JP3038370B2 - Method of creating suspension culture cell line - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a fish-derived suspension culture cell line, a fish-derived suspension culture cell line produced by the method, and a vaccine and / or diagnostic antigen for infectious fish disease using the cell line. More specifically, a method for producing a suspension culture cell line from a fish-derived adherent culture cell line, a carp epithelioma-derived suspension culture cell line (EPC / NIAH cells), and a Japanese eel kidney-derived suspension culture produced by the method The present invention relates to a cell line (EK-1 / NIAH cell) and a method for producing a vaccine for infectious fish disease and / or an antigen for diagnosis using the cell.

[0002]

2. Description of the Related Art The aquaculture fisheries in Japan have been making remarkable developments, such as an increase in fish species that can be cultured and an improvement in the stability of supply, with the progress of aquaculture technology. However, on the other hand, the mass mortality of farmed fish due to viral diseases has increased rapidly in recent years. Conventionally, as a method of coping with these diseases, only removal of diseased fish and prohibition of movement have been performed, and development of a rational method is desired. Therefore, development of vaccines and diagnostic solutions effective against these diseases is desired.

[0003] The raw material of vaccines and diagnostic solutions is viruses, but fish-derived cell lines such as suspension culture cell lines suitable for mass culture of the virus have not been developed yet. Moreover, even a method for developing a fish-derived suspension culture cell line has not been established.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to establish a technique for producing a fish-derived suspension culture cell line, and to use the technique to produce a fish-derived suspension culture cell such as EPC / NIAH cells and EK-1 / NIAH cells. Creating a stock. This facilitates large-scale culture of fish-derived cells and enables large-scale production of fish disease virus. As a result, it is possible to provide techniques and materials essential for the production and practical application of effective fish disease vaccines and the like.

[0005]

As a result of repeated studies to solve the above-mentioned problems, the present inventors have established a method for producing a fish-derived suspension culture cell line of interest, and have also developed a carp epithelioma-derived cell line. The suspension culture cell line (EPC / NIAH cell) was converted from the cell line (EPC cell) to the suspension culture cell line (EK-1 /
NIAH cells). Furthermore, a method for producing an infectious fish disease vaccine and / or a diagnostic antigen using the suspension culture cell line has been developed.

According to the first aspect of the present invention, the adherent cultured cell line derived from fish is subcultured while acclimating to a culture medium for suspension culture, and the resulting floating cells are removed from the culture medium for suspension culture.
Gather in the center of the bowl and gently float over a 2-3 cm width
This is a method for producing a suspension culture cell line characterized by culturing while stirring . The invention according to claim 2 provides a suspension culture cell line (EPC / NIAH cell) produced from a carp epithelioma-derived adherent culture cell line by the production method according to claim 1.
(FERMP-15793). The invention according to claim 3 provides a suspension culture cell line (EK-1 / NIAH cell) (FERM P-1579) produced from the adherent culture cell line derived from Japanese eel kidney by the production method according to claim 1.
2). The invention according to claim 4 is the invention according to claim 2 or 3
Infectivity characterized by using the suspension culture cell line described in the above.
This is a method for producing a vaccine for fish disease caused by a virus . A fifth aspect of the present invention is a method for producing an antigen for diagnosing fish disease caused by an infectious virus, comprising using the suspension culture cell line according to the second or third aspect.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION EPC cells derived from carp epithelioma and EK-1 cells derived from Japanese eel kidney are originally adherent cells, and adhere to a culture vessel such as a plastic culture bottle and proliferate in monolayer culture. I do. When the proliferating cells are infected with infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, or the like, they have the property of proliferating these viruses well. Thus, the present inventors have attempted to create a cell strain that can be cultured in suspension without impairing the virus propagation properties. That is, the above cells were cultured in a suspension culture medium, and the grown cells were subcultured. In this case, the cells adhered to the container wall and the like were treated with a mixed solution of trypsin and EDTA according to a conventional method and peeled off. Also,
When the cells proliferated to form a complete cell layer without any gaps on the vessel wall or the like, or when the cell density became excessive, the cells were diluted and transferred to a new medium to continue the culture.

