WO2023216104A1 - Préparation associée à une réparation endométriale, cellule endométriale, son procédé de préparation et son utilisation - Google Patents

Préparation associée à une réparation endométriale, cellule endométriale, son procédé de préparation et son utilisation Download PDF

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WO2023216104A1
WO2023216104A1 PCT/CN2022/091967 CN2022091967W WO2023216104A1 WO 2023216104 A1 WO2023216104 A1 WO 2023216104A1 CN 2022091967 W CN2022091967 W CN 2022091967W WO 2023216104 A1 WO2023216104 A1 WO 2023216104A1
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endometrial
cells
medium
cell
repair
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PCT/CN2022/091967
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Chinese (zh)
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张洪丹
黄仁杰
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上海赛立维生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to the field of biotechnology, and in particular to preparations related to endometrial repair, endometrial cells, preparation methods and applications.
  • Human uterine tissue contains endometrial epithelial stem cells (eESCs) and endometrial mesenchymal stem cells (endometrial mesenchymal stem cells, eMSCs).
  • eESCs endometrial epithelial stem cells
  • eMSCs endometrial mesenchymal stem cells
  • the proliferation and differentiation of adult stem cells play a very critical role in the damage repair of tissues and organs.
  • the damage repair of the endometrium also requires the participation of both epithelial and mesenchymal stem cells. Therefore, an endometrial epithelial stem cell and mesenchymal stem cell culture system is constructed in vitro. It is of great significance to study the pathophysiological repair of endometrium and the treatment of intrauterine adhesion (IUA).
  • IUA intrauterine adhesion
  • Endometrial mesenchymal stem cells have been shown to have the ability to differentiate into multiple lineages, while endometrial epithelial stem cells (eESCs) are difficult to culture for long periods of time in vitro using traditional two-dimensional culture methods, so their functional testing is limited in in vitro studies. have been restricted.
  • the purpose of the present invention is to provide an endometrial repair-related preparation, endometrial cells, preparation methods and applications, and to successfully achieve in vitro culture and differentiation of endometrial epithelial precursor-like cells that have a repair-promoting effect on endometrial damage.
  • Culture and expand the cell source of endometrial stem cells on the basis of successfully achieving in vitro culture of endometrial epithelial precursor-like cells and further obtaining the conditioned culture medium of the endometrial epithelial precursor-like cells, a method for endometrial epithelial precursor-like cells was constructed.
  • the in vitro co-culture system with positive effects on damage repair is of great significance to the study of pathophysiological repair of endometrium and the treatment of intrauterine adhesion (IUA).
  • Human uterine tissue contains endometrial epithelial stem cells (eESCs) and endometrial mesenchymal stem cells (endometrial mesenchymal stem cells, eMSCs).
  • eESCs endometrial epithelial stem cells
  • eMSCs endometrial mesenchymal stem cells
  • the proliferation and differentiation of adult stem cells play a very critical role in the damage repair of tissues and organs.
  • the damage repair of the endometrium also requires the participation of both epithelial and mesenchymal stem cells. Therefore, an endometrial epithelial stem cell and mesenchymal stem cell culture system is constructed in vitro. It is of great significance to study the pathophysiological repair of endometrium and the treatment of intrauterine adhesion (IUA).
  • IUA intrauterine adhesion
  • eMSCs have clonogenic and multi-lineage differentiation capabilities, and can be induced in vitro to differentiate into adipocytes, osteocytes, chondrocytes, smooth muscle cells, and skeletal muscle cells using different induction media.
  • adipocytes osteocytes
  • chondrocytes smooth muscle cells
  • skeletal muscle cells skeletal muscle cells using different induction media.
  • due to the difficulty in obtaining and culturing endometrial epithelial precursor cells in vitro there have been few relevant studies to date.
  • the present invention uses human endometrial cells as seed cells for in vitro culture, and the obtained endometrial epithelial precursor-like cells conform to the characteristics of human endometrial epithelial stem cells and achieve stable culture in vitro.
  • the endometrial repair preparation of the present invention contains a conditioned culture medium of endometrial-derived cells, which have precursor cell characteristics and positively express stage-specific embryonic antigens.
  • the beneficial effect is that the conditioned culture medium derived from endometrial-derived cells with precursor cell characteristics and positive expression of stage-specific embryonic antigens helps to construct an in vitro co-culture system that has a positive effect on endometrial damage repair. It is of great significance to study the pathophysiological repair of endometrium and the treatment of intrauterine adhesion (IUA).
  • the stage-specific embryonic antigen is SSEA-1.
  • the endometrial epithelial precursor-like cells positively express stage-specific embryonic antigens and the expression rate is not less than 80%.
  • the endometrial-derived cells are endometrial epithelial precursor cells or endometrial epithelial precursor-like cells.
