JP2859679B2 - New cell line - Google Patents

Description

The present invention relates to a novel Chinese hamster ovary cell (CHO cell) cell line that can be cultured in a serum-free and protein-free medium.
The present invention relates to a novel CHO cell which can be subcultured in a serum-free protein-free medium for 2 months or more and has no glutamine requirement.

The cell obtained in the present invention can be used as a host cell into which a gene encoding a useful protein is introduced.

2. Description of the Related Art Mass culture of animal cells is used to produce viruses and vaccines, and also to produce interferons and monoclonal antibodies from leukocytes and hybridomas. In recent years, with the development of genetic recombination technology, proteins can be produced as natural forms of biologically active substances present in living organisms. Sex is being recognized again.

One of the problems of producing useful substances by mass culture of animal cells is that serum must be added to a basal medium for the growth of animal cells. Serum is not only expensive but also contains a wide variety of proteins, which complicates the purification of the target protein. In addition, the quality varies from lot to lot, and the effect on cell growth is large. Therefore, development of cells capable of growing in a serum-free medium and further in a protein-free medium has been desired.

As a method using a serum-free medium, a method of culturing CHO cells in a serum-free medium containing insulin, transferrin, and selenium [in vitro cellular and developmental biology (in vitro Cellular &
Developmental Biology), Vol. 21, No. 10, 588, 1985
A method of culturing Hela cultivation in a serum-free medium containing insulin, hydrocortisone, transferrin, epidermal growth factor, and fibroblast growth factor [Procedure of the National Academy of Science (Proc. Natl. Acad. Sci. .) USA, Volume 75, Issue 2, 9
01, 1978], a method of culturing BHK cells in a serum-free medium containing insulin, transferrin, fibroblast growth factor, fibronectin, and oleic acid [Journal of Cellu
lar Physiology), 114, 215, 1983]. Cells that can be grown in a serum-free medium include monoclonal antibody-producing mouse hybridomas (JP-A-58-63).
No. 385), conditioned cells of Namaluba cells derived from human Burkitt tumor producing interferon (Japanese Patent Application Laid-Open No. 60-6190), and grown in a medium containing only insulin as a protein growth factor.
Adapted CHO cells (JP-A-63-192381) are known.

Although combinations of growth factors have allowed serum-free culture of various cells, some growth factors are expensive. Also, from the viewpoint of pharmaceutical production by mass culture, it is not preferable that a small amount of heterogeneous protein is mixed in the growth factor, and the growth factor to be used is required to be very high in purity.

Thus, there is a need for cells capable of growing in a serum-free medium and growing in a protein-free medium. It is difficult to culture the serum-free conditioned cells already reported in a protein-free medium, and the growth of the cells is stopped or the cell viability is remarkably reduced, resulting in poor substance production efficiency.

Various P3 strains adapted by Katsuta et al. As cell lines grown in a protein-free medium (Methods in Cell Biology, 6, 1973)
There is. However, none of them are CHO cells and are glutamine-requiring strains.

Furthermore, when culturing these cells, a filter is used to sterilize at least one of the components of the culture medium, and contaminants such as microorganisms and viruses cannot be strictly denied. In order to prevent the contamination of these microorganisms and viruses, it is necessary to use sterilization by heating as a sterilization method.If protein components or nutrients such as glutamine are contained, heat denaturation due to heating and sterilization will result. It cannot be heated and steamed because of decomposition. Glutamine non-requiring cells include cells derived from normal rat hepatocytes that grow in a protein-free and glutamine-free medium [Dokkyo J. Med. Sci, 9, 97-105, 1982),
Namalva cells that require insulin and transferrin as proteins but grow in glutamine-free medium [Cytotechnology, Vol. 151-158.
P. 1988], making glutamine non-requiring in the presence of serum
BHK21 cells African green monkey cells [Morden Approaches to Animal Cell Technology (Mo
dern Approaches to Animal cell technology)], and K562K ₁ cells [Fifth Next-Generation Industrial Technology Symposium Proceedings, pp. 129-141, 1987], which grow in a completely steam-sterilized medium (protein-free, glutamine-free medium). Are known.

However, there is no report that it is possible to grow a CHO cell substrain in a serum-free and protein-free medium and further to grow in a glutamine-free medium.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel cell line of CHO cells that can be subcultured for more than 2 months in a serum-free and protein-free medium, that can be subcultured for more than 2 months in a serum-free and protein-free medium, and that glutamine is not required. An object of the present invention is to provide a novel CHO cell having the property.