[0008] After obtaining the floating cells in this manner, the cells were cultured while being adapted to a medium for suspension culture. The culture at this time is preferably static culture. When free-floating cells formed, they were collected. At that time, the types and amounts of added serum and added components to the suspension culture medium were examined. Fetal bovine serum, newborn bovine serum, adult bovine serum,
The amount of serum or the like from which protein has been removed by adding 8% polyethylene glycol is changed, and the amount of trypsose phosphate broth (3%), lactalbumin hydrolyzate (2%), bactopeptone (1%), etc. The effect on cell proliferation, cell maintenance, etc., was examined by adding in combination with. As a result, the addition of adult bovine serum was effective for culturing EPC / NIAH cells, the cell proliferation was superior to the case where other serum was added, and cell aggregates were hardly formed. In addition, in the culture of EK-1 / NIAH cells, when commercially available fetal bovine serum was added, the cell proliferation was excellent. The amount of serum to be added is preferably 1 to 3% for obtaining floating cells, usually about 2%, and about 5% for EPC / NIAH cells during suspension culture.
In the case of -1 / NIAH cells, about 10% is appropriate.
However, addition of tryptose phosphate broth and the like did not show a favorable effect on cell proliferation. From these results, it was found that a suspension culture cell line derived from fish can be produced by the following method.

As the culture solution used for the suspension culture, known ones can be used. For example, Eagle MEM medium for suspension culture (manufactured by Nissui Pharmaceutical Co., Ltd.), Joklik's modified Eagle medium (manufactured by JRH Bioscience) Etc. are preferably used. In culturing the above-mentioned EPC cells and EK-1 cells, a culture vessel with low cell adhesion is preferable, and for example, a plastic culture bottle for suspension culture cells is suitable. The culturing of the cells is usually performed by stationary culture. The grown cells are peeled off from the container wall or the like using the mixed solution of trypsin and EDTA as described above and dispersed in the culture solution, and further when sufficiently grown, diluted and transferred to a new medium to continue the culture. By so-called subculture, floating cells are obtained.

Next, the cells are adapted to a culture medium for suspension culture (for example, Eagle MEM medium for suspension culture or Joklik's modified Eagle medium). At this time, EPC / NIAH
About 2% of adult bovine serum is effective for culturing cells, and about 2% of fetal bovine serum is effective for culturing EK-1 / NIAH cells. Each cell is adapted to these media. The acclimated cells are maintained by repeatedly exchanging the culture solution without subculture using trypsin and EDTA. The culture medium is appropriately exchanged for about 3 to 4 weeks in the case of culturing EPC / NIAH cells, and about 10 days in the case of EK-1 / NIAH cells. A suitable culture temperature is about 28 ° C. The cells continue to be maintained even after suspension cells start to be generated in the culture supernatant (according to microscopic observation), and when the number of suspension cells increases, the culture vessel is shaken gently so that the contents are not biased (for example, a shaker (eg, , The belly dancer shaker (manufactured by STOVALL Life Science) and gently perform suspension culture. That is,
The cells collect in the center of the suspension culture vessel,
Incubate with a shaking intensity that floats 3 cm wide. The temperature of the culture is suitably 25 to 30 ° C, usually about 28 ° C,
The culturing time is about 7 to 10 days.

[0011] The suspension culture cell line thus produced,
EPC / NIAH cells and EK-1 / NIAH cells have been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology. The deposit number is FERM P-1579
3. The latter is FERM P-15792.

Infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, and viral ascites of yellowtail using the EPC / NIAH cells and EK-1 / NIAH cells which are suspension culture cell lines thus produced. A known method may be applied to produce a vaccine such as a virus and / or a diagnostic antigen. For example, a method of producing a virus stock solution by growing a virus infected with the above-mentioned buoyant cell line using a cell culture method or a tank culture method using a roller bottle can be applied.

