JPS6237606B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a drug for the treatment of human granulocytopenia, and more specifically to a substance that directly acts on human granulocytic stem cells (hereinafter simply referred to as stem cells) to promote the differentiation and proliferation of these stem cells (colony stimulating substances).
The present invention relates to a method for producing factor (hereinafter abbreviated as CSF). In the hematopoietic developmental mechanism of human granulocytic cells, namely granulocytes, monocytes (cells that have not matured into macrophages), and macrophages, CSF in the human body is the mother cell of these cells. It was widely known that CSF plays a central role in this mechanism because it acts on certain stem cells and induces their division, proliferation, and differentiation (Metcalf, D.; Experimental
Haematology, vol. 1, pp. 185-201, 1973). CSF with such biological activity was expected to be useful as a medicine for treating granulocytopenia (Fumimaro Takahisa, History of Medicine, Vol. 95, 2).
No. 41-50, 1975). However, the mechanism for regulating the production of granulocytes, monocytes, and macrophages in vivo is complex, and the behavior of CSF within this mechanism remains unknown. Because it has been difficult to produce CSF in large quantities, its use as a medicine has not yet been put to practical use. Regarding the use of CSF diagnostic reagents, for example, measuring the number of CSF-reactive cells in the bone marrow cells of patients with myeloid leukemia has great significance for diagnosing the patient's prognosis (Nakao and Takahisa eds.; ``Differentiation and proliferation of blood cells - basic and clinical'', p. 29, Kagaku Hyoronsha, 1975), CSF as a reagent (standard).
was known to be used. However, it has not been put to practical use yet because it has been difficult to produce a large amount of CSF with a quality that can be used for diagnosis as well as for use as a medicine. On the other hand, as a method for producing CSF that directly acts on stem cells, human peripheral blood white blood cells (Price, GB
et al; Biochemical Journal, vol. 148, pp. 209-217,
1975), human placental cells (Burges, AW et al.;
Blood, Vol. 49, No. 4, pp. 573-583, 1977) or
A type of cancer cell called CSF-producing tumor (Nakaaki Osawa et al., Journal of the Japanese Society of Hematology, Vol. 42, No. 2, 237
Page, 1979) was known.
Of these, the first two are the ones that have the potential to be used as medicines, but the conventional manufacturing methods using these cells are laboratory methods, produce only a small amount of CSF, and do not produce large amounts of CSF. Cannot be manufactured. Furthermore, when producing CSF using these conventional methods, serum is essential as a medium component for culturing cells (CSF cannot be produced without serum), and bovine serum or fetal bovine serum is usually used. Ta. However, for pharmaceutical use, in order to avoid side effects caused by these foreign proteins in serum added to the culture medium, measures must be taken to remove them after culturing or to use human serum. be. It is complicated and difficult to remove these proteins from the CSF produced in the culture medium, and human serum is expensive, so the production cost is high. As described above, although CSF has been known to be used as a medicine and a diagnostic reagent, there was no known method for manufacturing it in large quantities at low cost and without side effects. An object of the present invention is to provide a method for producing a large amount of CSF that can be used as a drug for treating granulocytopenia in humans and as a reagent for diagnosing myeloid leukemia without causing side effects. The present inventors used a glycoprotein isolated from human urine that proliferates granulocytes in vivo or a fraction containing the glycoprotein as a medium component, and used this medium to grow monocytes and macrophages isolated from human peripheral blood. We have already filed an application for a method for producing CSF that directly acts on human stem cells, which is characterized by culturing human stem cells, producing active ingredients in the culture solution, and recovering the active ingredients (Japanese Patent Application No. 54-92355. ). After filing the earlier application, the present inventors conducted research on substances having the same effect as the above-mentioned glycoprotein, discovered a substance other than the above-mentioned glycoprotein, and completed the present invention. That is, the present inventors have discovered that a known sialic acid-containing glycoprotein (hereinafter simply glycoprotein) isolated from human urine, which has been thought to have no or almost no effect on human stem cells but has an effect on mouse stem cells. ) was found to be effective in producing CSF that directly acts on human stem cells. The present invention involves culturing monocytes and macrophages isolated from human peripheral blood in a tissue culture medium containing glycoproteins that promote colony formation of mouse granulocytes and macrophages isolated from human urine. The present invention relates to a method for producing a substance for differentiating and proliferating human granulocytic stem cells, which is characterized by producing an active ingredient and recovering the active ingredient. Next, the method of the present invention will be explained in detail. (1) Separation of monocytes and macrophages Blood was collected from the vein of a healthy person using a heparin-treated syringe, transferred to a sterile test tube, and incubated at room temperature for 1 hour.
