JPS6237605B2 - - 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 (Fumima 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 yet been put to practical use 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, volume 148, pages 209-217, 1975
), human placental cells (Burges, AW et al.; Blood,
49, No. 4, pp. 573-583, 1977) or a type of cancer cells called CSF-producing tumors (Nakaaki Osawa et al.;
Journal of the Japanese Society of Hematology, Volume 42, Issue 2, Page 237, 1979
A production method was known to cultivate the Among these, the first two are the ones that can be used as medicines, but the conventional manufacturing methods using these cells are:
It is a laboratory method, produces a small amount of CSF,
CSF cannot be produced in large quantities. 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. According to the present invention, monocytes and macrophages isolated from human peripheral blood are cultured in a tissue culture medium containing a glycoprotein that proliferates human granulocytes isolated from human urine, and active ingredients are produced in the culture medium, Provided is a method for producing a substance for differentiating and proliferating human granulocytic stem cells, which comprises recovering an active ingredient. The present invention relates to an invention for which a portion of the present inventors has already applied for a patent (Japanese Patent Application No. 53-31999 (hereinafter referred to as the "prior application")). (hereinafter simply referred to as glycoprotein) or a fraction contained in glycoprotein as a medium component,
This is a method for producing CSF that directly acts on stem cells. After completing the earlier invention, the present inventors conducted further research on glycoproteins. As a result, although the mechanism is not clear, this glycoprotein has some relationship with macrophages in vivo.
It was found that CSF was produced. Therefore, the present inventors used a medium containing glycoproteins, and even if macrophages were cultured in vitro, CSF
We thought that it might be possible to manufacture the same, and after conducting studies, we completed the present invention. Next, the method of the present invention will be explained in detail. (1) Separation of monocytes and macrophages Collect blood from the vein of a healthy person using a heparin-treated syringe, transfer the blood to a sterile test tube, and incubate for 1 to 2 hours at room temperature.
Leave it for a while. All subsequent operations are performed aseptically. After standing, the upper leukocyte layer was collected, the cells were washed once with synthetic tissue culture medium, and then subjected to density gradient centrifugation (Mahmood, T. & W.A.
Robinson; Blood, Vol. 51, No. 5, pp. 879-887.
(1978), the cells are fractionated into monocytes, macrophages, lymphocytes, and granulocytes, and the former are collected. The obtained cell fraction is suspended in a commercially available synthetic medium for tissue culture (hereinafter sometimes simply referred to as medium), centrifuged and precipitated, the supernatant is discarded, and the same medium is added to the obtained cells for washing. 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 cells are plated on glass or plastic culture dishes at a predetermined number of inoculated monocytes and macrophages, preferably 10 ⁵ to 10 ⁷ /1 culture dish, and then 5 to
Add a commercially available synthetic tissue culture medium containing 20% serum (volume %, the same applies below for the medium) and leave it at 37°C in a tissue culture incubator, preferably under humidification and aeration of carbon dioxide gas, for 1 to 2 hours. do. During standing, monocytes and macrophages adsorb to the bottom of the culture dish, and lymphocytes float in the medium. Then discard the medium and
Add serum-free medium or saline and wash the culture dish 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 a serum-supplemented synthetic medium or a serum-free synthetic medium containing 0.1 μg or more per culture dish into the culture dish, aerate carbon dioxide gas, and culture for 1 to 7 days in an incubator maintained at 37°C to remove CSF from the culture medium. to be produced. The synthetic medium used is a commercially available tissue culture medium. The optimal culture conditions for CSF production of the present invention, ie, 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 a medicine by the method of the present invention,
When producing a diagnostic reagent using a human serum-containing medium or a serum-free medium to avoid side effects caused by foreign proteins, a medium supplemented with bovine serum or fetal bovine serum may be used. 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 added to culture medium The glycoprotein used in the method of the present invention is the same as the glycoprotein or glycoprotein-containing fraction of the earlier invention, and the method is the same as the method of the earlier invention. It is prepared as follows. Add a dilute acid or alkaline aqueous solution to fresh urine collected from a healthy person to bring the pH to 6-9, preferably 7.