  • the preparation method of the endometrial repair preparation of the present invention includes: providing the endometrial-derived cells, using a conditioned medium to condition the endometrial-derived cells, and after the conditioned culture is completed, the obtained culture The conditioned culture supernatant of the endometrial-derived cells is obtained from the product.
  • the conditioned medium includes basal medium and serum-based substances.
  • the content of the serum-like substance does not exceed 20% based on the volume of the basal culture medium.
  • the basal culture medium is Hep-X basal culture medium, DMEM-high sugar culture medium, DMEM-low sugar culture medium, DMEM/F12 culture medium, MEM culture medium, William's Medium E culture medium, Ham's F-10 culture medium. base, Ham's F-12 medium, IMDM medium, McCoy'5A medium, RPMI-1640 medium, BME medium, M-199 Medium, Leibovitz Medium, CMRL1066 medium, Neurobasal medium and Fischers' Any kind.
  • the content of the serum-like substance does not exceed 20% based on the volume of the basal culture medium.
  • the serum substance is animal-derived serum.
  • the animal-derived serum is fetal bovine serum.
  • the animal-derived serum in the conditioned medium can be replaced with a serum substitute.
  • the serum substitute is platelets and derivatives thereof without animal components.
  • the serum replacement is sphingosine monophosphate and indoleacetic acid.
  • the endometrial-derived cells are obtained by in vitro culture of human primary endometrial cells that positively express epithelial cell-related markers in endometrial precursor cell culture medium.
  • the epithelial cell-related marker is any one of epithelial cell markers and epithelial precursor cell markers, and the expression rate is not less than 70%.
  • the epithelial cell-related marker is any one of EpCAM, E-Cadherin, CD9, CK19, CD24 and CD44.
  • the epithelial precursor cell marker is either CD24 or CD44.
  • the steps of obtaining human primary endometrial cells that positively express epithelial cell-related markers include: screening the primary cell suspension containing human endometrial cells, lysing and removing red blood cells, and cell sorting.
  • the human primary endometrial cells that positively express epithelial cell-related markers are obtained.
  • the cell sorting is immunomagnetic bead sorting.
  • the cell sorting is flow cytometric sorting.
  • the step of using the endometrial precursor cell culture medium to culture the human primary endometrial cells that positively express epithelial cell-related markers in vitro includes: using the endometrial precursor cell culture medium with 0.3 ⁇ 10
  • the human primary endometrial cells that positively express epithelial cell-related markers are cultured at a seeding density of 4-1 ⁇ 105 cells/cm2 until the cell confluence is not less than 80%, then digested, and then continue to use as described Intimal precursor cell culture medium was used for subculture.
  • the subculture ratio of the subculture is not less than 1:3.
  • the intima precursor cell culture medium is replaced every 24-48 hours.
  • the endometrial epithelial precursor-like cells that positively express stage-specific embryonic antigens are further expanded and cultured using the endometrial precursor cell culture medium.
  • the intimal precursor cell culture medium includes the basal culture medium, growth factors, TGF- ⁇ signaling pathway inhibitors, Wnt signaling pathway agonists, ROCK kinase inhibitors and serum substances.
  • the intimal precursor cell culture medium further includes at least one of N-acetyl-L-cysteine and ascorbic acid.
  • the content of the sodium pyruvate is 5-20 ⁇ g/ml
  • the content of the growth factor is 5-50 ng/ml
  • the Rock kinase inhibitor is based on the volume of the basal culture medium.
  • the content is 0.5-50 micromol/L
  • the content of the WNT signaling pathway agonist is 0.5-50 micromol/L
  • the content of the TGF- ⁇ signaling pathway inhibitor is 0.5-50 micromol/L
  • the The content of serum-like substances does not exceed 20%.
  • the basal culture medium is any one of MEM, DMEM, BME, DMEM/F12, RPMI1640, CMRL1066, WilliamE, Neurobasal or Fischers culture medium.
  • the basal culture medium is Hep-X basal culture medium, DMEM-high sugar culture medium, DMEM-low sugar culture medium, Ham's F-10 culture medium, Ham's F-12 culture medium, IMDM culture medium, McCoy'5A Medium, any of M-199 Medium, Leibovitz Medium, CMRL1066 medium, Neurobasal medium and Fischers.
  • the growth factor component is at least one of epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, interleukin-6 and oncostatin.
  • the Rock signaling pathway inhibitor is at least one of Y27632, Fasudil, Thiazovivin and SB-772077-B.
  • the WNT signaling pathway agonist is at least one of recombinant WNT protein, recombinant R-spondin protein, BIO, CHIR99021 and TWS119.
  • the TGF- ⁇ signaling pathway inhibitor is at least one of A8301, RepSox and SB431542.
  • the content of the serum-like substance does not exceed 20% based on the volume of the basal culture medium.