Let stand for ~2 hours. All subsequent operations are performed aseptically. After leaving it to stand, collect the upper white blood cell layer, wash the cells once with synthetic tissue culture medium,
Later, the density gradient centrifugation method (Mahmood, T. &
WARobinson;Blood, Volume 51, Issue 5, 879~
887, 1978), the cells are fractionated into a monocyte, macrophage, and lymphocyte layer, and a granulocyte layer, and the former is collected. The obtained cell fraction was used as a commercially available synthetic tissue culture medium (hereinafter sometimes simply referred to as the medium).
The cells are suspended in water, centrifuged to sediment, the supernatant is discarded, and the same medium is added to the resulting cells to wash them. Repeat washing at least twice. Next, cells were suspended in a small amount of the same medium, a portion of which was taken, and the number of cells was measured using an automatic blood cell counter, and the Wright-Giemsa stained smear was examined microscopically to determine the ratio of monocytes and macrophages to lymphocytes. The number of monocytes and macrophages inoculated is determined, preferably 10 ⁵ ~
10 ⁷ / 1 culture dish, sow on glass or plastic culture dish, then 5-20%
A commercially available synthetic tissue culture medium containing serum (volume %, the same applies hereinafter for the medium) is added, and the mixture is allowed to stand for 1 to 2 hours in a tissue culture incubator at 37° C., preferably under humidification and carbon dioxide aeration. While standing still, monocytes and macrophages adsorb to the bottom of the culture dish,
Lymphocytes float in the medium. The medium is then discarded, serum-free medium or saline is added, and the culture dish is washed several times. This treatment removes most lymphocytes. Monocytes and macrophages are adsorbed to the bottom of the culture dish. In this method, the adsorption surface was directly stained and microscopic findings showed that more than 95% of the cells adsorbed to the bottom were monocytes and macrophages, and the number was 10 ⁵ to 10 ⁷ per culture dish. know. (2) Culture of monocytes and macrophages Next, glycoproteins or fractions containing glycoproteins are added to at least 1 ml of the medium so that the number of monocytes and macrophages is at least 10 ⁵ per ml of the medium. Pour serum-added synthetic medium or serum-free synthetic medium containing 500 units or more (see (3) below for units) into the culture dish, aerate carbon dioxide gas, and incubate for 1 hour in an incubator maintained at 37°C. Culture for ~7 days to allow CSF to be produced in the culture medium. The synthetic medium used is a commercially available tissue culture medium. Optimal culture conditions for CSF production of the present invention,
That is, the number of culture days, the amount of glycoprotein added, the amount of cell inoculation, the amount of serum added, and the type of medium will be described later using test examples. When manufacturing pharmaceuticals using the method of the present invention, a human serum-supplemented medium or a serum-free medium is used to prevent side effects caused by foreign proteins, and when manufacturing diagnostic reagents, bovine serum or fetal bovine serum is added. You may also use a culture medium prepared for this purpose. Incidentally, a culture bottle can also be used instead of a culture dish. Monocytes and macrophages cultured once can also be used repeatedly. (3) Glycoprotein to be added to the medium The glycoprotein used in the method of the present invention can be prepared by a known method such as the method of Stanley and Metcalf (Stanley and Metcalf; Australian Journal of
Experimental Biological Medical Science
Volume 47, pages 467-483, 1969. For details, see Example 2), the method of Stanley et al.
Federation Proceedings, Volume 34, No. 13, 2272~
2278 pages, 1975. For details, see Example 1), Laukel et al.'s method (Laukel et al, Journal of
Cellular Physiology, vol. 94, pp. 21-30, 1978
Year. For details, see Example 3). The biological activity of the prepared glycoprotein on mouse bone marrow cells was measured by the following method and expressed as "units". 20% fetal bovine serum, 0.3%
Add 0.1 ml of the glycoprotein to be tested or a fraction containing it to 1 ml of Mc Coy's 5A medium containing agar and 1 x 10 ⁵ C ₅₇ Bl/6J mouse bone marrow cells, and prepare a 35 mm diameter
The cells were cultured at 37°C for 7 days under 5% _CO2 aeration, and then under an inverted microscope.
Count clumps of 50 or more cells as colonies. The biological activity of a sample that forms one colony is defined as one unit. Furthermore, in order to determine the degree of purification of the glycoprotein, the specific activity is calculated using the following formula. Specific activity = unit/glycoprotein amount of glycoprotein or fraction containing it (mg
) Specific activity increases as the glycoprotein becomes more highly purified. In the method of the present invention, as the glycoprotein added to the culture medium or the fraction containing the same, purified glycoprotein with high specific activity can be used, as is clear from the results of Test 6 described later, but A fraction with a low glycoprotein content is more desirable. The amount of glycoprotein added to the medium is at least 500 units, more preferably 1000 units or more per ml of medium. (4) Recovery of active ingredients from culture medium Collect the medium containing CSF obtained as above from the culture dish, and