-8, and then centrifuged to remove insoluble matter contained in the urine. The urine supernatant obtained here is treated with a silicon-containing adsorbent, such as silica gel, silica gel-magnesium silicate, diatomaceous earth,
Contact with silica glass, bentonite, etc. to elute adsorbed components. Elution is preferably performed with an alkaline aqueous solution having a pH of 9 or higher. The alkaline aqueous solution used for elution is not particularly limited, but preferably an aqueous solution of ammonium hydroxide, sodium hydroxide or the like having a concentration of 0.3 to 1.5M is used. The pH of the eluate thus obtained is adjusted to 7 to 8, and a neutral salt, such as ammonium sulfate, is added to 70% saturation to salt out the effective substance to obtain a crude fraction containing glycoproteins. Next, this crude fraction is dissolved in a small amount of alkaline aqueous solution, and low-molecular components with a molecular weight of 10,000 or less are removed using a molecular separation filter. ) to adsorb and remove impurities contained in the solution. The contact is carried out at approximately neutrality, and the crude glycoprotein fraction and ion exchanger are heated at pH 6 to
8, preferably with a 0.01-0.15M inorganic salt buffer. At this time, most of the glycoprotein passes through. After concentrating this, it is equilibrated with a dilute buffer solution of pH 6-8, and contacted with an equilibrated anion exchanger (e.g. DEAE cellulose) to adsorb glycoproteins, and with 0.1-0.3M inorganic salt, e.g. chloride. The salt concentration is varied using a sodium solution, and elution is carried out by a so-called linear concentration gradient elution method. At this time, glycoproteins are eluted at a salt concentration of 0.1M or higher, but complete separation is difficult. This elution fraction with a salt concentration of 0.1 to 0.3M is collected, desalted and concentrated if necessary (hereinafter referred to as fraction A). This fraction can be used in the method of the invention. In addition, before this step of elution by changing the salt concentration, the glycoprotein is adsorbed on an anion exchanger,
Purification may be carried out by elution with an aqueous solution having a salt concentration of 0.1 to 0.3M. Furthermore, for the purpose of molecular sieve chromatography, the fraction is passed through a column packed with a highly crosslinked polymer gel having a water absorption of 10 to 20 ml/g, such as Sephadex G-150 or Biogel P-100. Develop the active substance in a 0.05-0.1 molar salt buffer, separate fractions with a relative eluate volume of 1.11-1.60, preferably 1.11-1.45, desalt, and concentrate or freeze-dry. (hereinafter referred to as fraction B). The glycoprotein-containing fraction thus obtained can also be used in the present invention. The relative eluate volume is a value expressed as the ratio Ve/Vo (Ve indicates the volume of solvent required to elute the substance in the column, and Vo indicates the volume of solvent outside the gel particles in the column). ) To further purify the above crude product, the crude product is
Dilute buffer containing 1.0-2.0M salt, e.g. phosphate buffer PH6.0-8.0, preferably PH6.0-7.0
The glycoprotein is adsorbed onto a sugar-affinity adsorbent such as Concanavalin A-Sepharose 4B (manufactured by Fine Chemical Laboratories) which has been equilibrated in advance with the buffer, and then 20mM to
1.0-2.0M salt-added buffer containing 100mM sugars, such as α-methyl-D-glucoside, pH 6.0~
Elution is carried out at pH 8.0, preferably pH 6.0 to 7.0, and fractions containing glycoproteins are collected, desalted if necessary, and concentrated or lyophilized. This fraction can also be used in the present invention. Furthermore, in order to electrophoretically purify the fraction obtained here, it is subjected to preparative zone electrophoresis using, for example, acrylamide gel or agar gel pH 7.0 to 9.0 as a support, and then purified with a dilute salt solution under cooling. The glycoprotein is recovered from the support, desalted, and concentrated or lyophilized. The purified glycoprotein thus obtained can also be used in the present invention. However, as is clear from the test results described below,