  • the serum substance is animal-derived serum.
  • the animal-derived serum is fetal bovine serum.
  • the animal-derived serum in the intimal precursor cell culture medium can be replaced with a serum substitute.
  • the serum substitute is platelets and their derivatives without animal components.
  • the serum substitute is sphingosine monophosphate and indoleacetic acid.
  • the application of the endometrial repair preparation of the present invention in repairing endometrial damage includes co-culturing the endometrial repair preparation with endometrial stromal cells or vascular endothelial cells.
  • the endometrial cell preparation of the present invention includes endometrial epithelial precursor-like cells, the endometrial epithelial precursor-like cells positively express stage-specific embryonic antigens, and the endometrial epithelial precursor-like cells are composed of positive Primary endometrial cells expressing epithelial cell-related markers are cultured in vitro and have a repair-promoting effect on endometrial damage.
  • the endometrial cell preparation further includes endometrial stromal cells.
  • the combination of the endometrial stromal cells and the endometrial epithelial precursor-like cells that positively express stage-specific embryonic antigens has a further repair-promoting effect on endometrial damage.
  • the expression rate of the stage-specific embryonic antigen is not less than 80%.
  • the stage-specific embryonic antigen is SSEA-1.
  • the expression rate of epithelial cell-related markers is not less than 70%.
  • the epithelial cell-related marker is any one of EpCAM, E-Cadherin, CD9, CK19, CD24 and CD44.
  • the application of the endometrial cell preparation of the present invention in the repair of endometrial damage includes using the endometrial cell preparation to intervene in an in vivo animal model to investigate the repair effect of the endometrial cell preparation on endometrial damage.
  • the endometrial cell culture method of the present invention includes: obtaining primary endometrial cells that positively express epithelial cell-related markers; using endometrial precursor cell culture medium to culture the primary uterine cells that positively express epithelial cell-related markers. Endometrial cells were cultured in vitro to obtain endometrial epithelial precursor-like cells that positively expressed stage-specific embryonic antigens.
  • the method for culturing endometrial cells further includes using the endometrial precursor cell culture medium to continue to expand and culture the endometrial epithelial precursor-like cells that positively express stage-specific embryonic antigens.
  • the method for culturing endometrial cells further includes using an induction medium to differentiate and culture the endometrial epithelial precursor-like cells that positively express stage-specific embryonic antigens to obtain positive expression of the epithelial cell-related markers. and leukocyte differentiation antigens of epithelioid cells.
  • the induction medium consists of basal medium, estrogen, serum, human epidermal cell growth factor, transforming growth factor ⁇ 1 and recombinant human platelet-derived growth factor.
  • the estrogen content is 0.5 ⁇ 10 -7 -2 ⁇ 10 -6 mol/L
  • the human epidermal cell growth factor content is 5-50 nanometers.
  • the content of transforming growth factor ⁇ 1 is 5-15 ng/ml
  • the content of recombinant human platelet-derived growth factor is 5-50 ng/ml.
  • the serum is fetal bovine serum.
  • the estrogen is estradiol.
  • the induction medium is used to culture the endometrial epithelial precursor-like cells at a seeding density of 1 ⁇ 10 5 -1 ⁇ 10 6 cells/cm2 until the cell confluence is not less than 80% and then digested. , and then continue to use the induction medium for continuous culture for no less than 20 days.
  • the induction medium is replaced every 24-48 hours.
  • Figure 1 is a flow cytometric analysis result of verifying the magnetic bead sorting efficiency of the cell suspension to be tested in a centrifuge tube in Example 1-1 of the present invention
  • Figure 2 is a flow cytometry analysis result for verifying the magnetic bead sorting efficiency of the cell suspension to be tested in the sorting tube in Example 1-1 of the present invention
  • Figure 3 is a comparative diagram of the expression of EpCAM and SSEA-1 obtained after surface antibody flow cytometry detection of EpCAM + cells, P0 cells, P1 cells and P2 cells in Example 1-2 of the present invention
  • Figure 4 is a light microscope image of EpCAM + cells on the 4th day of amplification and culture using ECM in Example 1-2 of the present invention
  • Figure 5 is a light microscope image of EpCAM + cells on the second day of culture using TEM-1 medium in Example 1-2 of the present invention
  • Figures 6 and 7 are respectively OD value change trend diagrams of 1-P3-SSEA-1 + cells and 2-P3-SSEA-1 + cells in Examples 1-3 of the present invention
  • Figure 8 is a light microscope photo of clonal clumps formed by 1-P3-SSEA-1 + cells in Examples 1-3 of the present invention.
  • Figure 9 is a comparative diagram of the expression of EpCAM, SUSD2, CD34, CD45, CD90 and CD105 obtained by flow cytometric detection of 1-P3-SSEA-1 + cells in Examples 1-4 of the present invention.
