JPS6238325B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention provides colony formation stimulating factors (colony stimulating factors).
Stimulating factor (hereinafter simply referred to as CSF) relates to a method for producing CSF by adding a protease inhibitor to the culture medium when producing CSF by culturing producing cells. More specifically, the purpose is to use it to manufacture an industrial production medium for producing CSF containing protease inhibitors, or to establish an industrial mass production method for CSF, and apply the resulting CSF to medicines and disease diagnosis. It is related to a useful technical field. Conventional technology CSF is known as a humoral factor that acts on granulocytes and macrophage stem cells, which are classified as white blood cells derived from myeloid dry cells, and causes them to differentiate into mature white blood cells [Journal of Immunological
Methods, 42 , 253-284 (1981), etc.] CSF has the effect of inducing the differentiation of myeloid stem cells into white blood cells such as granulocytes and macrophages, so it is used as a therapeutic drug for leukopenia in cancer patients, etc. It is a physiologically active substance useful as a prophylactic agent for infectious diseases. Furthermore, this CSF
is useful as a diagnostic reagent for CSF increase or decrease caused by in vitro production. Conventionally, when attempting to produce CSF in a culture system using cells in anticipation of the usefulness of CSF, it is necessary to add animal serum or serum components to the culture medium. In some cases, the serum itself has the disadvantage of lack of lot identity, and furthermore, even if CSF-producing cells are cultured without using serum, CSF production hardly occurs or significantly decreases. . Problems to be Solved by the Invention When animal serum is added to a culture medium to increase CSF production, it is extremely difficult to completely separate the substances contained in the serum from the produced CSF. There was a problem that the serum added at the time of production varied widely depending on the product lot, making it impossible to obtain consistent culture results. If one is trying to isolate CSF more purly, cell culture methods that involve adding serum to the production medium are not preferred, but cell culture without the addition of serum has the disadvantage that CSF production is small or not produced at all. . Means for Solving the Problems The present inventors believe that if the chemical properties of additives to the culture medium are known, it will be fairly easy to separate and remove the additives from the culture medium containing CSF. Or, find a clear CSF production-promoting substance (protease inhibitor) with a composition other than serum components, and use it.
The problem was solved by adding it to the CSF production medium and culturing it. That is, the present invention relates to a method for producing CSF, which comprises adding a protease inhibitor to the culture medium of CSF-producing cells. Effect: When a protease inhibitor is added to a serum-free medium using the method of the present invention, CSF production increases to the same level or more than when serum is used in the medium.
CSF productivity became possible. Next, the raw materials used in the method of the present invention are as follows. (a) As the CSF-producing cell line, (1) human lung cancer cells (TLC-9A), (2) human renal cancer cells (TRC-29R), and (3) other CSF-producing lines can be used. First, TLC-9A is a human CSF-producing human cell line derived from human lung cancer tissue and having the following properties. Morphology: Epithelial cell-like Chromosome number: Chromosome number distribution mode characterized by a modal number of 117 chromosomes, which is a high tetraploid range Subculture: Infinite subculture Functional characteristics: Human CSF production Cell proliferation: Shows monolayer sheet-like proliferation,
In particular, it proliferates well in RPMI-1640 + 5-20% fetal bovine serum-containing medium, and the population doubling time (Population
Dublining Time) is 37±7 hours. Furthermore, TRC-29R is a human CSF-producing human cell line derived from human renal cancer tissue having the following properties. Morphology: Epithelial cell-like Chromosome number: Chromosome number distribution mode characterized by a modal number of 74 chromosomes, which is in the hypertriploid range Subculture: Infinite subculture Functional characteristics: Human CSF production Cell proliferation: As cells proliferate and reach saturation, they tend to proliferate in a layered manner. In particular, RPMI-1640 containing 5-20% fetal bovine serum
It grows well in the culture medium, and the population doubling time is 29±6 hours. (b) The following medium can be used as a medium for CSF production. Eagle MEM (E-MEM), Alpha MEM (α-MEM), Dulbecco MEM (DME), Iscobb's medium (IMDM), Fuitscher medium, Grace medium, Matsukoi 5A medium, 199 medium, Ham's F-10 medium , Ham's F-12 medium, RPMI-1640 medium, Waymouth medium, William's medium, and these mediums may be used by adding a small amount of an antibacterial substance such as penicillin G or dehydrostreptomycin sulfate. RPMI- as a medium for CSF production in the above medium.
1640 medium (Table 1) and α-MEM medium (Table 2) are suitable as a medium for measuring the activity of CSF. (c) Serum includes fetal calf serum (FCS) (U.S.
(manufactured by GIB Co.) and horse serum (manufactured by GIB Co., USA) are used. (d) Phosphate buffered saline (PBS) (free of magnesium and calcium) was used as the saline solution. (e) Culture conditions One type of CSF-producing cell line is prepared at a concentration of 10 ⁴ to 10 ⁶ cells per ml of CSF production medium, and 1 to 10 ml of this is incubated at a temperature of 37°C in a 5% Co ₂ mixed gas phase. ,
It is sufficient to culture for 2 to 10 days under 100% humidity culture conditions. (f) The following protease inhibitors are used: Derived from animal tissue: _α1 -proteinase inhibitor _α2 -microglobulin Ovoinhibitor Bovine pancreatic trypsin inhibitor Derived from plant tissue: Soybean trypsin inhibitor Lima bean inhibitor Peanut bean Kidney bean Mung bean Fuji bean Jack bean Broad bean, etc. Potato inhibitor Derived from microorganisms: Streptomyces subtilisin inhibitor Subtilisin Inhibitor, S
-SI) Plasminostreptin Among the above, particularly preferred protease inhibitors include the following. Soybean trypsin inhibitor (manufactured by Worthington) Lima bean inhibitor (manufactured by Worthington) Streptomyces subtilisin inhibitor Plasminostreptin (manufactured by Wako Pure Chemical Industries, Ltd.) _α1 -Proteinase inhibitor (manufactured by SIGMA) These inhibitors are added to the CSF at appropriate concentrations.
Production medium, such as RPMI-1640 medium (first
After dissolving the solution in Table 1), it was passed through a 0.22 μm membrane filter and sterilized before use. Furthermore, the amount of these inhibitors to be added is such that at least the addition of the inhibitor improves productivity, and is preferably 0.001.
It is sufficient to add % or more, and there is no limitation to adding more than this, but it is usually 5%.
It may be added to a certain extent. Furthermore, after culturing under such conditions, the target CSF may be purified and collected by a conventional method. For recovery, generally gel filtration,
This may be carried out using means such as ion exchange chromatography and adsorption chromatography.

