JP2021107385A - Pharmaceutical composition containing culture supernatant of menstrual blood-derived stem cell - Google Patents

Abstract

To provide a novel ART (Assisted Reproductive Technology) that can effectively treat endometrial dysfunction and infertility.SOLUTION: A pharmaceutical composition for treating endometrial dysfunction and/or infertility contains a culture supernatant of a menstrual blood-derived stem cell. The menstrual blood is, preferably, the self-menstrual blood of a patient in need of treatment and the culture supernatant is administered into the uterine cavity of a patient who desires treatment.SELECTED DRAWING: None

Description

The present invention relates to a novel treatment of endometrial failure and / or infertility with a culture supernatant of stem cells derived from menstrual blood.

The mainstream of infertility treatment is ART (Assisted Reproductive Technology) including in vitro fertilization, and 1 in 18 newborn babies is born by ART. Although recent advances in ART have been remarkable, the implantation rate and pregnancy rate of ART are still not sufficient. The main causes of infertility are considered to be decreased estrogen secretion due to aging, endometrial insufficiency, abnormal implantation of cytokines involved in implantation, and embryonic implantation failure due to the disruption of the immune system. Among them, the thickness of the endometrium is deeply related to the implantation rate, pregnancy and childbirth rate, and it is generally said that the thicker the endometrium, the higher the pregnancy rate.

Mesenchymal Stem Cell (MSC) has been confirmed to be present in various tissues, and is currently being attempted to be used in a wide range of fields from cosmetology to cardiovascular regeneration. The culture supernatant of mesenchymal stem cells contains various growth factors, cytokines, hormones, etc., and its effects on cell proliferation and intercellular signal transduction activity have been reported. Therefore, in recent years, not only cell transplantation but also regenerative medicine using a culture supernatant of mesenchymal stem cells has attracted attention.

It is known that the human endometrium contains a population of stem cells positive for mesenchymal stem cell markers called endometrial stem cells. Expecting the tissue regeneration ability of endometrial stem cells, a cell population containing endometrial stem cells is collected from the donor's uterus and transplanted into the recipient's uterus for endometrial function and fertilization. A method for enhancing the ability has also been proposed (Patent Document 1). However, collecting stem cells from the endometrium and transplanting the collected stem cells has a problem that the burden on the subject is heavy in addition to the problem of safety.

Menstrual blood includes cell groups such as stromal cells and epithelial cells, but it is also known that a small number of stem cells exist (Non-Patent Documents 1-3). Pluripotent stem cells are efficient from menstrual blood. There is also a report that it can be induced in (Non-Patent Document 4). Further, a method for efficiently obtaining stem cells (stem cells derived from menstrual blood) from menstrual blood has also been reported for the purpose of treatment and use in cosmetics (Patent Documents 2 and 3).

Special Table 2018-525356 Special table 2010-530214 Special Table 2011-501960

Patel et al., Cell Transplant. 2008; 17 (3): 303-11. Alcayaga-Miranda et al., Stem Cell Res Ther. 2015 Mar 17; 6:32 Mehrabani et al., Iran J Med Sci. 2016 Mar; 41 (2): 132-139. Li et al., Stem Cells Dev. 2013 Apr 1; 22 (7): 1147-58.

An object of the present invention is to provide a novel ART capable of effectively treating endometrial insufficiency and infertility.

The inventors have administered the culture supernatant of stem cells (such as endometrial stem cells) contained in menstrual blood into the uterine cavity to patients who require endometrial insufficiency or fertility treatment. It was found that the regeneration and proliferation of the uterus, the normalization of the immune response between the fertilized egg and the endometrium, and the development of the fertilized egg after transplantation are promoted, and pregnancy and healthy delivery are possible.
That is, the present invention relates to the following (1) to (10).
(1) A drug for improving the endometrial environment (for example, thickening the endometrium, activating the immune response, and improving the engraftment of fertilized eggs), which contains a culture supernatant of stem cells derived from menstrual blood. Composition; Specifically, a pharmaceutical composition for treating any disease or symptom selected from endometrial insufficiency, infertility, recurrent miscarriage, and habitual abortion.
(2) The pharmaceutical composition according to (1), wherein the menstrual blood is the autologous menstrual blood of a patient in need of treatment.
(3) The pharmaceutical composition according to (1) or (2), wherein the stem cells derived from menstrual blood contain cells that are positive for CD90, CD105, CD73, and CD146 and negative for CD34 and CD45. Optionally, menstrual blood-derived stem cells may further be negative for any one or more of CD11b, CD14, CD73a, and HLA-DR.
(4) The pharmaceutical composition according to any one of (1) to (3), wherein the culture supernatant is enriched with respect to GDF-15.
(5) The pharmaceutical composition according to any one of (1) to (4), wherein the culture supernatant is enriched with respect to IL-6 and / or GM-CSF.
(6) The pharmaceutical composition according to any one of (1) to (5), wherein the culture supernatant contains IGF-1. If desired, the culture supernatant may further contain any one or more of VEGF, Estradiol, bFGF, and IL-1β.
(7) The pharmaceutical composition according to any one of (1) to (6), which is administered into the uterine cavity of a patient 2 to 5 days before the estimated ovulation date. However, the administration time and the number of administrations can be appropriately changed and set depending on the disease and the patient's condition.
(8) The pharmaceutical composition according to any one of (1) to (7), wherein the culture supernatant contains components derived from allogeneic serum, preferably patient's autologous serum. That is, the culture supernatant is prepared by culturing stem cells derived from menstrual blood in a medium containing allogeneic serum, preferably the patient's autologous serum.
(9) A method for preparing a culture supernatant of stem cells derived from menstrual blood.
1) Cell fractionation of menstrual blood isolated from donors was performed by centrifugation, and the layer containing stem cells was collected.
2) The cells are cultured in a medium containing the donor's autologous serum, and the cells are cultured.
3) The method according to the above method, which comprises collecting a culture supernatant of cells 6 to 10 days after at least one passage. When a blood separating agent is used, the layer containing the target stem cells is contained in the layer containing mononuclear cells, which is the upper layer of the separating agent.
(10) The pharmaceutical composition according to any one of (1) to (8), wherein the culture supernatant of stem cells derived from menstrual blood is prepared by the method according to (9).

