JP3024430B2 - Method for producing bioactive substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for mass-producing a physiologically active substance.

[0002]

2. Description of the Related Art Physiologically active substances include floating cells typified by T cells, B cells and monocyte macrophages, fibroblasts,
It is produced by adhesion-dependent cell lines such as osteosarcoma cells, lung cancer cells, and vascular endothelial cells. Since these cells derived from living organisms produce only a very small amount of useful physiologically active substances, methods for introducing and producing genes of useful substances into Escherichia coli and animal cells have been widely used in recent years as more efficient production means. . When using cells derived from living organisms, since the amount of production is very small, the cells are stimulated with various inducers, or after stimulation, the cells are treated with metabolic inhibitors such as cycloheximide, actinomycin D, and verapamil to produce cells. A super-induction method that enhances (Vilkek, J., Hav)
ell, E .; A. Proc. Natl. Acad. S
ci. U. S. A. 70, 3909-3913, 1973; Wal
z, G .; et. al. , J. et al. Immunol. , 144,
4242-4248, 1990), but a production method that can increase the productivity is desired.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing useful natural physiologically active substances which can be applied to medicines from normal cells or cancer cells. I do.

[0004]

The above object has been achieved by the present invention described below. That is, the present invention is a method for producing a physiologically active substance, which comprises adding an interferon (hereinafter abbreviated as IFN) when culturing a producing cell to produce a physiologically active substance.

[0005] Adhesion-dependent cells are preferably used as the production cells of the present invention, but are not limited thereto. Preferably, human normal diploid fibroblasts or human osteosarcoma-derived MG63 cells are used for the production of a natural physiologically active substance.

[0006] As a method for culturing adhesion-dependent cells,
There are a method using a roux bottle and a roller bottle, a method of adhesive culture on microcarriers or hollow fibers, and a method of immobilization culture on microcapsules. A method using microcarriers, hollow fibers or microcapsules is preferably used.

As a microcarrier, the matrix material is composed of a synthetic resin such as collagen, gelatin, cellulose, cross-linked dextran, or polystyrene, and dimethylaminopropyl, dimethylaminoethyl, trimethylhydroxyaminopropyl, and a negative charge are added as charged groups. Are preferably used. Further, a matrix material coated with collagen or gelatin is also used. “Cytodex-1” as a commercially available product obtained by adding dimethylaminomethyl to cross-linked dextran
(Pharmacia) and "Cytodex-3" (Pharmacia) in which crosslinked dextran is coated with denatured collagen. Some hollow fibers use modified cellulose. A commercially available product is “Vitafiber” (Amicon). Microcapsules are prepared by embedding cells inside using collagen or sodium alginate which forms a gel having water permeability (A. Klausn).
er, Bio / technol. , 1,736, (198
3)).

When the producing cells are treated with an inducing agent to produce a physiologically active substance, an inducing agent such as natural or synthetic RNA, or interleukin 1, tumor necrosis factor, I
Cytokines such as FN-β, PDGF, TG
There is a method of inducing using a growth factor such as F-β, or PMA, PHA, lipopolysaccharide, cholera toxin, etc. (Science, 235, 731 (1987)). In the present invention, PolyIPolyC, PolyA
It is most effectively performed using double-stranded RNA such as PolyU. Further, a method of stimulating cells with an inducer and then treating the cells with a metabolic inhibitor such as verapamil, cycloheximide, actinomycin D to further enhance production (J. Immunol., 144, 4242).
-4248 (1990)) is also preferably used.

The physiologically active substances of the present invention include IFN,
Interleukin (hereinafter abbreviated as IL), colony stimulating factor (hereinafter abbreviated as CSF), tumor necrosis factor (hereinafter abbreviated as T
NF), hepatocyte growth factor (hereinafter abbreviated as HGF)
And the like. In IL, in particular, IL-1α, IL-
It is effective for producing 1β, IL-6, IL-8, IL-11, IL-7 and the like. In particular, in CSF, granulocyte macrophage colony stimulating factor (hereinafter, GM-CSF)
Abbreviated) and TNF is particularly effective in producing TN
Effective for the production of Fα.

As the INF to be added to the production cell, α-type or β-type is preferably used. The IFN of the present invention has I
Any of natural IFN obtained by culturing FN-producing cells, recombinant IFN produced by genetic recombination, and IFN produced by chemical synthesis can be used. IF
It is effective to add N at a concentration of 10 to 1000 IU / ml and culture for 18 to 28 hours after the addition. The timing of addition is not particularly limited, but is preferably added after the cells have grown to confluence. When stimulating with an inducing agent, it is preferably added after the cells have grown to confluence and before inoculating the inducing agent.

The optimal concentration of the inducer when producing a physiologically active substance using the inducer varies depending on the type of cells, the type of inducer, and the like. For example, when PolyIPolyC is used, human diploid fibroblasts About 10
A concentration of ５０50 μg / ml is sufficient. The physiologically active substance produced at this time is preferably collected (harvested) at this time because IFN-β reaches the peak of production 1-2 days after the addition of IFN. IL-1α, IL-1β, I
Production of L-6, GM-CSF, TNFα, etc., reaches the peak of production 3 to 7 days after the addition of IFN, so it is preferable to harvest at this time.

