JP2895641B2 - Animal cell growth promoter and animal cell growth method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 2- (9-methyldecyl) -5- (4-methylpentyl) animal cell growth promoting agent containing the resorcin and on animal cell growth method using the same.

[0002]

2. Description of the Related Art Cell growth cannot be achieved by merely supplementing nutrients, and cell growth factors must be present.
Conventionally, serum such as fetal calf serum has been mainly used for growing animal cells for this purpose. However, it is estimated that there are multiple cell growth factors in serum, the majority of which are unknown components. Therefore, it is not possible at present to determine the cell proliferation activity of a product by examining the content of a specific component in serum, and there is a problem that the quality is not constant depending on the product. There are also isolated and purified cell growth factors, which are extremely expensive,
It is not practical to use this in large quantities. Therefore, as a result of the fact that the chemical structure is specified, there is a demand for the development of a cell growth promoter that can be easily inspected for quality and can be supplied at low cost.

[0003] The subject of the present invention, 2- (9-methyldecyl) -5- (4-methylpentyl) resorcinol, is
m. Ber. 112, 1841-1848 (1979), but makes no mention of its cell growth promoting action.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an animal cell growth promoting agent having a stable quality and which can be supplied at a low cost, and an animal cell growing method using the same. It is in.

[0005]

The present invention SUMMARY OF] is 2- (9-methyldecyl) -5- (4-methylpentyl) animal cell growth promoting agent containing resorcinol, and in the medium 2- (9
The present invention relates to a method for growing animal cells, which comprises adding (-methyldecyl) -5- (4-methylpentyl) resorcinol.

The present inventors have intensively searched for a substance which can reduce the amount of serum used in place of or in combination with serum, and found that a known substance, 2- (9-methyldecyl) -5, was used. -It has been found that-(4-methylpentyl) resorcinol (hereinafter referred to as an active substance) has such an effect, and the present invention has been completed.
The active substance of the present invention has been conventionally studied for its action as an antibiotic, but it was extremely surprising that such substance was confirmed to have a cell growth promoting action.

Hereinafter, the present invention will be described in detail. Animal cells whose growth is promoted by the active substance of the present invention include:
For example, mouse-mouse hybridomas (4E2, NS
-1,2A5), mouse myeloma (NS-1, P3V
1), floating cells such as mouse leukemia cell P388, mouse fibroblast NIH3T3 cells, Hela cells, L-9
29, adherent cells such as BALB3T3 cells, KB cells,
Preferably, mouse fibroblast NIH3T3 cells are used.

[0008] The medium that can be used in the method of the present invention is not particularly limited as long as it has compatibility with animal cells to be grown. It may be modified. Specific basic media include, for example, MEM medium, Iskov medium, RPM11640 medium, Ham's F-12 medium, Dulbecco's modified Eagle medium, Dulbecco's modified MEM medium,
Iskov-modified Dulbecco's medium supplemented with 10% FBS, preferably Dulbecco's modified Eagle's medium, may be used alone or in combination of two or more. Usually, these media contain
Appropriate antimicrobial substances are added and appropriate buffers are added to maintain the pH constant. Specific examples of the added antimicrobial substance include, for example, penicillin, streptomycin and the like. The amount of addition is preferably 100,000 UNIT / L for penicillin and 100 mg / L for streptomycin. These may be used alone or in combination of two or more. Further, specific examples of the buffer include sodium hydrogen carbonate and the like.

The amount of the active substance to be added is preferably selected according to the animal cells to be cultured. Not limited. Generally, for example, an amount is added so that the concentration in the culture solution becomes 0.05 to 5 mg / ml, preferably 0.1 to 1.0 mg / ml. Mouse fibroblast NIH3 in Dulbecco's modified Eagle's medium
When growing T3 cells, 0.1 to 1.0 mg / ml
Gives good results. When adding the active substance,
The solvent is preferably added as a solution, and the solvent used is not particularly limited as long as it is a solvent that dissolves the active substance and is compatible with the culture solution. Specifically, methanol, dimethyl sulfoxide, acetone, and preferably acetone are mentioned. These may be used alone or in combination of two or more. Such a solvent is preferably used so as to have a concentration of 2% by weight or less based on the total amount of the culture solution.

The present invention relates to certain active substances, but as long as such substances are the main active ingredients,
Other auxiliary components may be optionally included as long as the object of the present invention is not impaired. Representative auxiliary ingredients include:
For example, nutrients such as low-density lipoprotein, insulin,
Nutritional supplements such as transferrin, linoleic acid, selenium, and bovine serum albumin.