According to the present invention, endometrial insufficiency and infertility can be effectively treated. The culture supernatant of stem cells derived from menstrual blood used in the present invention contains components such as IGF-1 and LIF, and is enriched with components such as GDF-15, GM-CSF, and IL-6. These promote cell proliferation activity of endometrial progenitor cells, contribute to the promotion of engraftment of fertilized eggs, and improve the immune response in the uterine cavity. The pharmaceutical composition of the present invention enables effective endometrial regeneration and healthy pregnancy / delivery without imposing an undue burden on the patient. In particular, the method using autologous menstrual blood does not have a risk of biorejection or sensibility, and self-derived component factors are excellent in self-biocompatibility, so that safe treatment is provided.

A: In the cell fractionation after menstrual blood centrifugation, the target cells can be confirmed in the middle layer (the layer above the middle-low layer separating agent). B: A microscopic image of the cells in which the cells in the middle layer were collected and cultured. From the top, magnification: x40, x100, x200. FIG. 2 is a cell proliferation curve of P1 cells and P2 cells. The cell doubling time is P1: 48.4 ± 1.8, P2: 48.1 ± 0.9. FIG. 3A shows the results of expression analysis of mesenchymal stem cell (MSC) markers in cultured cells by a flow cytometer (FCM). The positive rates for CD90, CD105 and CD73 are high. FIG. 3B shows the results of confirming the cell surface localization of CD90 and CD105 with a fluorescence microscope by antibody staining. Green (MSC surface marker staining), blue (nuclear staining). A: Oil Red O staining (red) results on day 16 of induction of fat differentiation. Fat differentiation was confirmed. B: Results of Alizarin Red S staining (red) on day 16 of induction of bone differentiation. Bone differentiation was confirmed. C: Results of Alcian Blue staining (blue) on the 15th day of cartilage differentiation induction. Cartilage differentiation was confirmed. FIG. 5 shows the results of FCM expression analysis of CD31, CD117, and CD146. CD31 and CD117 were negative, but CD146 had a positive rate of 80% or more. FIG. 6 shows the quantitative results of the components (IGF-1, bFGF, VEGF-A, IL-1β, IL-6, Estradiol) contained in the culture supernatant. The graph shows the basal medium (Control), 10% serum-containing medium, and menstrual blood-derived stem cell culture supernatant from the left. A comparison of the component contents of the 10% serum-containing medium and the menstrual blood-derived stem cell culture supernatant confirmed an increase or decrease in the contents of IGF-1 and IL-6. A: Results of confirming the cell surface localization of the IGF-1 receptor (IGF-IR) with a fluorescence microscope by antibody staining. IGF-1R staining: green, nuclear staining: blue. B: Results of FCM analysis of IGF-IR expression (n = 3). Isotype control: black, CD221: red. C: Results of confirming the proliferative ability of cells by an IGF-1R inhibitor (PPP: Picropodophyllin). The absorbance decreased depending on the amount of PPP added, and a decrease in proliferation ability was confirmed in each sample (Sample 1, Sample 2, Sample 3). (DMSO concentration <0.1%) D: The proliferative ability test results when PPP (1 μM) was added to the menstrual blood-derived stem cell culture supernatant and the cells were cultured are shown. It was confirmed that the proliferation ability was reduced by the addition of PPP, and it was found that IGF-1 was involved in cell proliferation. The contents of IGF-1 and IL-6 in the culture supernatant stored frozen for 1 year under the temperature conditions of -20 ° C, -80 ° C, and -196 ° C are shown (Control: before cryostorage). FIG. 9 shows the amount of factors (A: chemokine, B: inflammatory factor, C: growth factor, all of which show only factors within the range of the calibration curve) contained in the menstrual blood-derived stem cell culture supernatant. FIG. 10 shows the amount of factors (A: chemokine, B: inflammatory factor, C: growth factor, all of which show only factors within the range of the calibration curve) contained in the umbilical cord stem cell culture supernatant. FIG. 11 shows each factor (A: chemokine, B: inflammatory factor) in the culture supernatant before and after culturing (menstrual blood-derived stem cell culture supernatant) before culturing (10% serum-containing medium) and after culturing (menstrual blood-derived stem cell culture supernatant). , C: Growth factor) changes in amount. FIG. 12 shows the amounts of factors (LIF, GDF-15, VEGF, GM-CSF) effective for infertility in the menstrual blood-derived stem cell culture supernatant and the umbilical cord stem cell culture supernatant.

1. 1. Menstrual blood "Menstrual blood" is a bloody secretion released by physiological bleeding (menstruation) that occurs when the endometrium of the uterine wall is periodically exfoliated and shed. , Approximately 60-80% of blood components, exfoliated and shed endometrial cells / tissues, vaginal epithelial cells, etc. are included.

The menstrual blood used in the present invention is preferably human menstrual blood, which may be allogeneic or self-reliant, but is preferably self-menstrual blood of the patient to be treated. When collecting samples such as menstrual blood and peripheral blood, for safety, perform a blood test of the donor (patient) and confirm that HIV-1, HBV, HCV, HTLV-1, syphilis, etc. are negative. do.

Menstrual blood is collected vaginal from the uterus of the donor (preferably the patient being treated) during the menstrual period. Collection of menstrual blood is not particularly limited as long as it is possible, for example, 1 to 5 days after the start of menstruation, preferably within 3 days from the start of menstruation. The larger the amount of menstrual blood collected, the easier it is to collect stem cells and the higher the success rate of culture. The amount is 0.5-2.5 ml, preferably 1.0-2.0 ml, most preferably about 1.5 ml. The collected menstrual blood is controlled at about 4 ° C until the cell culture processing stage in order to maintain the quality.

In addition, in this specification, "about" means plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1 respectively with respect to a reference value. Indicates a value that fluctuates up to%. Preferably, the term "about" refers to a range of plus or minus 15%, 10%, 5%, or 1%, respectively, relative to the reference value.

2. Stem cells derived from menstrual blood (menstrual blood stem cells)
The cells used in the present invention are stem cells derived from menstrual blood and having self-renewal ability and pluripotency (hereinafter, also referred to as "menstrual blood stem cells"). These menstrual blood stem cells have a spindle-shaped fibroblast-like morphology as found in mesenchymal stem cells, are positive for mesenchymal stem cell markers such as CD90, CD105, and CD73, and are mesenchymal. Stem cells are characterized by negative CD34, CD45, CD11b, CD14, CD73a, and HLA-DR.