  • Figure 10 is a GO biological process cluster analysis diagram of the top 30 differential genes obtained by performing high-throughput transcriptome sequencing on 1-P3-SSEA-1 + cells in Examples 1-4 of the present invention
  • Figure 11 is a comparative photo of the tubule formation observed with a light microscope at 6 and 12 hours after using SSEA-1 + -CM to act on human vascular endothelial cells in Examples 1-5 of the present invention
  • Figure 12 is a comparative photo of the tubule formation observed with a light microscope at 6 and 12 hours after using SUSD2 + -CM to act on human vascular endothelial cells in Examples 1-5 of the present invention
  • Figure 13 is a comparison chart of the expression levels of VEGFA, TNF, IL-1A and IL-1B in SSEA-1 + cells and SUSD2 + cells in Examples 1-5 of the present invention
  • Figure 14 is a comparative photo of the scratch healing conditions observed with a light microscope at 0, 6 and 12 hours after using SSEA-1 + -CM to act on SUSD2 + cells to start the scratch experiment in Examples 1-5 of the present invention. ;
  • Figure 15 is a comparative photo of the scratch healing conditions observed with a light microscope at 0, 6 and 12 hours after the SUSD2 + -CM of Examples 1-5 of the present invention acts on SUSD2 + cells to start the scratch experiment;
  • Figure 16 is a comparison chart of the expression levels of ITGB2, ITGB4, ITGB7 and NOTCH1 in SSEA-1 + cells and SUSD2 + cells in Examples 1-5 of the present invention
  • Figure 17 is a schematic diagram of the expression of CD9 and EpCAM on the respective cell surfaces obtained by flow cytometric detection of 1-P3-SSEA-1 + cells and the obtained differentiated cells in Example 2-1 of the present invention;
  • Figure 18 is a photo of the uterus sample of SD rat in Example 2-2 of the present invention.
  • Figures 19 to 23 are respectively HE stained photos of the uterine samples from the normal side, the CG group surgical side, the SSEA-1 + group surgical side, the SUSD2 + group surgical side and the combined group surgical side in Example 2-2 of the present invention;
  • Figures 24 to 28 are respectively Masson stained photos of the uterine samples from the normal side, the CG group surgical side, the SSEA-1 + group surgical side, the SUSD2 + group surgical side and the combined group surgical side in Example 2-2 of the present invention.
  • cell culture was performed in a cell culture incubator at 37 degrees Celsius and with a carbon dioxide concentration of 5%.
  • the culture medium used for cell culture and various reagents used to process cells, such as buffers, are sterilized and filtered with a 0.22 micron filter to remove impurities before use.
  • Each embodiment of the present invention involves statistical analysis of data. Each set of experiments is repeated at least three times. The experimental result data is statistically analyzed using GraphPad Prism 8.0 software. The two-tailed unpaired t test was used to calculate statistical differences between two groups of data, and the ANOVA analysis of variance was used to calculate statistical differences between multiple groups of data. p ⁇ 0.05 was considered statistically significant.
  • Example 1-1 of the present invention provides a method for preparing primary endometrial cells that positively express the epithelial cell marker EpCAM.
  • Example 1-1 human endometrial tissue was used as a starting material to obtain human primary endometrial cells that positively express EpCAM.
  • the human endometrial tissue is a surgical sample derived from a female patient aged no more than 45 years old.
  • the patient was not found to have malignant endometrium, malignant tumors or malignant endometrial lesions after medical examination.
  • the patient was diagnosed before surgery. No steroid medication has been used within three months.
  • the patient was fully informed about the purpose of obtaining surgical samples before surgery and signed an informed consent form.
  • a volume of 1 cubic millimeter of endometrial tissue was washed and sterilized using sterile PBS buffer, and then 3 ml of cell digestion consisting of type I collagenase, sterile PBS buffer and trypsin digestion solution was used.
  • the endometrial tissue was digested with liquid at 37 degrees Celsius for 60 minutes to obtain a primary cell suspension containing human endometrial cells.
  • Sterile PBS buffer and trypsin digestion solution have the same volume, and the volume percentage of type I collagenase in the modified buffer is 1%.
  • the primary cell suspension was screen sorted with the assistance of sterile PBS buffer using a 100-mesh sterile screen, and the filtrate was collected and mucus and undigested tissue were removed to complete the screen sorting.
  • centrifuge the obtained filtrate and remove the supernatant add red blood cell lysis balance solution to the obtained precipitate, resuspend, centrifuge again, and repeat the above process until no red blood cells are observed in the cell pellet after centrifugation again to complete the red blood cells. Lytic removal. Specifically, the centrifugation rate for each time was 1000 g, and the centrifugation time was 3 minutes.