[0012]

The present invention will be specifically described below with reference to examples.

Example 1 Crosslinked dextran microcarrier with 5% fetal calf serum (FCS) and diethylaminoethyl 0.3 w /
v% of Eagle's MEM (200 ml) was inoculated with human normal diploid fibroblasts at a rate of 2 × 10 ⁵ cells / ml, and stirred at 37 ° C. with gentle stirring in a glass spinner flask.
Cultured for days. On the way, the medium was replaced every two days.
The number of cells reached was 1.2 × 10 ⁶ / ml.

When cells have reached growth saturation, the medium is changed to serum-free MEM and about 100 IU / ml of human native IF
Nβ (manufactured by Toray Industries, Inc.) was added, and the mixture was stirred and cultured at 37 ° C. for 24 hours. Thereafter, the medium was polished with 10 μg / ml of Pol.
Change to serum-free MEM containing yIPolyC and incubate for 2 hours, then remove serum-free MEM except for PolyIPolyC
And cultured for 2 to 6 days. At this time, among the physiologically active substances induced by PolyIPolyC, there was a difference in the production time course between IFNβ and other substances as shown in FIG.

Example 2 Human diploid fibroblasts were grown in a flask having a culture area of 150 cm ² using an Eagle MEM medium containing 5% FCS. The number of cells reached was 1.5 × 10 ⁷ cells / flask. The cells are washed with PBS (-), detached from the surface of the flask using 0.1% trypsin, sufficiently separated from the cells, and placed in a 6-well culture plate (Corning) at 1.0%.
Cells were inoculated at a rate of × 10 ⁵ cells / well. FCS
The cells were cultured in a MEM containing 5% for 7 days in a CO ₂ incubator, and when the cells became sufficiently confluent, the medium was added to each concentration of natural human IFNα (available from Nippon Chemical Co.) and human natural IFNβ (Toray Industries, Inc.) Was changed to a serum-free MEM medium containing the same, and the cells were cultured at 37 ° C. for 24 hours.

Thereafter, the medium containing IFN was removed and 10 μg
The cells were stimulated for 2 hours by changing to MEM containing 1 / ml PolyIPolyC. Then switch to serum-free MEM
After culturing for 6 days, sampling was carried out every two days over time to measure the activity of various physiologically active substances. 2 to 6 show the results of examining the activity of a physiologically active substance produced under these conditions by an enzyme immunoassay using a specific monoclonal antibody. Thus, pretreatment of cells with α-type and β-type IFN was performed using IFNβ, IL-1β, IL-6,
It was shown to significantly enhance the production of useful substances such as IL-8 and GM-CSF.

Example 3 Next, IFN required to exhibit the effect of enhancing the production of useful substances
The processing time after addition was examined. Cells were inoculated and grown to confluence and treated with 100 IU / ml of native human IFNβ for 0 to 46 hours as in Example 1. After stimulation with PolyIPolyC, serum-free M
After culturing in EM for 6 days, the amount of produced IL-6 is shown in FIG. The processing time with the highest production enhancing effect is
It became clear that it was around 22 hours.

[0018]

According to the present invention, useful physiologically active substances can be efficiently produced in large quantities.

[Brief description of the drawings]

FIG. 1 shows the results of production of various physiologically active substances by human fibroblasts to which IFNβ has been added.

FIG. 2 shows the effect of adding IFN on IFNβ production.

FIG. 3 shows the effect of adding IFN on IL-1β production.

FIG. 4 shows the effect of adding IFN on IL-8 production.

FIG. 5 shows the effect of adding IFN on IL-6 production.

FIG. 6 shows the effect of adding IFN on GM-CSF production.

FIG. 7 shows changes in culture time after addition of IFNβ on IL-6 production.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:91) (58) Field surveyed (Int.Cl. ⁷ , DB name) C12P 21/00-21/02 C12N 5 / 06 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for producing a physiologically active substance, which comprises adding an interferon when culturing a producing cell to produce a physiologically active substance. 2. A bioactive substance obtained by treating a producing cell with an inducing agent.
It is important to add interferon when producing
The method for producing a physiologically active substance according to claim 1, characterized in that: 3. After addition of interferon production method of a physiologically active substance according to claim 1 or 2, wherein the stimulation with an inducing agent. 4. The method according to claim 1, wherein the interferon is interferon α,
4. The physiological activity according to claim 1, wherein β is β.
Method for producing sex substances. 5. A producing cells to claim 1 fibroblasts
5. The method for producing a physiologically active substance according to 4 . 6. physiologically active substance is interferon, interleukins, colony stimulating factors, methods of producing bioactive substances of claims 1 to 5, wherein the tumor necrosis factor or hepatocyte growth factor.