The production of the active substance of the present invention can be carried out by either an organic chemical synthesis method or a biological method using a microorganism. As the former method, for example,
Chem. Ber. 112, 1841-1848 (1979). This involves subjecting 3,5-dimethoxybenzaldehyde to a Grignard reaction with 3-methylbutylmagnesium bromide and hydrogenating the product to produce 5- (4-methylpentyl) resorcinol dimethyl ether,
Next, this is formylated with N-methylformanilide to give 4- (4-methylpentyl) -2,6-dimethoxybenzaldehyde, which is subjected to a Grignard reaction with 8-methylnonylmagnesium bromide and a hydrogenation reaction of the present invention. This is a method in which an ether form of the active substance is obtained and hydrolyzed. On the other hand, the latter method is based on Cytophaga Jhnsonae (AJ1258).
9) This is a method in which (FERM P-12001) is used as a production bacterium, which is cultured in a kazitone medium, and the target substance is extracted from the obtained cells with an appropriate solvent. In any method, it is optional to further purify and use the obtained active substance.

The propagation of animal cells using the active substance of the present invention can be carried out, for example, as follows. First, cells to be proliferated using the active substance of the present invention in a medium containing serum are semiconfluent (Semiconfluent).
luent) (primary culture). Then, this was added to a medium containing the same serum for 5,000 to 5 times.
10,000 / ml, preferably 10,000-30,
The cells are seeded at a concentration of 000 cells / ml and subjected to a secondary culture.
Here, examples of the serum to be contained in the medium in the primary culture and the secondary culture include fetal calf serum, horse serum, bovine serum, human serum, and preferably fetal calf serum. The serum concentration in the medium in the primary culture was 5
-20%, preferably 8-12%, whereas
The concentration of serum used in the secondary culture is 5 to 20%, preferably 10%, of the amount used in the primary culture. For example, mouse fibroblasts N in Dulbecco's modified Eagle's medium
When the IH3T3 cells are expanded, the concentration of the fetal bovine serum in the primary culture is 10% and the concentration in the secondary culture is 1%.
% May be sufficient. At the stage where this secondary culture is carried out for about 2 to 24 hours by a conventional method, the active substance of the present invention is added, preferably in the form of a solution. When the solution is added as a solution, the amount of the active substance to be added and the solubility are adjusted so that the concentration of the solvent used for dissolving the active substance is 2% by weight or less based on the total amount of the culture solution as described above. It needs to be considered. The addition of the active substance may be either batch addition or sequential addition. The culture conditions after the addition of the active substance are not particularly limited.
6-37 ° C, pH 7.1-7.4, preferably 5% CO
_You can do it under ₂ . After culturing, the cells may be treated according to a conventional method according to the purpose of use. It goes without saying that the active substance of the present invention can be applied to any of monolayer culture, suspension culture, and further improved methods thereof.

[0013] The active substance of the present invention promotes the growth of animal cells.
The use of serum can be reduced. For example, in the case of the growth of mouse fibroblast NIH3T3 cells in the above Dulbecco's modified Eagle's medium, the use of the active substance of the present invention requires about 1/10 Of fetal calf serum.

[0014]

EXAMPLES Example 1 Production of Active Substance A 2% kadington medium (Difc) was placed in a 0.5 L Sakaguchi flask.
o 0.2% MgCl ₂ , pH 7.2)) 0.1 L
And the site ferga Jansonnier (AJ125
89) One loopful of (FERM P-12001) was inoculated and cultured with shaking at 28 ° C for 40 hours. Next, the entire amount of the culture was transferred to a 5 L Sakaguchi flask into which 1 L of the same medium had been placed, and cultured with shaking at 28 ° C. for 2 days. The obtained seed mother liquor was inoculated into a jar fermenter having a capacity of 30 L in which 20 L of the same medium had been placed, and the mixture was stirred at a rotation speed of 210 rpm.
While venting at a flow rate of 0.25 V / V / min.
After culturing for 4 hours, centrifuged and the cells 220 g (wet cell mass)
I got During this time, the pH of the medium rose from 7.2 to 8.3.

The obtained cells were immersed in acetone at room temperature overnight to extract the products in the cells. After extraction, this was filtered to remove insolubles, then acetone was distilled off under reduced pressure, and the residue was redissolved in ethyl acetate. This solution was filtered again,
Concentration gave about 2 g of crude extract. Then, the crude extract was subjected to two column chromatography using silica gel (Wakogel C-200; manufactured by Wako Pure Chemical Industries, Ltd.) as a carrier (developing solvent: first; chloroform, second; n-hexane: ethyl acetate = 15). : 1), and further subjected to column chromatography (developing solvent; chloroform: methanol = 1: 1) using Sephadex LH-20 to obtain about 30 mg of a pale yellow oil.