  • Example 1-1 after the centrifuge tube suspension and the sorting tube suspension obtained by immunomagnetic bead separation were centrifuged respectively, 100 ⁇ l of staining buffer and 5 ⁇ l of EpCAM were added to the obtained cell pellets. Resuspend the flow cytometry antibody and incubate it at 4 degrees Celsius for 30 minutes; then discard the supernatant and add 600 microliters of staining buffer to the resulting cell pellet and resuspend it into the flow tube to obtain a suspension of cells to be tested in a centrifuge tube. Liquid and sorting tube to be tested cell suspension.
  • Figure 1 is the flow cytometric analysis result of verifying the magnetic bead sorting efficiency of the cell suspension to be tested in a centrifuge tube in Example 1-1.
  • Figure 2 is the flow cytometric analysis result of verifying the magnetic bead sorting efficiency of the cell suspension to be tested in the sorting tube in Example 1-1.
  • the flow cytometry analysis results shown in Figure 1 show that in the cell suspension to be tested in the centrifuge tube, the EpCAM expression rate of the cells does not exceed 2%, which can be regarded as barely expressing EpCAM; as shown in Figure 2
  • the flow cytometry analysis results showed that the EpCAM positive expression rate of cells in the cell suspension to be tested in the sorting tube exceeded 97%.
  • Example 1-1 negatively express EpCAM (abbreviated as EpCAM - cells ); primary endometrial cells in the sorting tube suspension positively express EpCAM (abbreviated as EpCAM + cells), and the expression rate exceeds 97%.
  • Example 1-2 uses an endometrial precursor cell culture medium (abbreviated as TEM-1 culture medium) to culture the EpCAM + cells of Example 1-1 in vitro, and obtains endometrium that positively expresses SSEA-1.
  • TEM-1 culture medium an endometrial precursor cell culture medium
  • 1-SSEA-1 + cells Epithelial precursor-like cells
  • the TEM-1 medium used in Example 1-2 consists of DMEM/F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Y27632, CHIR99021, A8301, S1P and LPA.
  • the content of ascorbic acid is 10 ⁇ g/ml; the content of sodium pyruvate is 1 mmol/L; the content of HGF is 20 ng/ml; the content of EGF is 20 ng/ml. ml; the content of Y27632 is 10 micromol/L; the content of CHIR99021 is 3 micromol/L; the content of A8301 is 1 micromol/L; the content of S1P is 1 micromol/L; the content of LPA is 5 micromol/L Lift.
  • Example 1-2 the steps of using TEM-1 culture medium for in vitro culture are:
  • the EpCAM + cells and EpCAM + cells obtained after magnetic bead sorting in Example 1-2 were subjected to TEM
  • the primary cells (abbreviated as P0 cells) obtained after amplification and culture in -1 medium until the confluence rate is not less than 80%, and the first generation cells obtained by the first passage of P0 cells in TEM-1 culture (abbreviated as P1 cells), and the second-generation cells (abbreviated as P2 cells) obtained by the second subculture were subjected to surface antibody flow cytometry to examine the expression of EpCAM and SSEA-1 in different cells.
  • Figure 3 is a comparison of the expression of EpCAM and SSEA-1 in EpCAM + cells, P0 cells, P1 cells and P2 cells after surface antibody flow cytometry detection.
  • the proportion of cells that positively express EpCAM can reach more than 97%, and only about 8% of the cells positively express SSEA-1; after expansion in TEM-1 medium Among the P0 cells obtained by augmentation culture, the proportion of positive EpCAM-expressing cells dropped to about 50%, and the proportion of positive SSEA-1-expressing cells increased to about 50%; in the P1 cells and P2 cells formed after passage, the proportion of endometrial epithelial cells
  • the characteristic marker EpCAM was almost not expressed, while the proportion of positive SSEA-1 expressing cells further increased, and the proportion of positive SSEA-1 expressing cells in P2 cells increased to more than 80%. It can be seen that the TEM-1 culture system not only successfully cultured human endometrial epithelial cells in vitro, but also affected the expression of EpCAM and SSEA-1 in the cells, which was beneficial to the acquisition of SSEA-1 + cells.
  • Examples 1-2 also provide a routine solution consisting of DMEM F/12, double antibodies and fetal calf serum.
  • ECM culture medium
  • ECM consisted of 445 ml of DMEM F/12, 5 ml of double antibody and 50 ml of fetal calf serum.
  • Figure 4 is a light microscope image of EpCAM + cells on day 4 of expansion culture using ECM.
