JP2897043B2 - Complete synthetic medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention is a medium for culturing cells, which does not contain any proteins, hormones, steroid components and cell growth factors, and is composed of only known chemical substances. It relates to a completely synthetic medium to be used.

(Prior Art) For culturing animal cells, a culture solution obtained by adding about several to 20% of animal serum (for example, fetal bovine serum) to a basic synthetic medium is usually used. This serum medium contains high concentrations of serum proteins, and these proteins are
It has chemical properties and a very broad molecular weight distribution.

Therefore, depending on the animal cell culture, for example, from a hybridoma to a monoclonal antibody, or a recombinant (cell)
When a recombinant product is produced from E. coli, it is very difficult to purify the product with high purity in serum culture, and the purification step requires a great deal of labor and cost. In addition, it is inevitable that there is a difference in the quality of animal serum between production lots.
The disadvantage is that the supply is unstable and expensive. Therefore, when producing a monoclonal antibody or a recombinant product in animal cells, a serum-free medium is frequently used as a culture solution.

The serum-free medium, unlike the serum medium, is composed of all known components, has no quality difference between production lots, and has a stable supply. However, it usually contains proteins such as serum albumin and transferrin or growth factors with a molecular weight of about tens of thousands (eg, insulin), etc., and the total amount of protein in the medium reaches several tens μg / ml to several mg / ml. .
Although this concentration is sufficiently low as compared with the serum medium, the concentration is considerably high when the monoclonal antibody or the recombinant product is purified from the medium, and the purification process becomes complicated when the purification is performed with high purity. easy. Although the price of the medium is lower than that of the serum medium, the production cost is considerably higher than, for example, the method of producing a monoclonal antibody by culturing the hybridoma in the mouse intraperitoneal cavity.
When monoclonal antibodies (especially human antibodies) or recombinant products are directly administered to the human body, protein components other than the target substance and pyrogens (pyrogen)
Must be completely removed, requiring very sophisticated purification techniques.

Therefore, when producing useful biologically active substances using cells, the medium used does not contain proteins, cell growth factors, hormones and steroids, and all components are composed of known chemical products. It is ideal that all components are low molecular substances (for example, a molecular weight of 2000 or less). If such a medium can be developed, unlike a serum-free medium, it does not contain proteins, growth factors, etc., which are difficult to obtain in large quantities. It is also very easy to do.

Recently, few protein-free media have been reported (eg, Jan Kovar et. Al, Biotech.let., 9 (19
87) 259-264, Yves-Jacques Schneider, J. Immunol.Me
th., 116 (1989) 65-77), all of which use high concentrations of iron citrate, ethanolamine, ascorbic acid (vitamin C), trace elements, and hormones (such as estradiol) in conventional basal media. With the addition, mouse hybridomas can be cultured, but the composition is very complicated.

(Problems to be Solved by the Invention) An object of the present invention is to provide a medium for animal cells that does not contain any protein components, cell growth factors, and hormones, and is composed of only known chemical substances (chemical products). An object of the present invention is to provide a component of a complete protein synthesis medium. Also, to provide a method for preparing a protein-free medium which is inexpensive and suitable for various animal cells such as mouse hybridomas, human-human hybridomas, and human cancer cells, which can be used not only for medical and biological research but also for industrial use. It is in.

(Means for Solving the Problems) The present inventors simply add a newly known low-molecular-weight chemical substance (chemical product) to a conventional basic synthetic medium for cells, and can prepare a normal serum medium or serum-free medium. As a result of searching for a substance that constitutes a protein-free complete synthetic medium that can be used more satisfactorily than the above and examined a combination of compositions as simple as possible, the present invention was completed. That is, the present invention is a complete synthetic medium for cell culture containing a: iron citrate, b: ethanolamine, and c: linoleic acid, oleic acid or taurine and does not contain proteins, cell growth factors, hormones and steroids.

 The details of the present invention will be described below.

The medium for cell culture of the present invention is not particularly limited as long as it is a serum-free basic synthetic medium used for ordinary cell culture and does not contain proteins, cell growth factors, hormones, and steroids. In the case of long-term culture or high-density culture, a medium containing amino acids and vitamins at a high concentration, for example, E-
RDF medium (Hiroki Murakami, Nippon Nougeikagaku kaish
i 58 (1984) 575-583), RPMI-1640 / DME / F-12 (2: 1:
1) Medium, DME / F-12 (1: 1) medium is suitable.

The concentration of iron citrate added to the medium of the present invention was determined by Jan Kova.
r (HYBRIDOMA, 7 (1988) 255-263)
About 500 μM is suitable, and 25-100 μM is most desirable.

The concentration of ethanolamine to be added is different from the report of Murakami et al. (Patent Publication No. 63-18463), preferably about 20 to 500 μM, and most preferably 50 to 200 μM.

Furthermore, when added to the medium of the present invention, the concentration of linoleic acid or oleic acid is preferably about 0.1 to 1 μg / ml, most preferably 0.2 to 0.7 μg / ml.