It is more desirable to use fractions containing these glycoproteins than to use purified glycoproteins. (4) Recovery of active ingredients from culture medium The CSF-containing medium obtained as described above is collected from the culture dish and centrifuged at 1000-2000 xg for 5-10 minutes to obtain a clear supernatant. This supernatant contains CSF with extremely high activity. This supernatant is used for the preparation of clinical diagnostic reagents or reagents for colony formation tests with stem cells. That is, the supernatant of the culture solution is adjusted to contain CSF having the activity of forming at least 100 human granulocyte colonies per 0.1 ml of the supernatant of the culture solution, sterilized, filled aseptically into a container, and sealed, Manufacture liquid reagents. 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, the 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. Further, the dialysis fluid is aseptically filtered and freeze-dried aseptically,
Powdered medicaments can also be produced. In order to further purify it for pharmaceutical use, the supernatant is passed through an ultrafiltration membrane that separates molecules with a molecular weight of 5,000 to 10,000, and a high molecular weight fraction (more than 5,000 to 10,000) is obtained.
and a low molecular weight fraction (molecular weight 5,000 to less than 10,000). CSF is contained in both the high-molecular fraction and the low-molecular fraction, but more than 90% of CSF is contained in the high-molecular fraction. The low molecular weight fraction can be concentrated under reduced pressure to produce a product. The concentrate of the polymer fraction is dissolved in a buffer solution with a concentration of 0.01 to 0.1M, pH 6.0 to 8.0, and an anion exchange resin equilibrated in advance with the buffer solution, such as DEAE cellulose, DEAE
- Contact with Cephadex or QAE-Sephadex, etc. to adsorb CSF to the resin, and then with a buffer solution with a concentration of 0.1 to 0.3M and a pH of 6.0 to 8.0.
Elute and purify the CSF. 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,
and 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. As a developing buffer when performing gel perchromatography, a buffer with a concentration of 0.01 to 0.3M and a pH of 6.0 to 8.0 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. The liquid medicine thus obtained is used as it is, and the powder medicine is appropriately dissolved in sterile water, sterile physiological saline, etc. at the time of use, and used as an injection. The medicament produced by the method of the present invention has an effective dosage determined by the animal test of Test 7 described below.
It is administered intravenously to patients with granulocytopenia at a dose of 77.8 mg/1 kg body weight/day or more. [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 purified glycoprotein obtained in Example 1, two types of media containing it and two types of media not containing it were prepared. Medium does not contain serum
Contains McCoy's 5A medium and 20% fetal bovine serum.
McCoy's 5A medium 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 aspirated from the sternum of a healthy person, taken into a heparinized syringe, and centrifuged at 1000 g for 10 minutes to collect buffy coat. Next, this was washed with McCoy's 5A medium, suspended in McCoy's 5A medium containing 20% serum, and plated on a Petri dish. Several mg of iron carboxylate powder, which had been sterilized by dry heat, was added per ml of the medium. ℃, 1-2 in incubator
Let stand for a while. 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-digesting bone marrow cells, used to measure 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. The number of colonies was used to represent the activity of CSF, 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 0.1, 1.0, 10.0, and 100 μg of purified glycoprotein prepared in the same manner as in Example 1 was added per ml of the medium, and this was added to the culture dish on which monocytes and macrophages had been adsorbed. The cells were cultured for 3 days using the method described above. Each of the obtained culture solutions was tested in the same manner as Test 1 to measure CSF activity and test for CSF production. A sample cultured without adding purified glycoprotein was used as a control. The results are in Table 2
As shown below.

[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
1 μg of glycoprotein (purified product obtained in Example 1)
A culture solution was obtained in the same manner as in Test 1, except that the following was added at a ratio of 1.5% and the number of days of culture was 3 days.