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[Table] Examples Next, the present invention will be explained with reference to examples, but the present invention is not limited thereto. Example 1 (a) Culture method Human CSF producing cell TRC-29R strain was cultured for 10
Using RPMI-1640 medium containing % fetal bovine serum,
5 ml of cells were seeded in a Corning tissue culture flask (#25100) at a cell density of 1 x 10 ⁵ /ml, and 37
The cells were cultured for 4 days under the culture conditions of 5% Co ₂ mixed gas phase and 100% humidity to allow the cells to proliferate to saturation. After that, the used medium was removed, and the cells were washed twice with 5 ml of physiological saline. After that, 5 ml of RPMI-1640 medium containing lima bean inhibitor at various concentrations was added, and the cells were incubated at 37°C, 5% CO ₂ mixed gas phase, and 100% humidity. The cells were cultured for 6 days under the following culture conditions. Collect the culture solution,
After removing cells and cell debris through a 0.22 μm membrane filter (manufactured by Millipore), the CSF activity of the culture solution was measured. (b) CSF assay method Colony formation method using mouse bone marrow cells The following method using methylcellulose was performed according to Niho's method. 2.2% methylcellulose/α-MEM 1.6ml Horse serum 0.8ml Mouse bone marrow cell suspension/α-MEM 0.8ml Test sample or CSF standard solution 0.8ml Methylcellulose is Dow, α-
The MEM was manufactured by Flow, the horse serum was manufactured by Gibco (GIBCO) (#28K8024), and the CSF standard solution was GCT-CM manufactured by Gibco (GIBCO). For mouse bone marrow cells, mononuclear cells from femoral bone marrow were isolated using a 7-week-old male ICR purchased from the Shizuoka Experimental Animal Agricultural Cooperative Association, and suspended in α-MEM.
The concentration was adjusted to 10 ⁵ /ml. Dispense 1 ml of the above mixture into three 35 mm diameter plastic dishes and heat at 37°C in a 5% Co ₂ mixed gas phase.
After culturing for 7 days under 100% humidity, a cell population consisting of 20 or more cells was considered as one colony, and the CSF activity that caused one colony to form under the above conditions was defined as one unit (U). The activity of CSF was calculated from the average value of three trays. Lima bean on CSF productivity of TRC-29R strain
The experimental results showing the effect of adding the inhibitor are as shown in FIG. 1, and CSF productivity was sufficiently improved when 0.1% or more was added compared to the group without the inhibitor. Example 2 The same procedure as in Example 1 was carried out, except that the TLC-9A strain of human CSF producing cells was used and soybean inhibitor was used instead of adding lima main inhibitor to the culture medium. The experimental results showing the effect of adding soybean inhibitor on the CSF productivity of the TLC-9A strain are shown in FIG. Example 3 CSF production was carried out in the same manner as in Example 1 using the representative protease inhibitors listed in Table 3 in the culture medium of human CSF producing cell strain TRC-29R. Table 3 shows the relationship between the types and amounts of various protease inhibitors added and the amount of CSF produced.