Centrifuge for 10 minutes to obtain a clear supernatant. This supernatant contains CSF with extremely high activity. This supernatant is used to prepare reagents for clinical diagnostic reagents or stem cell colony formation tests. That is, the supernatant of the culture solution is adjusted to contain CSF having the activity of forming at least 100 colonies of human granulocytes per 0.1 ml of the supernatant of the culture solution, sterilized, filled aseptically into a container, sealed, and liquid-formed. Manufacture reagents for Furthermore, a powdered reagent can also be produced by aseptically freeze-drying the sterile supernatant. For pharmaceutical use, a culture solution prepared using a medium without serum or a medium supplemented with human serum is dialyzed against water, medium components are removed, and the dialyzed fluid is sterilized and used as necessary. Concentrate accordingly, fill containers aseptically, and seal them to produce liquid medicine. Alternatively, the dialysis fluid can be aseptically filtered and aseptically freeze-dried to produce a powdered medicine. In order to further purify the supernatant for pharmaceutical use, the supernatant is passed through an ultrafiltration membrane that separates molecules with a molecular weight of 5,000 to 10,000.
above) and a low molecular weight fraction (molecular weight 5,000 to less than 10,000). CSF is included in both high-molecular and low-molecular fractions, but 90% of CSF
% or more is contained in the polymer fraction. The low molecular weight fraction can be concentrated under reduced pressure to produce a product. Concentrate the high molecular fraction at a concentration of 0.01 to 0.1M.
An anion exchange resin dissolved in a buffer solution with a pH of 6.0 to 8.0 and equilibrated in advance with the buffer solution, for example,
Contact with DEAE cellulose, DEAE-Sephadex, QAE-Sephadex, etc., and CSF
is adsorbed onto the resin, and then the CSF is eluted and purified using a buffer solution with a concentration of 0.1 to 0.3M and a pH of 6.0 to 8.0. Furthermore, this eluate can be concentrated and purified by molecular sieve chromatography using gel filtration. In this case, the gel for gel passing is
Commercially available Cephadex G-150, Biogel P
-100, Ultrogel AcA-44, etc. can be used. In addition, when CSF activity is produced using a serum-free medium, the treatment by anion exchange resin chromatography may be omitted and purification may be performed by gel permeation chromatography. The developing buffer when performing gel perchromatography has a concentration of 0.01 to 0.3M and a pH of 6.0 to 8.0.
buffer solution is suitable. The CSF active fractions separated by gel filtration are collected, concentrated, desalted, and lyophilized to obtain purified CSF products. The purified CSF obtained here was analyzed for mixed proteins by immunoelectrophoresis using anti-human serum and anti-bovine serum, and it was found that the protein obtained from the bovine fetal serum medium was derived from fetal bovine serum. As a result, serum albumin and globulin-like proteins, which are presumed to be present, and trace amounts of human-derived globulin-like proteins are detected. On the other hand, those obtained from serum-free medium are not detected at all. Therefore, CSF produced in a serum-free medium can be used as a medicine without side effects. [Test 1] Test regarding culture days. (1) Separation of monocytes and macrophages, preparation of glycoproteins. The same method as in Example 1 described later was used. (2) Culture of monocytes and macrophages. Using the glycoprotein obtained in Example 1 (standard purified product; specific activity 180000), two types of media containing it and two types of media not containing it were prepared. The medium is McCoy's 5A medium that does not contain serum,
McCoy's 5A medium containing 20% fetal bovine serum was used. These four types of media were added to a culture dish with monocytes and macrophages adsorbed to the bottom at a ratio of 10 ⁶ monocytes and macrophages per ml of culture medium, and cultured in the same manner as in Example 1.
Then, a certain amount of each culture solution was collected before culturing and 1, 3, 5, and 7 days after culturing. (3) Measurement of CSF in culture medium. The activity of CSF in the culture solution was measured by colony formation method using human bone marrow cells. Bone marrow is punctured from the sternum of a healthy person, taken into a heparin-treated syringe, and centrifuged at 1000g for 10 minutes to collect buffycoat. This was then washed with McCoy's 5A medium,
Suspend in McCoy's 5A medium containing 20% serum,
Seed in a Petri dish, add several mg of dry heat sterilized iron carboxylic acid powder per ml of culture medium, and incubate at 37°C.
Leave in an incubator for 1 to 2 hours. After standing still, the cells that have absorbed the iron carboxylate are fixed to the bottom of the Petri dish using a magnet, and the supernatant cell suspension is collected.
The cells obtained here are non-adsorbed, non-deficient bone marrow cells, and are used for measuring CSF activity. The bone marrow cells are once centrifuged and washed, then suspended in a small amount of medium, and the number of nucleated cells in the suspension is counted by staining with acetic acid-gentian staining solution. Next, add 2× non-adsorbed, non-nucleated cells per ml.
Add ¹⁰ cells to McCoy's 5A medium supplemented with 0.3% agar and 20% fetal bovine serum, add 0.1 ml of culture solution per 1 ml of the medium, and add 5% carbon dioxide under aeration of 5% carbon dioxide gas.
Culture in a humidified incubator at 37°C for 10 days. The activity of CSF was determined by counting the number of colonies formed under a microscope, using clusters of 40 or more cells as colonies among the cell clusters formed after culturing. Then, the activity of CSF was expressed using this number of colonies, and CSF production was tested. The results are shown in Table 1.