These culture solutions were then tested in the same manner as Test 1 to measure SSF activity and test for CSF production. The results are shown in Table 3.

[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. No serum was added to these media, and 1.0 μg of glycoprotein (purified product obtained in Example 1) was added per ml of each medium.
and cultured in the same manner as in 3-eye 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.

[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
Added 30% and further added glycoprotein (purified product of Example 1)
The same procedure as in Test 1 was used, except that a medium containing 1 μg of 1 μg per 1 ml of medium was used, the inoculation amount of monocrystals and macrophages was 10 ⁵ per 1 ml of medium, and the number of days of culture was 3 days. Cultivated by method,
A culture solution was obtained. Then, the obtained culture solution was tested in the same manner as Test 1 to measure the activity of CSF,
CSF production was tested. The results are shown in Table 5.

[Test 6]

Test on the addition of fractions containing glycoproteins. Although tests 1 to 5 were conducted using purified glycoproteins, it is demonstrated that glycoprotein-containing fractions, so-called semi-purified glycoproteins, can also be used in the method of the present invention. The glycoprotein-containing fraction used was as described in Example 1.
Fractions A and B were prepared in the same manner as above. These fractions were added to serum-free McCoy's 5A medium at ratios of 0, 0.5, 1.0, 5.0 and 10 mg/ml, respectively. If this amount of addition is converted into the amount of glycoprotein added per ml of medium, fraction A is 0, 8.3, and 8.3, respectively.
16.6, 83.3 and 166.6 μg, and 0, 41.7, 83.4, 417 and 834 μg in fraction B, respectively. 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. Since these fractions contain human serum albumin excreted in urine, human serum albumin (manufactured by Sigma) was added at the same ratio as a control.
It was added to McCoy's 5A medium and cultured in the same manner. 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.

[Test 7]

Test Regarding Effective Dose, etc. The present inventors conducted the following animal test to test the effective dose and acute toxic dose (LD ₅₀ ) of CSF obtained by the method of the present invention. A culture solution was produced by the same method as in Example 3.
This culture solution was filtered aseptically, and the ultrafiltration membrane (molecular weight
It was concentrated and desalted by filtration (less than 10,000 filtration membrane), and then aseptically freeze-dried to produce powdered CSF. As a result of testing this powder in the same manner as Test 1, the number of colonies formed by human bone marrow cells per mg
There were 4,500 pieces. For comparison, the bone marrow cells of C3H/He mice were tested in the same manner as Test 1, and the number of colonies formed by these mouse bone marrow cells was 7000 per mg. 6-week-old male C3H/He mice (average weight 20
g) The 80 animals were randomly divided into 16 groups of 5 animals each, and each group was further randomly divided into 4 groups of 4 groups. The CSF was added at 1 mg per 0.1 ml of sterile physiological saline (
This solution was subcutaneously injected at a rate of 0.1 ml per mouse per day for 5 consecutive days, and 1, 3, 7, and 11 days after the start of administration, each mouse Blood was collected from 5 mice in each group (groups from which blood was collected were excluded from subsequent tests), and the number of white blood cells and granulocytes in peripheral blood were measured using an automatic hematology meter and Wright-Giemsa staining, and CSF The increase in leukocytes and granulocytes due to administration was tested. A group treated in the same manner by administering 0.1 ml of sterile physiological saline containing no CSF was used as a control. The results are shown in Table 7.

[Table] Among the CSF administration groups, the group results were as follows:
Compared to the group (control), on the 11th day of the study (6 days after the end of CSF administration), the white blood cell count was approximately 2
granulocytes showed an approximately 3-fold increase. Furthermore, on the same day 11, the group showed a remarkable increase of about 4.5 times in the number of white blood cells and about 8 times in the number of granulocytes compared to the group. Based on the above, the effective dose in mice is 50 g/kg body weight per day.