When added to the medium of the present invention, the concentration of taurine is preferably about 4 to 1000 μM, and most preferably 5 to 100 μM.

Among the above additional components, it is most preferable to add linoleic acid and taurine or oleic acid and taurine together with citric acid and ethanolamine. This is because it is most suitable for culturing cells.

In addition to these additional components, third components other than proteins, cell growth factors, hormones and steroids may be added.

(Effect of the Invention) As shown in the present invention, a protein-free complete synthetic medium suitable for cell culture can be easily obtained by adding iron citrate, ethanolamine, and linoleic acid, oleic acid, or taurine to a basic synthetic medium. Can be prepared. Moreover, unlike the conventional protein-free medium, the composition of the components is very simple, and various animal cells including human-derived cells can be cultured.

All of these additional components are inexpensive and very easy to obtain, and have low molecular weight as components constituting a protein-free medium, proteins contained in a serum-free medium,
It has many desirable properties not found in growth factors.

When animal cells are serum-free cultured to produce a bioactive substance, several tens μg / ml to several mg contained in the serum-free medium
It is necessary to purify a substance produced by cells from a protein having a concentration of / ml, and when purifying a product with high purity, there is a problem that the purification process is likely to be complicated. On the other hand, if a physiologically active substance is produced using a protein-free medium prepared by adding the components of the present invention, the medium itself is composed of low molecular weight components and does not contain any protein components. The product can be easily purified to high purity. When the produced substance is to be administered to the human body, it is necessary to completely remove protein components and pyrogens other than the target substance. In the case of a protein medium, unlike a serum-free medium, it is easy to prepare a medium that does not contain pyrogen in advance.

The use of the medium of the present invention makes it possible to easily prepare a very inexpensive protein-free medium as compared with a normal serum-free medium because all of these components are very inexpensive, and to culture cells in large quantities. When material production is carried out, the cost of raw materials can be significantly reduced.

Moreover, since the additive components of the present invention are all chemical products without any biological substances such as proteins and growth factors contained in the serum-free medium, protein-free complete synthesis prepared using these additives The medium does not cause a lot-to-lot difference between preparations, and a homogeneous medium can always be obtained.

Further, unlike the proteins and growth factors contained in the serum-free medium, these additional components are easy to obtain in large quantities, and are extremely simple in composition as compared with conventional media. It is very easy to prepare a large amount of medium even when culturing cells on a large scale on a large scale.

By using the medium of the present invention, it is possible to subculture various cells such as mouse hybridomas, human-human hybridomas, and human cancer cells, and it can be sufficiently used for production of monoclonal antibodies by the hybridomas.

(Example) An example is shown below. However, the present invention is not limited to only these examples.

Example 1 30 μg / ml human transferrin (Sigma) (hereinafter abbreviated as Tf), 20 μM ethanolamine (Nacalai Tesque),
25nM sodium selenite (Nacalai Tesque), 1.2g / l
E-RD prepared by adding sodium hydrogen carbonate
The effect of iron citrate on cell growth was examined using mouse hybridoma A2, C7, and 17 cells subcultured in F medium (Kyokuto Pharmaceutical) (hereinafter abbreviated as TES medium).

First, ferric citrate (Nacalai Tesque) was dissolved in pure water, and 4N sodium hydroxide was added to adjust the pH to 6 to 8 to prepare a 100 mM iron citrate solution. Then, a TES medium prepared by removing transferrin from the TES medium was supplemented with a 100 mM iron citrate solution containing 0.1 μg / ml of linoleic acid and a final concentration of 0 to 500 μM iron citrate. Serum or final concentration
Hybridomas subcultured with TES medium were inoculated into a dish containing 30 μg / ml Tf at a concentration of 1 × 10 ⁵ cells / ml, cultured at 37 ° C. and 5% CO ₂ , and grown on day 3 of subculture. The number of cells was counted to examine cell proliferation. The results are shown in FIG.

As is clear from FIG. 1, the addition of 10 to 500 μM iron citrate to the medium caused the hybridoma to grow sufficiently, and the optimal concentration was 50 μM.

Therefore, a medium prepared by adding 50 μM iron citrate and 0.1 μg / ml linoleic acid to the ES medium (hereinafter abbreviated as FES medium)
In the above hybridomas, A2 and C7 cells were placed at 37 ° C and 5% CO
After passage for one week at ₂ , the proliferative activity in the FES medium supplemented with a final concentration of 30 μg / ml Tf was compared with that without the supplement. 1 × 1
Inoculated at a concentration of 0 ⁵ cells / ml.
Shown in Clearly, all of the hybridomas grew well in the FES medium, showed no difference from the case where Tf was added, and showed growth comparable to the example in which 10% serum was added in FIG.

Example 2 The effect of ethanolamine on cell growth was examined using mouse hybridoma C7 and D3 cells subcultured in the FES medium of Example 1.