[Test 2]

Test regarding the amount of glycoprotein added. McCoy's5A medium supplemented with 20% fetal bovine serum and McCoy's5A medium without serum.
A medium was prepared in which 0 (control), 100, 500, 1000, and 2000 units of glycoprotein (standard purified product: specific activity 180000) prepared in the same manner as in Example 1 was added per ml of medium, and this was added to monocytes. and macrophages were added to the culture dish to which they had been adsorbed, and cultured for 3 days in the same manner as Test 1. Then, each culture solution obtained was tested in the same manner as Test 1 to measure the activity of CSF.
The production of was tested. A sample cultured without addition of glycoprotein was used as a control. The results are shown in Table 2.

[Test 3]

Tests on monocyte and macrophage inoculum. The number of monocytes and macrophages inoculated into the medium was 0, 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ per ml of medium, and McCoy's 5A supplemented with 20% fetal bovine serum.
1 ml of McCoy's 5A medium without medium or serum
A culture solution was obtained in the same manner as in Test 1, except that 500 units of per glycoprotein (standard purified product obtained in Example 1: specific activity 180,000) was added and the culture period was 3 days. Ta. These culture solutions were then tested in the same manner as Test 1 to measure CSF activity and test for CSF production. The results are shown in Table 3.
It is as follows.

[Test 4]

Testing on media type. There are currently four types of synthetic media commercially available for tissue culture or cell culture: McCoy's 5A medium (manufactured by Gibco), Nutrient Mixture HAMF-10
(manufactured by Gibco), RPMI-1640 (manufactured by Nissui Pharmaceutical), and Eagle MEM medium supplemented with amino acids (manufactured by Nissui Pharmaceutical) to compare the production of CSF. Without adding serum to these media, 500 units of glycoprotein (standard purified product obtained in Example 1: specific activity 180000) was added per ml of each medium, and cultured in the same manner as in 3-molecular test 1. Then, the obtained culture solution was tested in the same manner as in Test 1 to measure CSF activity and test for CSF production. The results are shown in Table 4.
It is as follows.

[Test 5]

Testing on the amount of serum added. Human serum (manufactured by Midori Juji) and fetal bovine serum (manufactured by Flow Laboratory) heated at 58°C for 30 minutes were added to McCoy's 5A medium at 0, 5, 10, 20, and
30% and glycoprotein (standard purified product of Example 1: specific activity 180000) was used at a rate of 500 units per ml of the medium, and the inoculation amount of monocytes and macrophages was 10 units per ml of the medium. A culture solution was obtained by culturing in the same manner as in Test 1, except that the number of cells was ⁵ and the number of days of culture was 3 days. Then, the obtained culture solution was tested in the same manner as Test 1.
CSF activity was measured and CSF production was tested. The results are shown in Table 5.