Means for Solving the Problems According to the present invention, a novel cell line of CHO cells that can be subcultured in a serum-free protein-free medium for 2 months or more, can be subcultured in a serum-free protein-free medium for 2 months or more, and does not require glutamine It is possible to provide a novel CHO cell having the property.

 Hereinafter, the present invention will be described in detail.

1) Method of habituation As a parent strain for habituation, Chinese hamster ovary cells (CHO cells) are used.
Any cell can be used as long as it is a cell derived from a cell that is difficult to propagate in a serum-free medium. As a preferred example, a dihydrofolate reductase-deficient CHO cell d ^- DXB11 strain derived from a CHO cell, which is a sub-strain of the CHO-K1 strain (ATCC CCL61) [Proceeding of the National Academy of Sciences (Proc. Natl. Acad. .Sci.) USA, 77
Vol. 4216-4220, 1980, DeNA (DN
A) Vol. 3, pp. 297-308, 1984].

Although CHO cells are inherently adherent cells, it is preferable to use a suspension culture method for acclimation.

In suspension culture, the cell seeding concentration is 0.5-8 × 10 ⁵ cells / m
It is preferable to maintain this concentration during the culture. Further, it is necessary to change the medium periodically so that the pH of the medium does not become 6.5 or less and the nutrient sources in the medium are not depleted. If the number of cells is significantly reduced at the time of replacing the medium, the cells are concentrated by centrifugation or removal of the supernatant. Conversely, if the number of cells is improved, the cells are diluted to adjust the number of cells. Particularly at the beginning of acclimation, the number of cells may be significantly reduced. The acclimatization period requires two months or more.

A specific adaptation method will be described using the CHO cell d ^- DXB11 strain as an example.

1-1) Method for adaptation to serum-free medium CHO cell d ^- DXB11 strain was used as a seed cell, and the number of cells (4 × 10 ⁵ cells / ml) that almost caused contact inhibition in the attached state was determined by using a serum substitute instead of serum. Seed in basal medium (40 ml) supplemented with. This is cultured at 37 ° C for 2 to 4 days. The cells growing in suspension are then centrifuged at 1500 rpm for 5 minutes and resuspended in fresh basal medium (40 ml).

By repeating such an operation, the growth of the conditioned strain is stabilized and the survival rate is improved. This adaptation operation is repeated for 2 months or more until the survival rate of the adapted strain approaches the survival rate when the parent strain is cultured in a medium that can normally grow.

As the basal medium, a general medium used for animal cell culture can be used. For example, RPMI-1640,
MEM, Dulbecco MEM, Ham F12, Ham F10, DM160, DM170
(Farto Pharmaceutical Co., Ltd.), Dulbecco MEM and Ham F12 1: 1
Mixed media, RPMI-1640, Dulbecco MEM and Ham F12
A 2: 1: 1 mixed medium can be used.

Transferrin 0.1-100 μg / m as serum replacement
l, preferably 1-10 μg / ml, insulin 0.1-100 μg / m
l, preferably 1-10 μg / ml, folic acid 0.1-100 μg / ml, preferably 1-10 μg / ml, selenous acid 0.1-100 × 10 ⁻⁸ M,
Preferably 1 to 10 × 10 ^-8 M, Pepol B-188 (manufactured by Toho Chiba Chemical Industry Co., Ltd.) 0.001 to 10%, preferably 0.01 to 1%,
Dimethyl-α or β-cyclodextrin (manufactured by Toshin Chemical Co., Ltd.) 1 μg to 10 mg / ml, preferably 10 to 100 μg / m
l, polyethylene glycol 20000 0.01-10%, preferably 0.01-0.1% in combination.

Further, as additives, Hepes buffer 1-300 mM, preferably 5-25 mM, glutamine 0.5-10 mM, preferably 1
~ 5 mM, ferrous sulfate 0.1 ~ 100μM, preferably 5 ~ 50μ
A mixture of non-essential amino acids for M and MEM is used in combination of 0.1 to 25 ml / 100 ml, preferably 1 to 10 ml / 100 ml.

Thus, CHO cells that can grow in a serum-free medium can be obtained. The obtained cells are designated as CHO cell KJM-1 strain.

The CHO cell KJM-1 strain is adhesion-independent and can grow in a serum-free medium in a suspended state.