  • Figure 5 is a light microscope image of EpCAM + cells cultured in TEM-1 medium on the second day. Referring to Figures 4 and 5, compared with the expansion culture of EpCAM + cells in TEM-1 medium, the growth status of cells cultured in ECM was obviously poor. The morphology of epithelial cell colonies is not obvious. When EpCAM + cells were cultured in TEM, the cells grew vigorously and formed typical nest-shaped or rosette-shaped epithelial cell colonies. The cells were large in size, oval in shape, with large nuclei and obvious nucleoli. In addition, epithelial cell colonies were visible. There are typical interstitial cells adherent between the colonies, which are spindle-shaped or fibrous, slightly wider in the middle and pointed at both ends.
  • the reprogramming substances provided by the embodiments of the present invention are crucial for the expansion of EpCAM + cells and the transformation into SSEA-1 + cells.
  • Example 1-3 used another endometrial precursor cell culture medium (abbreviated as TEM-2) to culture the EpCAM + cells of Example 1-1 in vitro, and also obtained in utero cells that positively expressed SSEA-1.
  • TEM-2 endometrial precursor cell culture medium
  • 2-SSEA-1 + cells Membrane epithelial precursor-like cells
  • the TEM-2 medium used in Examples 1-3 consists of DMEM/F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Thiazovivin, BIO, SB431542, S1P and LPA.
  • the content of ascorbic acid is 10 ⁇ g/ml; the content of sodium pyruvate is 1 mmol/L; the content of HGF is 20 ng/ml; the content of EGF is 20 ng/ml. ml; the content of Thiazovivin is 1 micromol/L; the content of BIO is 2 micromol/L; the content of SB431542 is 10 micromol/L; the content of S1P is 1 micromol/L; the content of LPA is 5 micromol/L Lift.
  • the difference is that the TEM-2 medium is used for Example 1 -2.
  • the third-generation cells obtained are cultured in TEM-2 medium until the confluence rate is not less than 80% and then flow sorted to obtain 2-SSEA-1 + cells.
  • the 1-SSEA-1 + cells and 2-SSEA-1 + cells of Examples 1-2 and 1-3 were continued to be subcultured to the third generation using TEM-1 medium and TEM-2 medium respectively, 1-P3-SSEA-1 + cells and 2-P3-SSEA-1 + cells were obtained respectively.
  • the steps of subculture are the same as those in Example 1-2.
  • Cell viability detection reagent (CCK-8) was used to examine the proliferation ability of the two P3-SSEA-1 + cells. Specifically, trypsin digestion solution was added to the two types of P3-SSEA-1 + cells for digestion, and then the cells were counted. After counting, the cells were digested and seeded into a 96-well plate at 5 ⁇ 10 3 /well, and each well was added 100 ⁇ l of the corresponding TEM-1 medium was cultured. Set up 9 duplicate wells and group them according to the number of days, with one plate in each group for 7 days, and detect continuously for 7 days at a fixed time point. During detection, add 10 microliters of CCK-8 detection solution to each well and mix well.
  • 1-P3-SSEA-1 + cells were cultured in the corresponding TEM-1 medium in a 6-well plate at a seeding density of 1 ⁇ 10 3 /well for 14 days. The medium was aspirated and the cell pellet was rinsed with PBS. After 3 times, use 4% paraformaldehyde to fix at room temperature, remove the paraformaldehyde and rinse the cell clumps with PBS. Add 0.1% crystal violet solution for room temperature staining and then rinse the remaining dye with PBS.
  • 1-P3-SSEA-1 + cells form single cell clones with larger cell sizes, polygonal or tadpole shapes, and are distributed in a rosette or nest-like cluster.
  • a clonal clump with a cell number of no less than 50 is defined as a large clonal clump. Counting showed that 1-P3-SSEA-1 + cells were able to form 134 ⁇ 6 large clonal clumps, showing good cloning ability.
  • Examples 1-4 examined the phenotypic characteristics of P3-SSEA-1 + cells, further proving that SSEA-1 + cells are adult stem cells derived from human endometrial epithelium.
  • 1-P3-SSEA-1 + cells see the comparison chart of EpCAM, SUSD2, CD34, CD45, CD90 and CD105 expression of 1-P3-SSEA-1 + cells shown in Figure 9.
  • SUSD2, CD34, CD45, CD90 and CD105 are characteristic markers of human endometrial mesenchymal stem cells eMSCs.
  • 1-P3-SSEA-1 + cells do not express SUSD2, EpCAM, CD34, CD45, CD90 and CD105, indicating that SSEA- 1+ cells are not interstitial cells.
  • P3-SSEA-1 + cells did not express EpCAM, further proving the important role of TEM culture in the transformation of EpCAM + cells into SSEA-1 + cells. More importantly, P3-SSEA-1 + cells do not express the mature somatic cell marker EpCAM, indicating that P3-SSEA-1 + cells are cells in a dedifferentiated state.
  • Examples 1-4 performed high-throughput transcriptome sequencing on 1-P3-SSEA-1 + cells, and obtained the GO biological process cluster analysis diagram of the top 30 differential genes as shown in Figure 10. As can be seen in Figure 10, SSEA-1 + cells expressed more genes related to epithelial differentiation.