"Mesenchymal stem cells" are somatic stem cells that can differentiate into mesenchymal lines including osteoblasts, muscle cells, chondrocytes, and adipocytes, and specific markers thereof are, for example, Vasileios Karantalis and Joshua M. Hare, Circ Res. 2015 April 10; 116 (8): 1413-1430, and Imran Ullah, et al., Biosci. Rep. (2015), 35 / art: e00191 et al.

In addition, menstrual blood stem cells are also characterized by being positive for the endometrial cell marker CD146. Based on these characteristics and the origin of menstrual blood, it is highly probable that the stem cells derived from menstrual blood are endometrial-derived stem cells (endometrial stem cells).

As used herein, the term "marker" means a cell antigen or a gene thereof that is specifically expressed by a predetermined cell type, such as "marker protein" or "marker gene", and includes both a positive marker and a negative marker. include. Preferably, the marker is a cell surface marker, allowing cell enrichment, isolation, and / or detection.

Detection of a marker protein can be performed using an immunological assay using an antibody specific for the marker protein, for example, ELISA, immunostaining, or flow cytometry. The marker gene can be detected by using a nucleic acid amplification method and / or a nucleic acid detection method known in the art, for example, RT-PCR, a microarray, a biochip, or the like.

In the present specification, "positive" marker expression means that the protein or gene is expressed in a detectable amount by a method known in the art. Protein detection can be performed using antibody-based immunological assays such as ELISA, immunostaining, and flow cytometry. In the case of a protein that is expressed inside the cell and does not appear on the cell surface (for example, a transcription factor or its subunit), the reporter protein is expressed together with the protein, and the target protein is detected by detecting the reporter protein. Can be detected. Gene detection can be performed using, for example, a nucleic acid amplification method and / or a nucleic acid detection method such as RT-PCR, microarray, biochip and RNAseq.

In the present invention, the "positive" marker has a positive rate of at least 70%, preferably 80%, more preferably 90%, 95%, or 98% positive rate, most preferably 99% positive rate. be. Further, the marker expression of "negative" means that the expression level of the protein or gene is below the detection limit or 1% or less by all or any of the above-mentioned known methods. ..

3. 3. Culture supernatant of menstrual blood stem cells The culture supernatant of menstrual blood stem cells is a culture supernatant obtained by culturing stem cells contained in the cell fraction obtained by separating blood components (blood cells) from menstrual blood. be. Since it is difficult to isolate, purify, and culture only menstrual blood stem cells from menstrual blood, the state of the cell fraction (cell group) containing other cells (leukocytes, granulocytes, squamous epithelial cells, etc.). Start culturing at. Therefore, in the present specification, the culture of menstrual blood stem cells means the culture of a cell group containing menstrual blood stem cells.

(1) Cell Separation The cell fractionation (cell group) containing menstrual blood stem cells can be separated from menstrual blood by centrifuging with a commercially available separating agent (using a commercially available separating agent). Any density gradient solvent capable of selectively separating blood cells) may be used, and for example, commercially available Histopaque® (Sigma-Aldrich) can be used. The layer containing the target menstrual blood stem cells is a separating agent. It is contained in the upper layer containing mononuclear cells.

(2) Cell culture (2-1) medium Menstrual blood stem cells are cultured in a medium containing the autologous serum of the donor (patient) from which menstrual blood was collected. Peripheral blood is collected from the same donor (patient) when menstrual blood is collected and is controlled at 4 ° C. Autologous serum is prepared from this peripheral blood by centrifugation according to a conventional method. The autologous serum is 10 to 30%, preferably 20 to 30%, more preferably 20 to 25%, and most preferably about 20% in the medium described later in the primary culture. During subculture, 5% to 20%, preferably 5 to 15%, more preferably 10 to 15%, and most preferably about 10% are added.

The basal media used for culturing include DMEM medium, BME medium, αMEM medium, serum-free DMEM / F12 medium, BGJb medium, CMRL 1066 medium, Glasgow MEM doubled medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, etc. Any medium that can be used for culturing animal cells, such as Eagle MEM medium, Ham's medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, McCoy's medium, and William's E medium, can be used. / F12 medium is preferred.

It is preferable that the medium does not contain pigments such as Phenol red, HEPES, components (factors) derived from animals or humans, components (factors) that cause tumorigenesis or neurotoxins, and the like.

The medium is composed of the above-mentioned components of the basal medium and autologous serum, but various nutrient sources necessary for cell maintenance and proliferation and each component necessary for differentiation induction are appropriately added as long as the object of the present invention is not impaired. You may. For example, nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch and dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin and alcohols, ammonium sulfate, ammonium nitrate and ammonium chloride. , Nitrogen sources such as urea and sodium nitrate, salt, potassium salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt and other inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride , Ferrous sulfate, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, amino acids and the like can be contained.

The pH of the medium obtained by blending the above components is in the range of 6.0 to 9.0, preferably 6.5 to 8.5, and more preferably 7.0 to 8.0.

(2-2) Culture conditions Menstrual blood stem cells are two-dimensionally cultured (planar culture). The container is not particularly limited as long as it is used for cell culture, and is not particularly limited. Flask, tissue culture flask, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multi-plate, multi-well Plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles can be used.

Cultures are at least 80% confluent with medium exchange under conditions of _{1% -25% O 2} and 1% -15% CO ₂ at 36 ° C-38 ° C, preferably 36.5 ° C-37.5 ° C. Do up to. Medium exchange is performed within 5 days, preferably once every 3-4 days, more preferably once every 2 days.

Typically, primary cultured cells have a slow growth rate and are cultured in the presence of about 20% autologous serum for 12 to 20 days until they reach at least 80% confluence. The cells after passage are cultured for 6 to 10 days in the presence of about 10% autologous serum.

(3) Separation of culture supernatant The cells after passage at least once are cultured in the culture supernatant according to the above procedure, the culture supernatant is collected, and the cells are sterilized with a 0.2 μm filter. Prepare.

For safety, it is desirable that the collected culture supernatant be subjected to endotoxin test, mycoplasma test, sterility test, and personal identification test again. The culture supernatant is cryopreserved at −20 ° C. or lower until clinical use.