1-2) Method for adaptation to serum-free protein-free medium The serum-free conditioned CHO cell KJM-1 strain obtained above was used as a seed cell at a cell density of about 4 × 10 ⁵ cells / ml in 1-1). The cells are seeded on a serum-free protein-free medium (40 ml) having a composition obtained by removing transferrin and insulin from the used serum-free medium. This is cultured at 37 ° C for 2 to 4 days. The cells growing in suspension are then centrifuged at 1500 rpm for 5 minutes and resuspended in the above fresh medium (40 ml).

By repeating such an operation, the growth of the conditioned strain is stabilized and the survival rate is improved. This adaptation operation is repeated for 2 months or more until the survival rate of the adapted strain approaches the survival rate when the parent strain is cultured in a medium that can normally grow.

As the basal medium, serum substitute and other additives, the same as those described above can be used except for transferrin and insulin.

In this way, CHO cells that can grow in a serum-free protein-free medium for two months or more can be obtained. The obtained cells are designated as CHO cell KJM-1PF strain.

2) Giving glutamine non-requirement The obtained serum-free protein-free conditioned CHO cell KJM-1PF strain was
Glutamine non-requirement can be imparted to the CHO cell strain KJM-1PF by culturing in a medium containing an excess of glutamic acid instead of the glutamine of the serum-free protein-free medium composition used in -2).

Excess glutamic acid is about 10 to 30 times the glutamic acid content contained in a general medium,
It is preferable that the medium be contained about twice as much.

In Example 3, instead of glutamine, the same amount of glutamic acid as glutamine (20 times the amount of glutamic acid generally contained in the medium) was contained.

In this way, CHO cells that can grow in a serum-free protein-free medium for two months or more and have no glutamine requirement can be obtained. The obtained cells are designated as CHO cell KJT-1 strain.

The CHO cell KJT-1 strain was
Deposited on February 27, 2012 at the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microfabrication Research Article No. 2776 (FERM BP-2776).

The growth of CHO cells KJT-1 strain in a completely steam sterilized medium and the confirmation of glutamine non-requirement were determined by using a glutamine in a culture solution as an amino acid automatic analyzer JLC as a linear verification method.
There is a method of analyzing by -200A (manufactured by JEOL Ltd.).
Glutamine, a component not included in advance, may be detected in the culture supernatant, but as a result of analyzing the culture solution obtained by culturing the CHO cell KJT-1 strain, the presence of glutamine was not confirmed. . As an indirect proof method, CH
There is a method that can be proved by the growth curve of the O cell KJT-1 strain and the induction of glutamine synthase.

These results demonstrate that the steam-sterilized medium does not contain any glutamine, and that the CHO cell strain KJT-1 can grow on a medium that does not contain any glutamine.

 Hereinafter, examples and reference examples of the present invention will be described.

Example 1 Growth of Strain-Free Medium-Adapted Strain Transferrin 10 μg / m
l, insulin 10 μg / ml, folic acid 10 μg / ml, selenite 1.2
5 × 10 ^-8 M, Pepol B-188 0.1%, Hepes buffer 7.5m
M, glutamine 2 mM, ferrous sulfate 10 μM, non-essential amino acid mixture for MEM 10 ml / 100 ml, and 40 ml of serum-free medium (hereinafter referred to as medium a) containing sodium bicarbonate 0.15% were placed in a 75 cm ² culture flask. CHO cell d ^- DXB11 strain (serum dependent)
Was seeded at a cell density of about 4 × 10 ⁵ cells / ml.

After static culture at 37 ° C. for 3 days, cells growing in suspension were collected by centrifugation at 1500 rpm for 5 minutes, and resuspended in 40 ml of a fresh serum-free medium having the same composition as described above.

The viability decreased as the cells were passaged. On the 7th day after the start of the culture, the viability decreased to 7%, but the subculture was repeated as it was, and the viability of the cells was improved. Survival rate 2 months after starting culture
It recovered to 80% or more, and the number of cells became proliferable to about 1 × 10 ⁶ cells / ml. Since then, stable growth has been observed for 17 months or more after the start of culture.

In this way, the CHO cell KJM-
One strain was obtained.