Recognized as mg. And used in this test
As mentioned above, the colony-forming ability of human and mouse bone marrow cells in CSF is about 1.556 times that of mouse bone marrow cells than that of human bone marrow cells. It is 77.8 mg per 1 kg. Therefore, the effective dose for granulocytopenic patients is at least 77.8 mg/Kg body weight/day. Regarding the acute toxicity of CSF obtained by the method of the present invention, the CSF used in the above-mentioned dose test was used to examine C3H/He mice (average body weight) aged 6 to 8 weeks.
20.4g), but the mice weighed 1Kg.
There were no deaths in the 4.0g/dose group (5 females and 5 males each). Therefore, it was not possible to determine the acute toxic dose. 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, the number of cells was counted using an automatic blood cell counter (manufactured by Tor), and a smear of this suspension was also collected. were prepared, subjected to Wright-Giemsa staining, and the numbers of lymphocytes, monocytes, and macrophages were morphologically measured under a microscope, and their ratios were determined. As a result, the ratio of monocytes and macrophages was 25.5%. Pour 5 ml of this into four 15 cm diameter glass Petri dishes, and add 30 ml of 10% fetal bovine serum.
Add McCoy's 5A medium, leave for 2 hours in a humidified incubator (37℃) under 5% carbon dioxide aeration, then discard the medium, add 30 ml of McCoy's 5A medium, shake vigorously and wash. This was discarded and lymphocytes were removed. When the ratio of monocytes and macrophages was measured using the same test method as above, both were 95%. 2 Preparation of Glycoprotein Glycoprotein was prepared as follows according to the method of Example 1 of the previous application. Add 10% sodium hydroxide to 400ml of fresh urine collected from healthy people, adjust the pH to 8, and store at 0°C.
The mixture was centrifuged in a continuous centrifuge at 15,000 xg while cooling to remove insoluble materials to obtain a supernatant. Next, adjust the pH of this supernatant to 7 with 10% hydrochloric acid,
The solution was passed through a column (10 x 80 cm) packed with silica gel, and the components adsorbed on the silica gel were eluted from the column with 40 ml of 5% aqueous ammonia. The pH of the eluate thus obtained was adjusted to 7.5 with 1N sulfuric acid, powdered ammonium sulfate was added to make it 70% saturated, and the mixture was allowed to stand at 0°C overnight, and the precipitate formed was separated. This precipitate was dissolved in 5% ammonia water 2 and placed in a dialysis tube (manufactured by Visking).
Dialyze thoroughly against 0.05M phosphate buffer (PH6.5), add the buffer to the dialysate, and dilute the total volume to 10%.
CM Sephadex C, which was adjusted to
-50 ion exchange column (40 x 40 cm),
Impurities were adsorbed onto the ion exchange resin to obtain a permeate. This permeate 10 was concentrated using a diaflow hollow fiber concentrator (manufactured by Amicon, model DC-30), and the concentrated solution was mixed with 0.1M Tris in the same manner as above.
Dialysis was performed against a hydrochloric acid buffer (PH7.0) overnight at 5° C., and the buffer solution was added to the dialyzed solution to adjust the concentration to 1. This solution was equilibrated and activated with the buffer beforehand on a DEAE cellulose column (4.0
x 40cm), and thoroughly wash the column with 0.1M Tris-HCl buffer (PH7.0), then add 0.1M Tris-HCl buffer (PH7.0) containing 0.3M NaCl.
Elution was performed with and collect the eluate,
Dialysis was performed against 0.1M Tris-HCl buffer (PH7.0) to obtain a dialysate solution. DEAE cellulose column (4.0 x 40 cm) in which this dialysis solution was equilibrated and activated with the buffer solution again.