3. 3. Pharmaceutical composition containing a culture supernatant of menstrual blood stem cells The culture supernatant of menstrual blood stem cells of the present invention is administered as it is or mixed with a pharmaceutically acceptable carrier after thawing, and is administered to a recipient (patient). Improves the intrauterine environment, promotes implantation of fertilization and maintains pregnancy. The present invention provides a pharmaceutical composition and a therapeutic method using a culture supernatant of such menstrual blood stem cells.

Diseases or diseases to which the pharmaceutical composition of the present invention can be applied include, but are not limited to, endometrial insufficiency, infertility, recurrent miscarriage, and habitual miscarriage. Endometriosis includes endometriosis, uterine fibroids, uterine adenomyosis, endometrial cancer, endometrial polyps, endometrial adhesions, endometrial adhesions (Asherman syndrome), uterine dysgenesis, uterus Includes diseases such as intimal fibroids and intimal disorders associated with their treatment. Infertility includes infertility of uterine factors such as unexplained infertility, elderly infertility, and infertility of infertility of the endometrium.

Pharmaceutically acceptable carriers include medical gels such as hyaluronic acid, gelatin, collagen, carboxymethyl cellulose, alginic acid, pectin, sulfated dextran; hyaluronic acid hydrogels, gelatin hydrogels, and collagen hydrogels.

The pharmaceutical composition of the present invention is administered into the uterine cavity of the recipient (patient). The administration is carried out by a method generally applied in the art, for example, using an injection needle for artificial insemination in a general 1CC syringe.

The administration time of the pharmaceutical composition of the present invention is appropriately determined according to the disease to which it is applied and the condition of the patient. The number of administrations is also appropriately determined according to the disease to be applied and the condition of the patient, and is administered once or multiple times at necessary intervals.

In the case of patients with endometrial insufficiency, the culture supernatant is generally administered intrauterinely 1 to 6 days, preferably 2 to 5 days before the estimated ovulation date, but is not limited to this.

In the case of infertility treatment, the culture supernatant is administered intrauterinely before ovulation in the patient's menstrual cycle, preferably 1 to 6 days, more preferably 2 to 5 days before the estimated ovulation date.

The culture supernatant of the menstrual blood stem cells of the present invention is characterized by being enriched with respect to GDF-15. Preferably, the culture supernatant is also enriched for IL-6 and / or GM-CSF. "Enriched" means that by culturing menstrual stem cells, the content is higher than before culturing. Preferably, the "enriched" component is contained in a higher content than other cell culture supernatants cultured under the same conditions.

GDF-15 (Growth and Differentiation Factor-15) is a growth factor that is highly expressed in the brain and placenta, also known as MIC-1 (macrophage inhibitory cytokine-1), but is a tree present in the endometrium. It is known to contribute to the maintenance of pregnancy through immune tolerance of dendritic cells (Tong S, et al. Lancet. 2004; 363 (9403): 129-130, Segerer SE, et al. Hum Reprod. 2012; 17 (1): 200-209. ).

IL-6 (Interleukin-6) is an inflammation-related cytokine produced by cells such as T cells and macrophages, and can contribute to the activation of immune response and angiogenesis in the intrauterine environment. Known (Park HY et al. Mol Reprod Dev. 2013; 80 (12): 1035-47., Herrmann JL et al. Shock. 2011; 35 (5): 512-6).

GM-CSF (Granulocyte Macrophage Colony-Stimulating Factor) functions as a hematopoietic growth factor and immunomodulator, but is involved in embryo protection, endometrial environment regulation, and implantation promotion. (Ziebe S, et al. Fertil Steril. 2013; 99: 1600-1609, Spandorfer SD, et al. Am J Reprod Immunol. 1998; 40: 377-381., Eftekhar M, et al. J Res Med Sci. 2018; 23: 7).

The culture supernatant of menstrual blood stem cells of the present invention contains IGF-1 (Insulin-like growth factor 1: insulin-like growth factor 1) derived from serum or menstrual blood. It also contains a small amount of LIF (Leukemia Inhibitory Factor) and the like. These components help menstrual blood stem cells maintain their undifferentiated state and proliferate, resulting in a culture supernatant enriched for useful factors such as GDF-15, GM-CSF, and IL-6. Allows the provision of. At the same time, IGF-1 and LIF, even in trace amounts, may promote endometrial cell proliferation and aid implantation when administered intrauterinely (Kondera-Anasz Z, et al). Am J Reprod Immunol. 2004; 52 (2): 97-105., Aghajanova L. Ann NY Acad Sci. 2004; 1034 (1): 176-183.).

Furthermore, the culture supernatant contains useful components / factors derived from donor menstrual blood and serum such as VEGF, Estradiol, bFGF, IL-1β, and these components / factors interact with each other. By adding to the effects of GDF-15, GM-CSF, IL-6, IGF-1, etc., it is expected that the intrauterine environment will be further improved, and it will be possible to promote the implantation of fertilization and maintain pregnancy.

From the above characteristics, the pharmaceutical composition containing the culture supernatant of menstrual blood stem cells of the present invention can be expected to be effective in treating endometrial insufficiency, infertility, recurrent miscarriage, habitual miscarriage, and infertility of unknown cause.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

[Example 1]
Preparation of stem cell culture supernatant from menstrual blood 1. Materials and methods 1.1 Collection of sample information and menstrual blood
Among infertility treatment patients aged 30 to 45 years, blood tests (HIV, HBV, HCV, HTLV-1, syphilis) were performed in advance, and only patients with a negative test were targeted. All samples were obtained from patients with informed consent.

Menstrual blood excreted within 3 days of the day of ovulation in the menstrual cycle was collected directly from the uterus using a heparin-coated syringe. The collected menstrual blood is Penicillin-Streptomycin Solution (100 unit / ml penicillin 100 ug / ml Streptomycin), Amphotericin B Solution (2.5 ug / ml), Phosphate Buffer Saline (Antibiotics and Reagent: GE Healthcare Lifesciences HyClone Inc. USA), It was diluted with a solution prepared with Heparin (10unit / ml) (NIPRO. JPN). In addition, about 100 ml of peripheral blood of the same patient was collected. Menstrual blood and peripheral blood were controlled at 4 ° C.