Example 2 Breeding of a serum-free protein-free medium-conditioned strain Using ham F12 as a basal medium, selenite 1.25 × 10 ⁻⁸ M, Pepol B-188 0.1%, Hepes buffer 7.5 mM, glutamine 2 mM, ferrous sulfate 10 μM Non-essential amino acid mixture for MEM
40 ml of a serum-free protein-free medium (hereinafter, referred to as medium b) to which 10 ml / 100 ml and sodium bicarbonate 0.15% are added is placed in a 75 cm ² culture flask, and CHO cell KJM-1 strain is about 4 × 10 ⁵ cells / ml. At a cell density of After culturing at 37 ° C for 3 days,
The cells growing in suspension were collected by centrifugation at 1500 rpm for 5 minutes and resuspended in 40 ml of a fresh serum-free and protein-free medium having the same composition as described above.

The viability decreased as the cells were passaged. On the 7th day after the start of the culture, the viability decreased to 20%, but by repeating the subculture as it was, the viability of the cells was improved. Two months after the start of the culture, the survival rate recovered to 80% or more, and the number of cells could be expanded to about 1 × 10 ⁶ cells / ml.

Thus, the CHO cell strain KJM-1PF, which is a serum-free and protein-free medium-conditioned strain, was obtained.

Example 3 Glutamine Non-Requirement Addition Glutamine of Serum-Free Protein-Free Medium Composition Used in Example 2
Medium (40 ml) supplemented with 4 mM glutamic acid instead of 2 mM
Then, the CHO cell KJM-1PF strain obtained in Example 2 was seeded, and 3
The cells were cultured at 7 ° C for 3 days.

In this way, CHO cells KJT-1 which can be subcultured for more than 2 months in a serum-free protein-free medium and have no glutamine requirement.
A strain was obtained.

Reference Example 1 Confirmation of impartation of glutamine non-required ham F12 was used as a basal medium, selenite 1.25 × 10 ⁻⁸ M, Pepol B-188 0.1%, Hepes buffer 7.5 mM, ferrous sulfate 10 μM, glutamic acid 4 mM, A 40 ml serum-free protein-free medium containing 10 ml / 100 ml of a mixture of non-essential amino acids for MEM is steam-sterilized, and a boiled soda that is separately steam-sterilized to a concentration of 0.15% when used is used (hereinafter referred to as medium c). , CH
The O cell KJT-1 strain was cultured at 37 ° C.

 FIG. 3 shows the results.

Reference Example 2 Comparison of Intracellular Glutamine Synthetase Activity The CHO cell d ^- DXB11 strain and the CHO cell KJT-1 strain were each cultured at 37 ° C. for 4 days in the medium c.

CHO cells d ^- DXB11 strain in medium b
CHO cells KJM-1PF were cultured at 37 ° C. for 4 days.

After the culture, the glutamine synthetase activity in each cell was compared. The results are shown in Table 1.

Effect of the Invention According to the present invention, a novel cell line of CHO cells that can be subcultured for more than 2 months in a serum-free protein-free medium and a novel cell line that can be propagated in a serum-free protein-free medium for more than 2 months and that does not require glutamine CHO cells can be provided.

[Brief description of the drawings]

FIG. 1 shows a cell growth curve when the CHO cell KJM-1 strain was cultured in the medium a at 37 ° C. and the CHO cell d ^- DXB11 strain at 37 ° C.
The lower part shows a growth curve when cultured in a medium containing 5% fetal bovine serum instead of transferrin and insulin in the composition of medium a. In the figure, a solid circle represents a CHO cell KJM-1.
The solid line with a solid circle represents the growth curve of the CHO cell d ^- DXB11 strain. FIG. 2 shows a growth curve when the CHO cell KJM-1PF strain was cultured at 37 ° C. in medium b. FIG. 3 shows a growth curve when the CHO cell KJT-1 strain was cultured at 37 ° C. in medium c. In each of the figures, the vertical axis represents the number of living cells (1 × 10 ⁵ cells / ml), and the horizontal axis represents the number of days of culture.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 5/10 C12R 1:91)

Claims (3)

(57) [Claims] 1. A serum-free and protein-free medium (hereinafter referred to as "medium").
It is called a serum-free protein-free medium. ), A new Chinese hamster ovary cell (CHO cell) that can be propagated for more than 2 months
Cell line. 2. A novel CHO cell which can be subcultured for more than 2 months in a serum-free and protein-free medium and has no glutamine requirement. 3. The novel CHO cell according to claim 2, wherein the serum-free protein-free medium is a completely steam-sterilized medium.