  • Examples 1-5 prepared an endometrial repair preparation, namely a conditioned culture medium of 1-P3-SSEA-1 + cells (abbreviated as SSEA-1 + -CM), and examined its effects on human vascular endothelial cells and human The role of endometrial stromal cells proves that SSEA-1 + cells can induce the formation of new blood vessels and promote the migration of endometrial stromal cells, showing the therapeutic potential for the regeneration and repair of the endometrium. It has been applied in the preparation of injury-related drugs.
  • the preparation method of SSEA-1 + -CM is as follows: 1-P3-SSEA-1 + cells are seeded on a 6-well plate at a seeding density of 6 ⁇ 10 5 cells/well, and each well is cultured with 2.5 ml of TEM-1 culture medium. After the confluence reached 80%, the culture medium was replaced with ECM medium for culture. The ECM medium was completely replaced every 24 hours and the supernatant was collected as SSEA-1 + -CM.
  • This example also examined the effects of the conditioned culture medium (abbreviated as SUSD2 + -CM) of endometrial stromal cells (abbreviated as SUSD2 + cells , sourced from Shanghai Selevi Biotechnology Co., Ltd.) on human vascular endothelial cells. effects on endometrial stromal cell migration.
  • SUSD2 + -CM conditioned culture medium
  • SSEA-1 + -CM a preparation method of SSEA-1 + -CM.
  • human vascular endothelial cells were seeded on a 6-well plate at a seeding density of 6 ⁇ 10 5 cells/well, and 2.5 microns were used in each well.
  • liter of ECM-1 culture medium until the confluence reaches 80%, then digested with trypsin digestion solution, resuspended the digestion solution with SSEA-1 + -CM, and then inoculated on a Matrigel-coated 96-well plate to start the small tube experiment.
  • the formation of small tubes was observed with a light microscope and photographed and recorded.
  • Figure 11 is a comparative photo of the tubule formation observed under a light microscope at 6 and 12 hours after SSEA-1 + -CM was used to act on human vascular endothelial cells.
  • obvious blood vessel formation can be observed at the 6th hour after SSEA-1 + -CM acts on human vascular endothelial cells.
  • the blood vessel formation at the 12th hour is more intensive and significant. It can be seen that SSEA-1 + -CM can Effectively promotes tubule formation.
  • Figure 12 is a comparative photograph of the tubule formation observed under a light microscope at 6 and 12 hours after SUSD2 + -CM was used to act on human vascular endothelial cells.
  • SUSD2 + -CM acts on human vascular endothelial cells
  • the blood vessel formation at 12 hours is further intensive and significant.
  • the average length of tubules formed per unit area at the 6th hour after SUSD2 + -CM acts on human vascular endothelial cells is 2 mm on average, while the length of the tubules formed at 6 hours after SSEA-1 + -CM acts on human vascular endothelial cells It can reach 18 mm on average, with a significant increase (P ⁇ 0.05).
  • the average length of tubules formed per unit area at the 12th hour after SUSD2 + -CM acts on human vascular endothelial cells is 15 mm, while the length of the tubules formed at the 12th hour after SSEA-1 + -CM acts on human vascular endothelial cells It can reach 25 mm on average, with a significant increase (P ⁇ 0.05).
  • this example performed qPCR detection on SSEA-1 + cells and SUSD2 + cells to examine the expression of factors related to blood vessel formation in the two cells, and obtained the results of SSEA-1 + cells and SUSD2 + cells shown in Figure 13 Comparison of expression levels of VEGFA, TNF, IL-1A and IL-1B.
  • the relative expression levels of vascularization-related factors VEGFA, TNF, IL-1A and IL-1B in SSEA-1 + cells were significantly increased (P ⁇ 0.001).
  • SSEA-1 + -CM has a stronger ability to promote tubule formation than SUSD2 + -CM.
  • SSEA-1 + -CM The effect of SSEA-1 + -CM on SUSD2 + cells was examined by scratch experiment. Specifically, SUSD2 + cells were seeded on a 6-well plate at a seeding density of 1 ⁇ 10 6 cells/well, and 2.5 ⁇ l of SSEA-1 + -CM was added to each well and cultured for 24 hours until the cell confluence was greater than 90%; Use a microliter pipette tip to make 3 scratches in each well of the 6-well plate, rinse twice with PBS, and then incubate with SSEA-1 + -CM to start the scratch experiment.
  • this example performed qPCR detection on SSEA-1 + cells and SUSD2 + cells to examine the expression of pro-migration related factors in the two cells, and obtained ITGB2 of SSEA-1 + cells and SUSD2 + cells as shown in Figure 16 ,Comparison of expression levels of ITGB4, ITGB7 and NOTCH1.