[Test 6]

Test on addition of fractions and highly purified products containing glycoproteins. In Tests 1 to 5, a standard purified product (specific activity: 180000) was used as the glycoprotein, but fractions containing glycoproteins, semi-purified glycoproteins or highly purified products can also be used in the method of the present invention. Prove what you can do. The glycoprotein-containing fractions and highly purified products used were fraction A, which was prepared in the same manner as in Example 1;
B (specific activity: 21000, 54000) and highly purified product (specific activity: 1240000). These fractions are serum-free
0, 100, 500, 1000 in McCoy's5A medium, respectively.
and 2000 units/ml. The culture was carried out in the same manner as in Test 1, except that these media were used, the inoculation amount of monocytes and macrophages was 10 ⁵ per ml of the culture medium, and the number of days of culture was 3 days. Obtained. Then, the obtained culture solution was tested in the same manner as in Test 1 to measure CSF activity and test for CSF production. The results are shown in Table 6.

[Table] As is clear from Table 6, the amount of CSF produced increases in proportion to the increase in the amount added in all cases. Comparing the test results of the serum-free medium shown in Table 2 with those of fractions A and B, the latter two produced a larger amount of CSF despite the same amount of glycoprotein added. This is presumed to be due to the influence of human urine-derived serum albumin contained in fractions A and B. The results of adding highly purified products clearly show that the increase in the amount of glycoprotein added
Although the amount of CSF produced increases, it is inferior to that of fractions A, B and standard purified products (see Table 2). These results indicate that various components derived from human urine, such as serum albumin, have the same effect on CSF production as serum in serum-free media. Therefore, in order to carry out the method of the present invention advantageously, it is more desirable to add crude glycoprotein to the medium rather than adding purified glycoprotein to the medium. And when adding crude glycoprotein to the medium, at least
Add at the rate of 500 units. Example 1 1 Separation of monocytes and macrophages 200 ml of peripheral blood from a healthy person was taken into a blood collection bottle containing 1000 units of heparin, mixed gently with heparin, transferred to a sterilized 200 ml glass cylinder (diameter 20 mm), and kept at room temperature. Let stand for 2 hours.
After standing still, the upper leukocyte layer was gently collected with a pipette, diluted 2 times with serum-free McCoy's 5A medium, and centrifuged at 1500 x g for 15 minutes. The supernatant was discarded and the precipitate was collected using McCoy's ’ Float in 20 ml of s5A medium. Next, the suspension was layered on top of a centrifuge tube containing sodium metrizoate solution, specific gravity d = 1.077, and centrifuged at 400 x g for 30 minutes. Collect the layer with a pipette and
Add medium, wash, centrifuge at 1500 xg for 10 minutes, and discard the supernatant. Repeat this twice. The obtained cells were suspended in approximately 20 ml of McCoy's 5A medium, a portion of this suspension was taken, and the number of cells was counted using an automatic blood cell counter (manufactured by Tor).
A smear of this suspension was prepared, stained with Wright-Giemsa, and the number of lymphocytes, monocytes, and macrophages was morphologically measured under a microscope.
The ratio was calculated. As a result, the ratio of monocytes and macrophages was 25.5%. This is 5
ml each into four 15 cm diameter glass Petri dishes, and then add 30 ml of McCoy's5A with 10% fetal bovine serum.
Add the culture medium and leave it in a humidified incubator (37℃) under 5% carbon dioxide aeration for 2 hours, then discard the culture medium, add 30ml of McCoy's 5A medium, shake slightly vigorously, wash, and discard. to remove lymphocytes. When the ratio of monocytes and macrophages was measured using the same test method as above, both were 95%. 2. Preparation of Glycoproteins Glycoproteins and fractions containing them were prepared as follows using the method of Stanley et al.
400ml of fresh urine collected from healthy individuals was dialyzed against water using an ultrafiltration membrane, and then the pH was adjusted to 7.4, which was equilibrated in advance with 0.03M Tris-HCl buffer, pH 7.4. The liquid was passed through a DEAE cellulose column (20 x 15 cm), the active ingredient was adsorbed onto the column, and then 20 0.04M
Washed with 0.1 Tris-HCl buffer, PH 7.0 containing NaCl, then eluted with 20 portions of 0.1 M Tris-HCl buffer, PH 7.0 containing 0.15 M NaCl, and dialyzed against distilled water. (Fraction A). Next, calcium phosphate gel was added to the dialysate in an amount of 58 ml per 1 g of protein, the active ingredient was adsorbed to the calcium phosphate gel, and the calcium phosphate gel was collected by filtration. Wash twice with 20, then 0.025M
Elute with phosphate buffer 5 and centrifuge (12,000 xg, 10 minutes) to obtain a supernatant. This was dialyzed against distilled water and concentrated under reduced pressure to about 50 ml. Next, the concentrated solution was equilibrated with 0.1M Tris-HCl buffer, and the concentrated solution was equilibrated with the buffer beforehand.
0-0.15M in 0.1M Tris-HCl buffer on DEAE cellulose column (2.5 x 90cm)
Concentration gradient elution with NaCl was performed, and fractions containing glycoproteins were collected and concentrated using an ultrafiltration membrane (fraction B). The concentrate was applied to Biogel P-100 (2.5 x 110 cm) equilibrated with 0.03M Tris-HCl buffer and gel-filtered to obtain about 230 mg of glycoprotein (standard purified product). Next, add 100mg of the standard purified product to 1.0M NaCl,
0.001M MgCl ₂ , 0.001M MnCl ₂ and 0.001M
Con A dissolved in 0.1M acetate buffer containing _CaCl2 , pH 6.0 and equilibrated with this buffer in advance
- Apply to Sepharose 4B column (36 x 1.0 cm),
It was eluted with 0.1 M α-methyl-D-glucose to obtain about 8 mg of glycoprotein (highly purified product). The biological activity of each of these samples using mouse bone marrow cells was measured using the same test method as described above, and the results were as shown in Table 7.

