FIG.

(1) Hepatitis A virus culture (comparison with microcarriers)
An experiment was conducted to compare culture using a fiber material support and suspension culture using a microcarrier as a support. The culture using the fiber material support was performed in the same manner as in Example 1 except that the initial cell number was 3.6 × 10 ⁷ cells. The suspension culture using a microcarrier (trade name: Cytodex 1, GE Healthcare) was performed using 140 mL of fetal bovine serum-added MEM medium with an initial cell number of GL37 cells of 5.2 × 10 ⁷ cells. Until the second day of culture, culture for attaching GL37 cells to microcarriers was performed, and hepatitis A virus (moi 0.1) was inoculated on the third day of culture, followed by suspension culture. During the culture period, 70% of the medium was changed every 7 days to maintain the culture.

(2) Cell number measurement and virus total antigen amount measurement The results of cell number measurement during the culture period are shown in FIG. In addition, Table 4 shows the results of measuring the total virus antigen amount on the 21st day of culture. As apparent from FIG. 4 and Table 4, in suspension culture using a microcarrier as a support, the cells cannot endure during the culture period of 3 weeks and are detached from the microcarrier. It was found that the number of cells was not increased and the total antigen amount was significantly reduced compared to the conventional culture.

  According to the above Examples 1 to 4, it is possible to cultivate and maintain hepatitis A virus for a long time without peeling adhesive cells by controlling the discharge and addition of the medium using a fiber material support. confirmed.

  INDUSTRIAL APPLICABILITY The present invention can be used as a method for producing a vaccine using an adhesive cell sensitive virus, particularly a hepatitis A vaccine.

Claims (9)

It includes adhering virus-infected adhesive cells to a fiber material support, filling the support in a culture vessel, and culturing the cells, and in this case, culturing is started while controlling the discharge and addition of the medium. A virus culturing method characterized in that the adhesion rate of the adherent cells on the 20th day after is maintained at 90% or more, wherein the adherent cells infected with the virus are adhered to a support made of a fiber material. Including the following steps (a) to (b), wherein controlling the discharge and addition of the medium comprises the following step (c);
(A) A step of adhering a cell containing virus-infected cells to a culture vessel filled with a support made of a fiber material, and then adhering the cell to the support by taking in and out a medium corresponding to 10% of the culture vessel volume;
(B) a step of ensuring adhesion by taking in and out almost the entire amount of the medium a plurality of times;
(C) A step of culturing while replacing the medium with a fresh one by repeating discharge of the old medium and addition of a new medium. The method according to claim 1, wherein the fiber material is polyethylene terephthalate. The method according to claim 1 or 2, wherein the support is a single-use product. 4. The method according to claim 1, wherein the virus is a Picornaviridae virus. 5. The method according to claim 4, wherein the Picornaviridae virus is hepatitis A virus. The method according to any one of claims 1 to 5, wherein the adherent cell is an African green monkey kidney-derived cell. The method according to claim 6, wherein the African green monkey kidney-derived cell is a GL37 cell. A method for producing an immunogenic composition, wherein a virus is cultured by the method according to any one of claims 1 to 7, and the obtained virus is used. A method for producing a hepatitis A vaccine, wherein hepatitis A virus is cultured by the method according to any one of claims 5 to 7, and the obtained hepatitis A virus is used.

Images