[Table] Effects of the invention The effects obtained by implementing the method of the present invention are as follows. (1) Among the methods for producing large amounts of human CSF, culturing large quantities of CSF-producing cells and using the culture medium as raw material is considered the most effective method, but serum must be added to the medium. An unavoidable problem arose. However, according to the present invention, it is not necessary to add serum.
In addition to making it possible to produce CSF, it has become dramatically easier to separate and purify CSF from the culture solution.
It has become possible to produce CSF with higher purity. (2) CSF is a substance that acts on bone marrow leukocyte precursor cells (CFU-C) and promotes the differentiation and proliferation of these cells into granulocytes or macrophages. It is an essential factor for simultaneous proliferation and colony formation. Human CSF is expected to be used as a therapeutic agent for leukopenia, a condition in which the number of white blood cells decreases due to various infectious diseases, radiation exposure, administration of anticancer drugs, etc. Experimental attempts have also been made to completely cure leukemia by differentiating leukemia cells into mature leukocytes using human CSF, and this is expected to have potential as an anticancer drug.

[Brief explanation of the drawing]

Figure 1 is a graph showing the effect of adding lima bean inhibitor on CSF productivity of TRC-29R strain.
FIG. 2 is a graph showing the effect of adding a soybean inhibitor on the CSF productivity of the TLC-9A strain.

Claims (1)

[Scope of Claims] 1. A method for producing a colony-forming stimulating factor, which comprises adding a protease inhibitor to the culture medium of cells producing the colony-forming stimulating factor. 2. The protease inhibitor is derived from animal tissue,
The manufacturing method according to claim 1, which is a protease inhibitor derived from a plant tissue or a microorganism. 3 The animal tissue-derived protease inhibitor is α ₁ -
The method according to claim 2, which is a proteinase inhibitor, α ₂ -macroglobulin, ovo inhibitor or bovine pancreatic trypsin inhibitor. 4. The production method according to claim 2, wherein the plant tissue-derived protease inhibitor is a soybean trypsin inhibitor, a lima main inhibitor, or a potato inhibitor. 5. The production method according to claim 2, wherein the microorganism-derived protease inhibitor is Streptomyces subtilisin inhibitor or plasminostreptin. 6. The manufacturing method according to any one of Items 1 to 5 above, which contains 0.001 to 5% by weight of the protease inhibitor.