[Table] 3 Culture of monocytes and macrophages The above standard purified product was added with 20% fetal bovine serum.
30 ml of McCoy's 5A medium was added at a rate of 1000 units per ml of medium, and 30 ml of medium was added to each Petri dish. The number of monocytes and macrophages per ml of medium was ¹⁶⁶ . This was cultured for 3 days in a humidified incubator (37°C) under 5% carbon dioxide aeration to obtain a culture solution containing CSF. 4. Purification of culture medium The culture medium was collected and centrifuged at 2°C, 2000 xg for 10 minutes to obtain about 120 ml of clear supernatant. The obtained supernatant liquid was filtered and concentrated through an ultrafiltration membrane (manufactured by Amicon) that removes molecular weights of less than 10,000, and then
100 ml of 0.05M Tris-HCl buffer (PH7.2) was added, concentrated again, and the concentrate was dissolved in 5 ml of the buffer. Next, this solution was mixed with 0.05M Tris-HCl, PH
Adsorb onto a DEAE cellulose column (2.0 x 60 cm) equilibrated with 7.0 buffer, and
CSF was eluted by concentration gradient elution using a buffer solution containing 0.3M sodium chloride. The eluate was again concentrated using the ultrafiltration membrane device and equilibrated with 0.05M Tris-HCl, PH7.0 buffer.
This concentrated solution was passed through a Sephadex G-150 column (2.0 x 90 cm), developed with the buffer solution, and the molecular weight
Fraction equivalent to 65000-90000 and molecular weight 30000
Fractions corresponding to ~60,000 were collected. These fractions were combined and concentrated using the ultrafiltration device, and then distilled water was added to perform desalting and concentration treatment to approximately 5 ml.
A solution containing purified CSF was obtained. As a result of testing in the same manner as Test 1, this solution had the activity of forming 35,000 human granulocyte colonies per ml. Example 2 Monocytes and macrophages were separated from human peripheral blood in the same manner as in Example 1. Then, the glycoprotein prepared as follows according to the method of Stanley and Metcalf was added to the serum-free protein.
Approximately 5 ml of a purified CSF-containing solution was obtained in the same manner as in Example 1 using a medium added to McCoy's 5A medium at a rate of 1000 units per ml of the medium. As a result of testing in the same manner as Test 1, this solution had the activity of forming 9800 human granulocyte colonies per ml. 20 human urine was dialyzed against tap water for 8 to 12 hours at room temperature, and the dialysate solution was mixed with 75 g of DEAE cellulose equilibrated with water and 1.0 M Tris-HCl buffer (PH
7.0) 100 ml was added and mixed to adsorb the glycoprotein, and the supernatant was discarded. Then DEAE cellulose
0.1M Tris-HCl buffer (PH
7.0) three times, and then 300 ml of 0.1M Tris-HCl buffer (PH7.0) containing 0.5M NaCl was added for elution (this operation was repeated 6 times). The eluate is concentrated under reduced pressure at 40°C and dialyzed against 0.1M Tris-HCl buffer (PH7.0). The dialysed fluid was passed through a DEAE cellulose column (2.3 x 44 cm) equilibrated with 0.1M Tris-HCl buffer (PH7.0) to adsorb glycoproteins, and the column was injected with the same buffer containing 0.05M NaCl. was washed, and then the adsorbed glycoprotein was eluted by a salt concentration gradient elution method using 0.1 to 0.5 M sodium chloride in the same buffer. Dialyze the eluate against water,
The dialyzed fluid was concentrated under reduced pressure and lyophilized. This dried product was dissolved in 0.1M Tris-HCl buffer (PH7.0), and Sephadex G-