First, ethanolamine (Nacalai Tesque) was dissolved in pure water to prepare an 80 mM ethanolamine solution. Incidentally
The hybridoma subcultured in the FES medium was inoculated into a dish at a concentration of 1 × 10 ⁵ cells / ml in a medium in which an 80 mM ethanolamine solution was added to a FES medium to a final concentration of 20 to 420 μM, and the mixture was cultured at 37 ° C., 5 ° C. On day 3 of the passage with% CO ₂ , the number of viable cells was counted to examine cell proliferation. The results are shown in FIG. As is clear from FIG. 2, the hybridoma showed sufficient growth in all the added concentration ranges, and the concentration was 10
0 μM was optimal.

Example 3 The effect of linoleic acid on cell growth was examined using mouse hybridoma A2, C7, and 17 cells subcultured in the FES medium of Example 1.

First, linoleic acid (Nacalai Tesque) was dissolved in ethanol to prepare a 200 mg / ml linoleic acid solution. Then
The hybridoma subcultured in the FES medium was inoculated in a dish at a concentration of 1 × 10 ⁵ cells / ml in a medium in which a linoleic acid solution was added to the FES medium to a final concentration of 0.02 to 20 μg / ml. On day 3 of the passage with% CO ₂ , the number of viable cells was counted to examine cell proliferation. The results are shown in FIG. The hybridoma showed sufficient growth, and the concentration was optimally 0.5 μg / ml.

Example 4 The effect of taurine on cell proliferation was examined using mouse hybridoma A2, C7, and 17 cells subcultured in the FES medium of Example 1. First, taurine (Nacalai Tesque) was dissolved in pure water to prepare a 200 mM taurine solution.
Then, a final concentration of 0 to 50 μM in ES medium supplemented with citrus iron was added.
In a medium to which a taurine solution was added to be 1000 μM,
Hybridomas that had been subcultured in FES medium were collected at 1 × 10 ⁵ cells /
Plant in dish at a concentration of ml and subculture 3 at 37 ° C, 5% CO ₂
On the day, the number of viable cells was counted to examine cell proliferation. FIG. 4 shows the results. Obviously, at all concentrations added, the hybridomas showed sufficient growth, with a concentration of 10
μM was optimal.

Example 5 In the E-RDF medium of Example 1, 50 μM ferric citrate, 100
μM ethanolamine, 0.5 μg / ml linoleic acid, 10 μM
Taurine, 25 nM sodium selenite, and 1.2 g / l sodium bicarbonate were respectively added to prepare a human-human hybridoma C5 cell (Murakami, H. et.al.In Vitro
Cell. Develop. Biol., 21: 593-596; 1985).
Proliferation on PFC medium supplemented with 5% fetal bovine serum was compared with that without the addition.

1 × 10 ⁵ cells / ml at 37 ℃
When the number of cells on the third day of culture was counted with 5% CO ₂ , the cell count was 8.0 × 10 ⁵ cells / ml in the serum-supplemented medium, and 5.1 × 10 5
It was 10 ⁵ cells / ml. The growth of C5 in the PFC medium was shown to be sufficient, though slightly inferior to that when 10% serum was added.

Example 6 In order to examine whether a monoclonal antibody can be produced by a hybridoma using the medium of the present invention, the PF of Example 5 was used.
Using the C medium, mouse hybridoma D3 cells were cultured.

D3 cells passaged for 1 week in PFC medium are seeded at 1 × 10 ⁵ cells / ml in a dish, and cultured for 4 days at 37 ° C. and 5% CO ₂ , and the antibody concentration in the medium produced by the cells is increased. Was measured over time by enzyme-linked immunosorbent assay. FIG. 5 shows the results.

As is clear from FIG. 5, both the cell number and the antibody amount increased.

Example 7 Human colon cancer cells COLO as cells other than hybridomas
Growth of 201 in PFC medium was examined. COLO201 cells subcultured using a DME medium supplemented with 10% fetal bovine serum were converted into single cells with a trypsin solution, and 1 × 10 ⁵ cells were added to a PFC medium.
The cells were implanted at a concentration of s / ml and cultured for 4 days at 37 ° C. and 5% CO ₂ to examine cell proliferation. FIG. 6 shows the results.

COLO201 adhered to the dish and grew in the serum-supplemented medium, but grew in suspension in the PFC medium. As shown in FIG. 6, the cells were able to grow in the PFC medium and were able to be passaged for a long period of time.

[Brief description of the drawings]

1 to 4 are diagrams showing the relationship between the concentration of each component measured in Examples 1 to 4 and the number of cells. FIG.
FIG. 9 is a graph showing changes over time in the amount of antibodies and the number of cells in Example 6. FIG. 6 is a diagram showing the change over time in the number of cells in Example 7.

Claims (2)

(57) [Claims] 1. A completely synthetic medium for cell culture containing a: iron citrate, b: ethanolamine and c: linoleic acid, oleic acid or taurine, and free of proteins, cell growth factors, hormones and steroids. 2. The medium according to claim 1, wherein the medium contains linoleic acid and taurine, or oleic acid and taurine.