¹ H-NMR, ¹³ C-NMR,
Each spectrum of IR, FAB-MS, and EI-MS confirmed that it was 2- (9-methyldecyl) -5- (4-methylpentyl) resorcinol. Measurement results: ¹ H-NMR solvent; deuterated chloroform, first
See FIG. ¹³ C-NMR solvent; deuterated chloroform, see FIGS. 2 and 3 IR KBr disk method, see FIG. 4 High resolution FAB-MS: m / z = 349.3095 (M
+ H) EI-MS: m / e = 348 Example 2 Culture of animal cells Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a 50 ml sterile bottle [manufactured by Nissui Pharmaceutical Co., Ltd.] 5
ml of mouse fibroblast NIH3T3.
50,000 cells were inoculated at 37 ° C., 5% CO ₂ ,
Culture was performed for 48 hours under the condition of pH 7.2. Then, cells grown to semi-confluency were placed in a Petri dish filled with 5 ml of the same medium containing 1% fetal calf serum for 20,000 hours.
Seed at a concentration of 0 cells / ml, 37 ° C, 5% CO ₂ , pH
The cells were cultured under the conditions of 7.2. Two hours later, this petri dish has 2
-(9-methyldecyl) -5- (4-methylpentyl)
0.1 ml of a methanol solution containing resorcin at a concentration of 50 microgram / ml was added in an upper volume. This is 3
The cells were cultured at 7 ° C., 5% CO ₂ , and pH 7.2 for 20 hours. After the cultivation, the tritium thymidine in PBS was adjusted to a concentration of 1 microCurie / ml in the total amount of the culture solution.
(-) A buffer solution was added, and further, at 37 ° C., 5% C
The cells were cultured for 1 hour under the conditions of O ₂ and pH 7.2. After the culture, the medium was removed, and 2.5 ml of 1N NaOH was added to lyse the cells. After standing for 1 hour, 0.5 ml of 6N HCl was added to make the solution acidic (pH 3). The precipitate was collected by a Whatman GF / c glass filter, and 5% TCA,
Then washed with ethanol and dried. Put the resulting precipitate in a liquid scintillator cocktail of toluene,
The amount of tritiated thymidine incorporated into the cells was measured.

FIG. 5 shows the relationship between the concentration of the active substance in the culture solution and the amount of tritiated thymidine when the mouse fibroblasts NIH3T3 cells were grown in Dulbecco's modified Eagle's medium.

[Brief description of the drawings]

FIG. 1: 2- (9) measured using deuterated chloroform as a solvent
^{1 is} a ¹ H-NMR spectrum chart of (-methyldecyl) -5- (4-methylpentyl) resorcinol.

FIG. 2: 2- (9) measured using deuterated chloroform as a solvent
It is a ¹³ C-NMR spectrum chart of (-methyldecyl) -5- (4-methylpentyl) resorcin.

FIG. 3 shows 2- (9) measured using deuterated chloroform as a solvent.
It is a ¹³ C-NMR spectrum chart of (-methyldecyl) -5- (4-methylpentyl) resorcin and a partially enlarged chart thereof.

FIG. 4. I of 2- (9-methyldecyl) -5- (4-methylpentyl) resorcinol measured by KBr disk method
It is an R spectrum chart.

FIG. 5 shows the results obtained by growing mouse fibroblast NIH3T3 cells in Dulbecco's modified Eagle medium in culture medium.
-(9-methyldecyl) -5- (4-methylpentyl)
2 is a diagram illustrating the relationship between the concentration of resorcinol and the amount of tritiated thymidine incorporated.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kotaro Fujioka 1-5-7 Mikitei Sakaecho, Higashiosaka-shi, Osaka House Foods Industry Co., Ltd. (72) Inventor Haruo Seto 100 Uenocho, Hachioji-shi, Tokyo (56) References Chemische Berichte, Vol. 112, no. 5, p. 1841 -1848 (1979) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 5/06

Claims (5)

(57) [Claims] (1) 2- (9-methyldecyl) -5- (4-
Animal cell growth promoting agent containing methylpentyl) resorcinol. 2. 2- (9-methyldecyl) -5- (4-
Mouse fibroblast NIH3T3 cell growth promoting agent containing methylpentyl) resorcinol. 3. 2- (9-methyldecyl) -5 in a medium.
-A method for growing animal cells, wherein (4-methylpentyl) resorcin is added. 4. The method according to claim 3, wherein the medium is Dulbecco's modified Eagle medium. 5. The medium according to claim 3, wherein the concentration of 2- (9-methyldecyl) -5- (4-methylpentyl) resorcin in the medium is 0.1 mg / ml to 1.0 mg / ml.
Or the method of 4.