[0013]

Next, the present invention will be described in detail with reference to examples. Example 1 EPC cells as parent cells were cultured at 28 ° C. for 3 to 4 weeks using an Eagle MEM medium for suspension culture containing 5% of adult bovine serum. After completion of the culture, the culture supernatant was removed, and the grown cells were treated with trypsin and EDTA. That is,
A mixed solution of 0.1% trypsin and 0.02% EDTA was added, and the mixture was kept warm for 28 minutes in a 28 ° C. flannel. After confirming that the cells had become rounded and began to peel off, a new culture solution was added. In addition, the cells were adjusted so that the cell density became 4 to 5 times, and then dispensed into a new culture vessel. This passage 2
After two passages, floating cells were generated in the culture supernatant. The suspension cells are placed in a plastic culture bottle for suspension culture cells, and the shaker (The Belly Dancer Shaker: ST) can be gently agitated so that the contents are not biased.
OVALL Life Science Co., Ltd.) at 28 ° C 7-10
Cultures were made at daily intervals.

The cells cultured in this manner are subjected to suspension culture using a spinner flask or high-speed rotation culture (8 rpm).
Proliferation was also possible in suspension culture with E. coli, and this property did not change even after passage for 57 generations or more. These cells were designated as EPC / NIAH cells as a fish-derived floating cell line. As described above, this cell has been deposited with the National Institute of Advanced Industrial Science and Technology, and the accession number is FERM.
P-15793.

Example 2 The proliferation of the EPC / NIAH cells obtained in Example 1 was examined. That is, the cells were inoculated into Eagle's MEM medium for suspension culture to which 5% of adult bovine serum was added, and high-speed rotation suspension culture was performed at 28 rpm at 8 rpm for 18 days. The growth curve at this time is shown in FIG. When the proliferation of the cells is expressed in terms of parts by weight of the centrifuged sedimented cells in the culture solution, as is apparent from the figure, the cells spread at 1.0 g / L in 3 to 8 days.
It grew to 5-4 g / L.

Example 3 In Example 2, the culture temperature of EPC / NIAH cells was set to 1
The procedure was performed in the same manner as in Example 2 except that the temperature was changed to 3 ° C. The result at this time is shown in FIG. As is clear from the figure, 2r
Even if suspension culture by pm rotation culture was continued for one month, the cell survival rate was as high as 80% or more.

Example 4 The susceptibility of the EPC / NIAH cells obtained in Example 1 to infectious hematopoietic necrosis virus was examined. That is, EP
C / NIAH cells were infected with the virus at a multiplicity (mo
i.) = inoculated with 0.1, 2% fetal bovine serum pressurized suspension culture for the Eagle's MEM medium with shaking culture in addition, measurement of the virus infection titer in the culture supernatant, about two days after the 10 ⁹ TCI
Proliferated to a level of D ₅₀ / ml and maintained this level after 10 days. This was at the same level as the parent EPC cells. The result is shown in FIG. ○ in the figure is EP
The virus infectivity titer of the virus IHNV N-4 strain in C cells and the black circles are the virus IHNV in EPC / NIAH cells.
The virus infectivity of the N-4 strain is shown, and □ is the virus infectivity of the virus IHNV Y-2 in EPC cells.
Is the virus IHNV Y-2 in EPC / NIAH cells
Indicates the virus infectivity of the strain, Δ indicates the virus infectivity of the virus IHNV Y-6 in EPC cells, and は indicates the EPC.
14 shows the viral infectivity of the virus IHNV Y-6 strain in S./NIAH cells. As is clear from the figure, EPC / N
The growth of IHNV in IAH cells was 10 ^9.5 TCID ₅₀ / 2-4 days after inoculation for viruses of strains N-4 and Y-2.
ml, a 10 ^6.25 TCID ₅₀ / ml in Y-6 strain,
It was almost the same as the virus multiplication in the parent cell line. This revealed that the cells were useful as cells for producing vaccines and diagnostic antigens. Furthermore, it is considered that the cell is also useful as a cell for producing a vaccine for a disease caused by a virus susceptible to the cell.