1.2 Menstrual blood separation and cell culture, preparation of autologous serum Add the separating agent Histopaque-1077 (Sigma-Aldrich Co. USA) to menstrual blood and fractionate the blood by centrifugation (400 g, 30 min, RT). I let you. The boundary between the separating agent and the supernatant was collected and washed with PBS (300 g, 10 min, 4 ° C). DMEM / F12 without DMEM / F12 without the supernatant removed from the collected fraction and containing medium (20% autologous serum, Penicillin-Streptomycin Solution (100 unit / ml penicillin 100 ug / ml Streptomycin), Amphotericin B Solution (2.5 ug / ml). It was suspended in HEPS, Phenol Red (GE Healthcare Lifesciences, HyClone Inc. USA), seeded in T-25 Flask (IWAKI Co. JPN), and cultured in a _{CO 2 incubator.} Medium exchange and cell morphology observation were performed once every two days.

Peripheral blood is centrifuged (1000 g, 10 min, RT) to collect only the supernatant, zosyn (450 μg / ml) (Taisho Toyama Pharmaceutical Co. JPN) is added, and the bacteria are eradicated with a 0.2 μm filter (Pall Corporation. USA). It was deactivated (56 ° C, 30 min) and used for cell culture as autologous serum.

Primary cultured cells (P0) are cultured to 80% confluence, washed with PBS, exfoliated with TrypLE Express without Phenol Red (Thermo Fisher Scientific, Life Technologies. USA), and the cells are collected and centrifuged (300 g). , 5 min, 4 ℃). Then, the cells were suspended in a medium (10% serum medium) containing 10% of autologous serum prepared from the patient's peripheral blood ^{, and seeded and cultured in 3.0 × 10 4 to} 5.0 × 10 ⁴ cells / T-25 Flask. did.

1.3 Preparation of autologous menstrual blood-derived stem cell culture supernatant The passaged cells were cultured until they became 80% confluent, and then the culture supernatant was collected and sterilized with a 0.2 um filter. The collected culture supernatant was subjected to sterility test, mycoplasma test, endotoxin test, and personal identification test, and controlled at -20 ° C or lower until use in medical institutions.

1.4 Evaluation of cell proliferation ability The culture period was set to 4,6,8,10 days, ^{and cells were seeded on a 24-well plate (IWAKI Co. JPN) at 2.26 × 10 3} cells / well, and after each culture period. The number of cells was measured and a proliferation curve was created. In addition, the cell doubling time (Td) was calculated. The proliferative capacity of the first passage cells (Passage1: P1) and the second passage cells (Passage2: P2) was evaluated. For culturing, 10% serum medium was used, and the medium was changed once every two days.

1.5 Measurement of mesenchymal stem cell markers For measurement of mesenchymal stem cell markers, cultured P1 cells were collected, washed, and then incubated with FcR brocking reagent (Miltenyi Biotec Inc USA) (30 min. 4 ° C). ). Then, antibody staining (30 min, 4 ° C.) was performed, and the measurement was performed with a flow cytometer (FCM) (Becton Dickinson Co. USA). Antibodies are CD90 PE, CD105 FITC, Isotype IgG1 PE, Isotype IgG1 FITC (Becton Dickinson Co. USA), CD73 APC, CD34 FITC, CD45 PE, CD14 APC, CD11b PE, CD79α PE, HLA-DR FITC, Isotype IgG1 PE. , Isotype IgG1 FITC, Isotype IgG1 APC, Isotype IgG2 FITC (Thermo Fisher Scientific. USA) were used. After antibody staining, measurement was performed by FCM.

Cell surface markers were measured with a fluorescence microscope. P1 cells were ^{seeded with 2.0 × 10 4} cells / 35 mm dish (SARSTEDT AG & Co. Germany), and after confirming the adhesion of the cells to the dish, the cells were fixed with 4% Paraformaldehyde, and the primary antibody mouse anti-human CD90: 5.0 ug / ml and mouse anti-human CD 105: 4.0 ug / ml (Thermo Fisher Scientific. USA) were added, respectively, and the cells were reacted overnight at 4 ° C. After washing with PBS, secondary antibody goat anti-mouse IgG FITC: 10.0 ug / ml (Thermo Fisher Scientific, USA) was added, reacted at 37 ° C for 1 hour, stained with DAPI (Sigma-Aldrich Co. USA), and then fluorescent. It was observed with a microscope (KEYENCE Co. JPN).

1.6 Confirmation of differentiation potential
P0 cells were seeded on a 24-well plate at 1 × 10 ⁴ cells / well and pre-cultured in 10% serum medium. After that, it was replaced with each differentiation medium (MesenCult Adipogenesis Differentiation kit, MesenCult Osteogenesis Differentiation kit (VERITAS Co. JPN), MSC Go Chondrogenic XF ^TM , MSC Go Chondrogenic XF ^TM Supplement Mix (Biological Industries Ltd. USA), and once every two days. The medium was changed at the same frequency as, and the cells were cultured for 20 days. For staining, the cells were fixed with 4% Paraformaldehyde (Sigma-Aldrich Co. USA). For adipogenic cells, Oil Red (Sigma-Aldrich Co. USA) was used. Stained with and washed with 60% Isopropanol (Sigma-Aldrich Co. USA). Bone differentiated cells were stained with Alizarin Red S (Sigma-Aldrich Co. USA) and washed with DW. Chondrogenic differentiated cells Was stained with Alcian Blue (Merck Co. GER) and washed with 3% Acidic acid (Sigma-Aldrich Co. USA) and DW. Cells after induction of differentiation were observed under a microscope.

1.7 Measurement of other cell surface markers The cell surface markers of cultured cells were measured according to the procedure of 1.5. Antibody staining was performed using CD31 APC, CD117 PE, CD146 PE, Isotype IgG1 APC, Isotype IgG1 PE (Thermo Fisher Scientific. USA).

1.8 Measurement of components in culture supernatant Measure the components (IGF-1, bFGF, VEGF-A, IL-1β, IL-6, Estradiol) in the culture supernatant, and measure the autologous serum-containing medium and menstrual blood. Comparison was made with the derived stem cell culture supernatant. Specifically, each component is Quantikine ELISA kit human IGF-1, human FGF basic, human IL-6, Estradiol (R & D systems Inc. USA), human VEGF-A ELISA kit, human IL-1 beta ELISA kit (Ray). It was measured with a microplate reader (Thermo Fisher Scientific. USA) using Biotech Inc. USA) and quantified with the Standard included in the ELISA Kit.