The solution was passed through a 150 column (2.3 x 150 cm), and the glycoprotein fraction was collected, dialyzed, and freeze-dried to obtain about 12 mg of powder. The specific activity of this powder was approximately 36,000. Example 3 Monocytes and macrophages were separated from human peripheral blood in the same manner as in Example 1. And said Laukel
The glycoprotein-containing fraction (fraction A) prepared as follows by the method of et al.
Using a medium added to McCoy's 5A medium at a rate of 2000 units per ml of the medium, culture was carried out in the same manner as in Example 1 to obtain about 120 ml of a culture solution. This culture solution was dialyzed against distilled water, and the dialyzed solution was concentrated under reduced pressure at low temperature to obtain about 5 ml of a CSF-containing solution.
As a result of testing in the same manner as Test 1, this solution had the activity of forming 29,000 human granulocyte colonies per ml. Dialyze 50% of human urine with an ultrafiltration membrane device (manufactured by Asahi Kasei, CL100) under running tap water to obtain a dialysate of 0.05M.
Equilibrated with Tris-HCl buffer (PH7.3)
Pass the liquid through a DEAE cellulose column (10 x 30 cm),
Adsorbed glycoprotein. and column 0.05M
0.05M Tris-HCl buffer containing salt (PH7.3)
and then eluted with the same buffer containing 0.3M NaCl. The eluate was dissolved in 0.05M Tris-HCl buffer (PH) containing 0.5M NaCl, 2mM _CaCl2 and _MgCl2 each.
8.0). The dialysate was equilibrated with the same buffer using a Con A-Sepharose 4B column (2.6×
40 cm) to adsorb glycoproteins. Then, wash the column with the same buffer, and then use 0.15Mα
The eluate was eluted with the same buffer containing -methyl-D-mannoside, and the eluate was concentrated by ultrafiltration to obtain about 7 ml of a glycoprotein-containing fraction containing 6 mg/ml of protein. The specific activity of this fraction was approximately 20,000. Example 4 Monocytes and macrophages were separated from human peripheral blood in the same manner as in Example 1. Then, a fraction containing glycoprotein (fraction B) prepared in the same manner as in Example 1 was mixed with human serum at a concentration of 10%.
Approximately 5 ml of a purified CSF-containing solution was obtained in the same manner as in Example 1 using a medium added to McCoy's 5A medium at a rate of 2000 units per ml of the medium. As a result of testing in the same manner as Test 1, this solution had the activity of forming 24,000 human granulocyte colonies per ml.

Claims (1)

[Scope of Claims] 1. Monocytes and macrophages isolated from human peripheral blood are cultured in a tissue culture medium containing a glycoprotein that promotes colony formation of mouse granulocytes and macrophages isolated from human urine. A method for producing a substance for differentiating and proliferating human granulocytic stem cells, characterized by producing an active ingredient in a liquid and recovering the active ingredient. 2. The method according to claim 1, wherein the culturing is carried out in the presence or absence of serum. 3. The method according to claim 2, wherein the serum present is human serum. 4 The amount of serum is at least 5% based on the volume of the medium.
The method according to claim 3, wherein: 5. The method according to claim 1 or 2, characterized in that the content of said glycoprotein is at least 500 units per ml of medium. 6. The method for producing a substance for differentiating and proliferating human granulocytic stem cells according to claim 1, wherein the glycoprotein is a partially purified product accompanied by urine protein. 7. The method according to claim 1, characterized in that the inoculated amount of monocytes and macrophages is at least 10 ⁵ cells/ml of medium.