Example 5 The parental cell, EK-1 cell, was prepared by adding 5% fetal bovine serum and L-1
5 medium was cultured at 28 ° C. for 7 to 10 days using an Eagle MEM medium for suspension culture containing 20% of the 5 medium, and the grown cells were treated with trypsin and EDTA in the same manner as in Example 1 to obtain 4 to 4 days.
After a 5-fold dilution, the cells were passaged for 8 passages, resulting in floating cells in the culture supernatant. Put these buoyant cells into a plastic culture bottle for suspension culture cells, and raise the temperature to 28 ° C on a shaker (The Belly Dancer Shaker: STOVALL Life Science) that can gently agitate so that the contents do not become uneven. For 7 to 10 days.

The cells cultured in this manner are subjected to suspension culture using a spinner flask or high-speed rotation culture (8 rpm).
Proliferation was also possible by suspension culture with E. coli, and this property did not change even if the cells were passaged for 27 or more passages. These cells were designated as EK-1 / NIAH cells as a fish-derived suspension culture cell line. As described above, this cell has been deposited with the National Institute of Advanced Industrial Science and Technology,
FERM P-15792.

Example 6 The proliferation of the EK-1 / NIAH cells obtained in Example 5 was examined. That is, 10% fetal bovine serum and 20% L-15 medium were used.
% Of the cells were inoculated into Eagle's MEM medium for suspension culture, and high-speed rotation suspension culture was performed at 28 rpm at 8 rpm for 6 days. The growth curve at this time is shown in FIG. When the proliferation of the cells is expressed in terms of parts by weight of the centrifuged sedimented cells in the culture solution, as apparent from the figure, the cells spread at 1.0 g / L grew to 3.5 to 4 g / L in 4 to 6 days. .

Example 7 The procedure of Example 6 was repeated, except that the culture temperature of EK-1 / NIAH cells was changed to 13 ° C. The result at this time is shown in FIG. As is clear from the figure, 2
Even if suspension culture by rotation culture at rpm was continued for 1 month or more, the survival rate was 80% or more.

Example 8 The sensitivity of the EK-1 / NIAH cells obtained in Example 5 to infectious pancreatic necrosis virus was examined. That is, EK
-1 / NIAH cells were inoculated with the virus at moi = 0.1, Eagle's MEM medium for suspension culture containing 2% of fetal calf serum and 20% of L-15 medium was added, and the cells were shake-cultured. When the virus infection titer was measured, it was 1 in about 4 days.
0 ⁹ 10 ¹⁰ grow to the level of the TCID ₅₀ / ml, up to 10 days later, keeping the same level. This is the parent stock E
It was at the same level as K-1 cells. The result is shown in FIG. The circle in the figure indicates the virus IPNVA in EK-1 cells.
b, the virus infectivity of the virus IPNV Ab strain in EK-1 / NIAH cells, □, the virus infectivity of the virus IPNV SP strain in EK-1 cells, and ■, EK -1 / NIAH cells show the virus infectivity of the virus IPNV SP strain, △ shows the virus infectivity of the virus IPNV VR299 strain in EK-1 cells, and ▲ shows the virus IPNV VR299 strain in EK-1 / NIAH cells. FIG.
As is clear from the figure, I in EK-1 / NIAH cells
The growth of PNV was between 10 ^{9.0 and} 10 ^10.0 TCID ₅₀ / ml at 4 to 6 days after inoculation for the Ab strain and SP strain virus, VR
For 299 strains of virus, 10 ^9.0 TCI 10 days after inoculation
D ₅₀ / ml, which was almost the same as that of the parent strain.