1.9 Action of IGF-1 The localization of IGF-1 receptor (IGF-1R) in cultured cells was investigated according to the procedure of 1.5. Mouse anti-human IGF-1R: 25.0 ug / ml (R & D systems Inc. USA), goat anti-mouse IgG FITC: 13.6 ug / ml (Jackson Immuno Research Inc. USA), and DAPI were added to the cells for reaction, and a fluorescence microscope was used. Observed at. In addition, antibody staining was performed using CD221 (IGF-1R) APC and Isotype IgG1 APC (Thermo Fisher Scientific. USA), and observation was performed by FCM.

A growth inhibition test with an IGF-1R inhibitor (Picropodophyllotoxin: PPP) was conducted. The cells were seeded on a 96-well plate (IWAKI Co. JPN) at 5 × 10 ³ cells / well, _{subjected to CO 2} incubation for about 12 hours, the medium was removed, and PPP 1 μM (Cayman Chemical) diluted with 10% serum medium was used. Co. USA), PPP 1 μM diluted with the culture supernatant, and the culture supernatant were added to each well and cultured for 48 hours. After culturing, WST-8 (Dojindo Molecular Technologies Inc. JPN) was added, CO ₂ incubation was performed for 2 hours, and the absorbance was measured with a microplate reader.

1.10 Examination of optimum storage conditions for culture supernatant
The culture supernatant was cryopreserved for 1 year under the temperature conditions of -20 ° C, -80 ° C, and -196 ° C, and IGF-1 and IL-6 were quantified according to the procedure of 1.8. The measured value after preparing the culture supernatant was set to 100% (Control), and the content rate after storage for 1 year was calculated.

2. Results 2.1 Menstrual blood separation and cell culture As a result of centrifugation, the menstrual blood sample should have red blood cells in the lower layer, a separating agent in the middle and lower layers, cell groups such as lymphocytes and granulocytes in the middle layer, and a diluent in the upper layer. It was fractionated into (Fig. 1A). Only the cells in the middle layer were collected and cultured. Observation of cell morphology was performed with objective lenses: × 4, × 10, × 20, eyepieces: × 10, and no abnormal cell morphology was confirmed (FIG. 1B). The cells contained spindle-shaped fibroblast-like cells and the like. As for the culture period, 80% confluence was confirmed within about 20 days for primary cultured cells (P0) and within about 8 days for P1.

2.2 Evaluation of cell proliferation ability
Cell culture was performed for 10 days, and the cell proliferation curve was prepared and the doubling time was determined. From the cell proliferation curve, it was judged that it was optimal to perform cell passage and collection of culture supernatant on days 6 to 8. The doubling time was about 48 hours, and it was considered appropriate to change the medium once every two days.

2.3 Measurement of mesenchymal stem cell markers The positive rates of the mesenchymal stem cell (MSC) markers CD90, CD105, and CD73 tended to be high in the cultured cells. CD34, CD45, CD11b, CD14, CD73a, and HLA-DR, which are negative for mesenchymal stem cells, were as low as 1% or less and were judged to be negative (Fig. 3A, Table 1). As a result of confirming the cell surface localization of CD90 and CD105 with a fluorescence microscope, fluorescence was confirmed in both (Fig. 3B). From these results, it was confirmed that the cultured cells were close to mesenchymal stem cells.

2.4 Confirmation of differentiation potential As a result of culturing in a differentiation-inducing medium, adipocyte differentiation (15th day), cartilage differentiation (16th day), and bone differentiation (16th day) were confirmed, respectively (Fig. 4). It was confirmed that the cells isolated from the menstrual blood had pluripotency.

2.5 Measurement of other cell surface markers As a result of measurement of cell surface markers other than mesenchymal stem cell markers, CD31 and CD117 were negative, and CD146 had a positive rate of 80% or more (Fig. 5, Fig. 5,). Table 2).

2.6 Measurement of components of culture supernatant The growth factors (IGF-1, bFGF, VEGF-A), cytokines (IL-1β, IL-6), and hormones (Estradiol) contained in the culture supernatant were measured. .. As a result of comparison with the culture supernatant using the serum-containing medium (10% serum medium) before culturing as a control, a significant increase or decrease in IGF-1 and IL-6 was confirmed (FIGS. 6 and 3). Since IGF-1 decreased after culturing (P <0.01), it was predicted that the cultured cells would proliferate via IGF-1 derived from autologous serum. Since IL-6 increased after culturing (P <0.01), it was considered that IL-6 was produced by cultured cells and was involved in the promotion of immune response between cells. Regarding other factors, no significant difference was confirmed between the control and the culture supernatant.

2.7 Action of IGF-1 Cell surface localization of IGF-1R (CD221) was confirmed by both fluorescence microscopy and FCM (FIGS. 7A and 7B). In the growth inhibition test, it was confirmed that the addition of PPP reduced the absorbance and the growth ability (P <0.05) (Fig. 7D), and that the decrease in the absorbance (proliferation ability) was PPP concentration-dependent. (Fig. 7C). From this, it was considered that IGF-1 is important for cell proliferation.

2.8 Examination of optimum storage conditions for culture supernatant
No significant difference was observed in the component factor content at each temperature of -20 ° C, -80 ° C, and -196 ° C, and no significant decrease in component factor was confirmed during the storage period of 1 year (Fig. 8). ). Therefore, it was judged that the culture supernatant can be cryopreserved for one year if the temperature is -20 ° C or lower.

3. 3. Discussion The amount of cells recovered from menstrual blood depends on the amount of menstrual blood, regardless of the age of the subject. Therefore, it was considered desirable to collect 1 ml or more of menstrual blood in order to obtain the necessary cell culture supernatant.

In the case of P0 cells, the culture period until reaching 80% confluence was within 20 days, and 20% of autologous serum was considered appropriate. On the other hand, in P1 cells, the culture period until reaching 80% confluence was within 8 days, and the autologous serum volume of 10% was considered to be sufficient. From these results, it was considered appropriate that the cell culture period should be 28 days, which is the same as the human menstrual cycle.

Although there are individual differences in the cell proliferation ability of P1 and P2 cells, it was considered desirable to collect the culture supernatant and subculture cells within 6 to 8 days from the start of culture. In addition, since the doubling time was about 48 hours, it was considered optimal to change the medium once every two days.

Considering the sterility test, mycoplasma test, endotoxin test, personal identification test, and securing the sampling required for treatment, it is difficult to recover the culture supernatant from P0 cells, and the culture supernatant derived from P1 cells is used clinically. It was considered appropriate to do so.