0.1M to 0.3M Cl ^- ions were eluted using a linear concentration gradient elution method, a fraction of 0.15 to 0.25M was collected, and powdered ammonium sulfate was added to this fraction to achieve 70% saturation, followed by precipitation. This precipitate was added to a small amount of 0.1M Tris-HCl buffer (PH
7.0) and dialyzed against the buffer to obtain a dialysate solution. (Fraction A). Next, add 20ml of this dialysis fluid to 0.1M in advance.
Developed on a Sephadex G-150 column (4.0 x 60 cm) equilibrated with Tris-HCl buffer (PH7.0), collected fractions with a relative eluate volume of 1.11-1.45, and added this fraction to distilled water. After thorough dialysis, the dialyzed fluid was freeze-dried to obtain about 500 mg of powder (Fraction B). Next, add 200mg of the above powder containing 1.0M salt.
The solution was passed through a column containing 100 ml of Concanavalin A-Sepharose 4B dissolved in 0.02 M phosphate buffer (PH7.0) and equilibrated with the buffer, and then 0.02 M phosphate buffer (PH7.0) containing 1.0 M NaCl was passed through the column. PH
Wash the column thoroughly with 7.0), then add 50mMα-
Contains methyl-D-glucoside and 1.0M salt
Elute with 0.02M phosphate buffer (PH7.0),
Collect the eluate, dialyze it against distilled water,
The dialyzed fluid was freeze-dried. Furthermore, about 50mg of the freeze-dried powder obtained here
was dissolved in 1 ml of 0.125M Tris-glycine buffer (PH6.8) containing 10% glycerin, and prepared using an 8% acrylamide gel (PH8.9, 20 mm x 25 mm) using a preparative electrophoresis device (Fuji Riken, Using a Fuji Coverer type), a current of 10 mA was applied while running cooling water, electrophoresis was performed, and a fraction with a relative mobility of 0.46 was collected in 0.025 M Tris-glycine buffer (PH8.3), and then added to distilled water. Dialysis was performed, and the dialyzed fluid was freeze-dried to obtain approximately 10 mg of glycoprotein. This method was repeated to obtain about 30 mg of glycoprotein. 3 Culture of monocytes and macrophages The above glycoprotein was added with 20% fetal bovine serum.
30 ml of McCoy's 5A medium was added at a rate of 100 μg 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 ¹⁰⁶ . This was cultured for 3 days in a humidified incubator (37°C) under 5% carbon dioxide aeration.
A culture medium containing CSF was obtained. 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 41,000 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 purified glycoprotein prepared in the same manner as in Example 1 was added to serum-free McCoy's 5A medium at a concentration of 100 μl per ml of the medium.
Approximately 5 ml of a purified CSF-containing solution was obtained in the same manner as in Example 1 using the culture medium added at a ratio of 5 ml. As a result of testing in the same manner as Test 1, this solution was 1
It had the activity of forming 16,000 human granulocyte colonies per ml. Example 3 Monocytes and macrophages were separated from human peripheral blood in the same manner as in Example 1. Then, the glycoprotein-containing fraction (fraction A) prepared in the same manner as in Example 1 was added to McCoy's 5A medium containing no serum.
Using a medium added at a rate of 5 mg (83.3 μg as glycoprotein) per ml of the medium, culturing was carried out in the same manner as in Example 1 to obtain about 120 ml of 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 36,700 human granulocyte colonies per ml. 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%.
Using a medium added to McCoy's 5A medium at a rate of 1 mg (83.3 μg as glycoprotein) per ml of medium,
Approximately 5 ml of purified CSF was prepared in the same manner as in Example 1.
A containing liquid was obtained. Tested in the same manner as Test 1, this solution yielded human granulocyte colonies per ml.
It had the activity of forming 43,200 pieces.

Claims (1)

[Claims] 1. Monocytes and macrophages isolated from human peripheral blood are cultured in a tissue culture medium containing a glycoprotein that proliferates human granulocytes isolated from human urine, and an active ingredient is added to the culture medium. A method for producing a substance for differentiating and proliferating human granulocytic stem cells, characterized by producing the substance 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 0.1 μg 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.