Example 9 The sensitivity of EK-1 / NIAH cells to viral ascites virus YAV strain of yellowtail was examined. That is, the virus YAV strain was inoculated at moi = 0.1 and bovine fetal serum 2
%, Eagle ME for suspension culture containing 20% L-15 medium
Shake culture by adding M medium was measured viral infection titer in the culture supernatant, approximately 4 days at 10 ⁸ ~10 ⁹ TCID ₅₀ /
Proliferated to the ml level and remained the same until day 10 onwards. It is as sensitive as the parental EK-1 cell. The result is shown in FIG. In the figure, ○ indicates the infectivity of the virus YAV strain in EK-1 cells,
Indicates the infectivity of the virus YAV strain in EK-1 / NIAH cells. As is clear from the figure, EK-1 / NIA
Propagation of the virus YAV strain in H cells was 1 day 8 days after inoculation.
^09.0 TCID ₅₀ / ml, which was almost the same as that of the parent strain. From the results of Examples 5 to 9, E
It has been elucidated that K-1 / NIAH cells are useful as cells for producing vaccines and diagnostic antigens. further,
It is also considered useful as a cell for producing a vaccine for a disease caused by a virus susceptible to the cell.

[0024]

According to the present invention, the following effects can be obtained. Since a fish-derived suspension cell line can be produced, a large-scale culture of fish-derived cells and fish disease-causing virus can be easily performed. Infectious hematopoietic necrosis virus proliferates well in EPC / NIAH cells, and the cells are used for the production of biological products such as vaccines for infectious hematopoietic necrosis, thereby realizing the vaccine. be able to. In EK-1 / NIAH cells, infectious pancreatic necrosis virus and yellowtail viral ascites virus proliferate well, and these cells can be used for biological treatment of infectious pancreatic necrosis and yellowtail viral ascites vaccine. The vaccine can be put to practical use by using it for production of a preparation. EPC / NIAH cells and EK-1 / NIAH cells
It is applicable to the production of biological products such as vaccines against other viral fish diseases.

[Brief description of the drawings]

Fig. 1. EPC / suspension culture by high-speed rotation culture
It is a proliferation curve of a NIAH cell.

FIG. 2 shows the storage stability of EPC / NIAH cells at 13 ° C.

FIG. 3 is a growth curve of infectious hematopoietic necrosis virus on EPC / NIAH cells.

FIG. 4. EK-1 in suspension culture using a high-speed rotating culture gun
9 is a proliferation curve of the / NIAH cells.

FIG. 5 shows the storage stability of EK-1 / NIAH cells at 13 ° C.

FIG. 6 is a growth curve of infectious pancreatic necrosis virus in EK-1 / NIAH cells.

FIG. 7 is a growth curve of yellowtail viral ascites virus on EK-1 / NIAH cells.

────────────────────────────────────────────────── ─── Continued on the front page (56) References Fish Disease Research, 1990, Vol. 25, No. 2, p. 69-79 Fish Disease Research, 1995, Vol. 30, No. 1, p. 47-52 Fish Disease Research, 1996, Vol. 31, No. 2, p. 59-63 “Summary of Special Research / Research Results”, Secretariat of the Agriculture, Forestry and Fisheries Technology Council, November 1994, p. 78-80 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 5/00 JICST file (JOIS)

Claims (5)

(57) [Claims] 1. A subculture is carried out while acclimating a fish-derived adherent culture cell line to a suspension culture medium, and the resulting suspension cells are collected at the center of a suspension culture container,
A method for producing a suspension-cultured cell line of fish cells, which comprises culturing while gently stirring to float a 3 cm width . 2. A suspension culture cell line (EPC / NIAH cell) (FERM P-1579) produced from a carp epithelioma-derived adherent culture cell line by the production method according to claim 1.
3). 3. A suspension culture cell line (EK-1 / NIAH cell) (FERMP-15) produced from the adherent culture cell line derived from Japanese eel kidney by the production method according to claim 1.
792). 4. A fish disease caused by an infectious virus, wherein the suspension culture cell line according to claim 2 or 3 is used.
A method for producing Kuching . 5. The suspension culture cell line according to claim 2 or 3.
Diagnosis of fish disease caused by infectious virus, characterized by using
A method for producing a sever antigen .