It was confirmed that the surface markers (positive rate) of the cultured cells were almost the same as those of mesenchymal stem cells. However, the positive rate of CD105 was about 80%, and although the positive rate of 95% or more, which is generally reported in mesenchymal stem cells, could not be confirmed, cell surface localization could be confirmed under a fluorescence microscope. rice field. In addition, the induction of differentiation into fat, bone, and cartilage was confirmed. From the above results, it is highly possible that menstrual blood-derived cells are a type of mesenchymal stem cells.

The cultured cells were negative for CD31, which is a marker for vascular endothelial cells and vascular endothelial progenitor cells, and CD117, which is a marker for myocardial stem cells. On the other hand, the positive rate for CD146, which is specific to endometrial progenitor cells, was 80% or more. From this, it seems that the cells derived from menstrual blood are cells close to endometrial cells. Since it is a type of somatic stem cell similar to endometrial cells, it is highly possible that menstrual blood-derived cells are endometrial stem cells.

In the culture supernatant, an increase / decrease tendency was confirmed in IGF-1 and IL-6 before and after cell proliferation. It has been reported that IGF-1 is involved in the proliferation and protection of endometrial cells, and IL-6 is involved in the immune response and activity of the intrauterine environment or angiogenesis. Although there was no significant difference, an increase in VEGF-A was confirmed in the culture supernatant. Since VEGF-A is involved in angiogenesis and the like, it is expected to promote cell proliferation in the uterine environment. In addition, although not derived from cells, the culture supernatant contains estradiol, bFGF, and IL-1β, which are involved in promoting implantation of fertilization and maintaining pregnancy, and IGF-1 and IL-6. At the same time, it was expected to contribute to the activation of the intrauterine environment. As described above, menstrual blood-derived cells are considered to be endometrial stem cells, and the effect of menstrual blood-derived stem cells on proliferation suggests an effect on endometrial stem cells. IGF-1 enriches useful components derived from stem cells such as IL-6 by promoting the proliferation of menstrual blood stem cells. Various components and factors in the culture supernatant thus obtained were considered to be important for the treatment of endometrial insufficiency and infertility.

[Example 2]
1. 1. Materials and methods 1.1 Cells and serum (1) Menstrual blood-derived stem cells (M-1, M-2, M-3)
Cells containing menstrual blood-derived stem cells were prepared from the menstrual blood of infertility patients according to Example 1 (3 samples). Serum prepared from the peripheral blood of the same patient was used for culturing menstrual blood-derived stem cells.
(2) Umbilical cord-derived mesenchymal stem cells (U-1, U-2, U-3)
Umbilical cord matrix-derived mesenchymal stem cells (hereinafter referred to as umbilical cord-derived stem cells) purchased from PromoCell were used (3 samples). Pooled serum was used to culture umbilical cord stem cells.

1.2 Cell culture The cryopreserved cells were thawed, medium was added, and the supernatant was removed by centrifugation (4 ° C., 5 min, 300 g). A medium containing 10% serum was added to the pellets, seeded in a T-25 flask, and cultured in a 5% CO ₂ incubator at 37 ° C. until reaching 80% confluence while changing the medium every 2 days. The cells were further subcultured and cultured until they became 80% or more confluent, and the supernatant was collected (stored at -196 ° C).

1.3 Measurement of components in culture supernatant The components in the culture supernatant were measured using Quantibody Human Cytokine Array 2000 (Ray biotech), and menstrual blood-derived stem cell culture supernatant and umbilical cord stem cell culture supernatant were measured. Compared with.
Measurement sample:
・ 3 samples of menstrual blood-derived stem cell culture supernatant (M: M-1, M-2, M-3)
・ Patient-derived 10% serum-containing medium 3 samples (Mp: Mp-1, Mp-2, Mp-3)
・ Umbilical cord stem cell culture supernatant 3 samples (U: U-1, U-2, U-3):
・ 10% pool serum-containing medium (P)
-Medium (N): Basic medium used for culturing various cells (DMEM / F12 (Hyclone))
Measurement item:
(1) chemokines, (2) growth factors, (3) inflammatory factors (Inflammation)

2. Results 2.1 Factors contained in the culture supernatant Table 5 shows the number (types) of factors within the range of the calibration curve contained in each culture supernatant. The types of chemokines and growth factors were more in the umbilical cord stem cell culture supernatant, but the types of inflammatory factors were more in the menstrual blood-derived stem cell culture supernatant.

2.2 Amount of each factor contained in the culture supernatant The factors contained in the menstrual blood-derived stem cell culture supernatant (factors within the calibration curve shown in Table 5) are shown in FIG. 9, umbilical cord stem cell culture supernatant. (Factors within the range of the calibration curve shown in Table 5) included in FIG. 10 are shown in FIG. Each supernatant contained many factors, but the types and amounts were different.

2.2 Changes in each factor before and after culturing The changes in the amount of each factor in the supernatant after culturing menstrual blood-derived stem cells are shown in FIG. In the menstrual blood-derived stem cell culture supernatant, chemokine: Axl, MIF, growth factors: GDF-15, IGFBP-2, OPG, inflammation-related factors: IL-13, TIMP-1, TNF RI increased after culture. Admitted. On the other hand, chemokines: LIF, MDC, MSPα, growth factors: BDNF, EG-VEGF, PDGF-AA, TGFα, and inflammation-related factors: IL-1α, IL-1ra, PDGF-BB were decreased.

2.3 Comparison of effective factors for infertility Factors (LIF, GDF-15, VEGF, GM-CSF) effective for infertility in menstrual blood-derived stem cell culture supernatant and umbilical cord stem cell culture supernatant ) Is shown in FIG. The menstrual blood-derived stem cell culture supernatant contains abundant factors that are considered to be effective against infertility, and GDF-15 is particularly significant in the menstrual blood-derived stem cell culture supernatant compared to the umbilical cord stem cell culture supernatant. Was included a lot in.

3. 3. Discussion The menstrual blood-derived stem cell culture supernatant and the umbilical cord stem cell culture supernatant contained many factors, but the types and amounts were different. The menstrual blood-derived stem cell supernatant contained abundant factors effective for infertility, and in particular, GDF-15 was characteristically enriched in the menstrual blood-derived stem cell supernatant.

GDF-15 (Growth and Differentiation Factor-15 / MIC-1 (Macrophage Inhibitory Cytokine-1)) is a growth factor highly expressed in decidualized intimal stromal cells and placenta, and is found in the endometrium. It has been reported that it contributes to the maintenance of pregnancy through the immune tolerance of existing dendritic cells (Tong S, et al., Segerer SE, et al., Supra). ).

LIF (Leukemia inhibitory factor) has been reported to be involved in implantation promotion (see above (Kondera-Anasz Z, et al. And Aghajanova L.), but on the other hand, stem cells are undifferentiated (proliferative) and polymorphic. It is also an important factor for maintaining potency. LIF is considered to be a component derived from the patient's serum or menstrual blood, and its amount is very small (outside the calibration line range), but together with IGF-1 (see Example 1). , Helped menstrual blood-derived stem cells to maintain undifferentiated and pluripotent and proliferate, enriched with useful factors such as GDF-15, GM-CSF, IL-6 (see Example 1) At the same time, these components act on affected cells such as intrauterine stem cells to normalize damaged endometrial membranes when administered to patients. It is thought that this will contribute to the treatment of infertility and endometriosis.

[Test Example 1] Treatment of endometrial defect intractable infertility The case was a 40-year-old infertility patient with endometrial defect, and the history and treatment history was endometrial cancer (3 years ago). So far, he has performed frequent endometrial cysts, chemotherapy, and hormone therapy, and tried frozen embryo transfer at another hospital, but abandoned the transfer due to the intima thickness of several millimeters, and PRP (platelet rich plasma purple therapy). ), But no change was observed in the amount of menstruation and the endometrium, and the transplantation was postponed.

Two days after menstruation, menstrual blood and autologous serum were collected, and a culture supernatant derived from menstrual blood was prepared according to Example 1. On Day 13 of the hormone replacement cycle, 1 ml of menstrual blood stem cell culture supernatant was injected into the uterine cavity. On Day 20, l frozen embryos transferred from another hospital were transplanted. Blood hCG32.38 IU / ml was confirmed on Day 27. It was a turning point for miscarriage, but it is miraculous that the endometrium was regenerated despite six endometrial detachments and chemotherapy for endometrial cancer.

[Test Example 2]
The case is a 45-year-old elderly infertile patient. So far, 3 artificial inseminations and 24 in vitro fertilizations have not led to pregnancy. Two days after menstruation, menstrual blood and autologous serum were collected, and a culture supernatant derived from menstrual blood was prepared according to Example 1. On Day 11 of the hormone replacement cycle, 1 ml of menstrual blood stem cell culture supernatant was injected into the uterine cavity. One frozen embryo was transplanted on Day 19. Blood hCG 53.94 IU / ml was confirmed on Day 26. The fetal sac was confirmed at 5 weeks and 2 days of gestation. The heartbeat was confirmed at 7 weeks and 0 days of pregnancy, and the pregnancy is continuing.

[Test Example 3]
The case is 40 years old. He has a history of surgery for endometriosis and hydrosalpinx. He still has a history of adenomyosis and uterine fibroids. So far, 3 cycles of timing therapy, 1 artificial insemination, and 5 in vitro fertilizations have not led to pregnancy.

After 4 days of menstruation, menstrual blood and autologous serum were collected, and a menstrual blood-derived supernatant was prepared according to Example 1. On Day 11 of the hormone replacement cycle, 1 ml of menstrual blood stem cell culture supernatant was injected into the uterine cavity. One frozen embryo was transplanted on Day 18. Blood hCG 99.31 IU / ml was confirmed on Day 31. The fetal sac was confirmed on the 0th day of the 5th week of pregnancy. The heartbeat was confirmed at 6 weeks and 3 days of pregnancy, and the pregnancy is continuing.

[Test Example 4]
The case was a 38-year-old infertile patient of unknown cause. So far, I have performed artificial insemination 7 times and in vitro fertilization 2 times, but I have not become pregnant. Three days after menstruation, menstrual blood and autologous serum were collected, and a menstrual blood-derived supernatant was prepared according to Example 1. On Day 12 of the hormone replacement cycle, 1 ml of menstrual blood stem cell culture supernatant was injected into the uterine cavity. One frozen embryo was transplanted on Day 19. Blood hCG451.80 IU / ml was confirmed on Day 29. The fetal sac was confirmed at 5 weeks and 1 day of pregnancy. The heartbeat is confirmed at 7 weeks and 1 day of pregnancy, and the pregnancy is continued.

According to the present invention, endometrial insufficiency is improved, and healthy pregnancy and delivery become possible even in patients who have conventionally had difficulty in pregnancy. Therefore, the present invention is useful in the medical field, especially in the field of infertility treatment.

Claims (10)

A pharmaceutical composition comprising a culture supernatant of stem cells derived from menstrual blood for treating any disease or symptom selected from endometrial insufficiency, infertility, recurrent miscarriage, and habitual miscarriage. The pharmaceutical composition according to claim 1, wherein the menstrual blood is the autologous menstrual blood of a patient in need of treatment. The pharmaceutical composition according to claim 1 or 2, wherein the stem cells derived from menstrual blood contain cells that are positive for CD90, CD105, CD73, and CD146 and negative for CD34 and CD45. The pharmaceutical composition according to any one of claims 1 to 3, wherein the culture supernatant is enriched with respect to GDF-15. The pharmaceutical composition according to any one of claims 1 to 4, wherein the culture supernatant is enriched with respect to IL-6 and / or GM-CSF. The pharmaceutical composition according to any one of claims 1 to 5, wherein the culture supernatant contains IGF-1. The pharmaceutical composition according to any one of claims 1 to 6, which is administered into the uterine cavity of a patient 2 to 5 days before the estimated ovulation date. The pharmaceutical composition according to claim 1, wherein the culture supernatant contains a component derived from allogeneic serum. A method for preparing a culture supernatant of stem cells derived from menstrual blood.
1) Cell fractionation of menstrual blood isolated from donors is performed by centrifugation, and the layer containing the target stem cells is collected.
2) The cells are cultured in a medium containing the donor's autologous serum, and the cells are cultured.
3) The method according to the above method, which comprises collecting a culture supernatant of cells 6 to 10 days after at least one passage. The pharmaceutical composition according to any one of claims 1 to 8, wherein the culture supernatant of stem cells derived from menstrual blood is prepared by the method according to claim 9.

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