  • the relative expression levels of pro-migration-related factors ITGB2, ITGB4, ITGB7 and NOTCH1 in SSEA-1 + cells were significantly increased (P ⁇ 0.01).
  • SSEA-1 + -CM has a stronger promoting effect on endometrial stromal cell migration.
  • SSEA-1 + -CM was used to culture SUSD2 + cells, and the effect of SSEA-1 + -CM on fibrosis-related factors in SUSD2 + cells was further investigated through qPCR testing, proving that SUSD2 + after culture with SSEA-1 + -CM
  • the mRNA expression of TGF- ⁇ 1, ⁇ -catenin, COL1A and ⁇ SMA in cells was significantly down-regulated compared with SUSD2 + cells (P ⁇ 0.05). It can be seen that SSEA-1 + cells have the ability to inhibit the expression of fibrosis-related factors in SUSD2 + cells.
  • SUSD2 + cells were seeded into a 6-well plate at 6 ⁇ 10 5 cells/well, and 2 ml of TEM was added to each well and cultured until the confluence reached about 80%, then they were divided into two groups, and one group continued to use ECM. culture, and the other group was cultured with SSEA-1 + -CM, with full medium replacement every day, and RNA was extracted for qPCR testing after 7 days.
  • Table 1 for the specific down-regulation degree of fibrosis-related factors. It was demonstrated that the mRNA expression of TGF- ⁇ 1, ⁇ -catenin, COL1A and ⁇ SMA in SUSD2 + cells cultured with SSEA-1 + -CM was significantly down-regulated compared with SUSD2 + cells. It can be seen that SSEA-1 + cells have the ability to inhibit the expression of fibrosis-related factors in SUSD2 + cells.
  • the internal standard used in the qPCR test process of this example is ⁇ -actin, and each primer is from Shanghai Sangon Bioengineering Co., Ltd.
  • Example 2-1 provides a process for using induction medium (respectively abbreviated as RCM-1 medium) to differentiate and culture 1-P3-SSEA-1 + cells to obtain endometrial epithelial-like cells expressing CD9 and EpCAM, and further It illustrates that P3-SSEA-1 + cells are endometrial epithelial precursor stem cells.
  • RCM-1 medium induction medium
  • P3-SSEA-1 + cells are endometrial epithelial precursor stem cells.
  • RCM-1 medium consists of DMEM F/12, estradiol, fetal bovine serum, human epidermal cell growth factor, transforming growth factor ⁇ 1 and recombinant human platelet-derived growth factor.
  • the volume percentage of fetal bovine serum is 5%
  • the content of estradiol is 1 ⁇ 10 –6 mol/L
  • the content of human epidermal growth factor is 10 ng/ml. Transformation
  • the content of growth factor ⁇ 1 is 10 ng/ml
  • the content of recombinant human platelet-derived growth factor is 10 ng/ml.
  • the steps for differentiation culture are: inoculate cells on a 6-well plate at a seeding density of 6 ⁇ 10 5 cells/well, add 2.5 ml of induction medium to each well, and culture until the confluence reaches 90%, then replace with the same volume of induction medium.
  • the culture medium was used for culture, and the entire medium was changed every 2 days. Differentiated cells were obtained after continuous culture for 20 days.
  • the differentiated cells obtained by differentiation and culture in RCM-1 medium are abbreviated as DC-1.
  • Example 2-1 also performed qPCR detection on 1-P3-SSEA-1 + cells and differentiated cells, and found that compared with 1-P3-SSEA-1 + cells, differentiated cells significantly expressed the gene E- representing somatic cells.
  • the mRNA expressions of cadherin and CD9, Oct-4, Sox9 and Nanog representing stem cells were significantly reduced, and SSEA-1 + cells successfully transformed into epithelial cells after being treated with RCM medium. Please see Table 2 for specific relative gene expression levels.
  • Example 2-2 establishes a SD rat endometrial injury model, uses SSEA-1 + cells to act on the SD rat endometrial injury model, and uses SSEA-1 + cells and SUSD2 + cells to jointly act on SD rats. Endometrial injury model to evaluate the function and efficacy of SSEA-1 + cells, the results show:
  • the application of SSEA-1 + cells or the combined application of SSEA-1 + cells and SUSD2 + cells transplantation treatment can significantly increase the number of endometrial glands, reduce the area of endometrial fibrosis, and promote endometrial regeneration in IUA rats.
  • the endometrium has a good repairing effect.
  • SSEA-1 + cells may promote endometrial repair in IUA rats by promoting neovascularization, endometrial stromal cell migration, endometrial epithelial cell regeneration, and inhibiting the expression of fibrosis-related factors.
  • the construction method of the SD rat endometrial injury model is as follows: select female SD rats aged 7 to 8 weeks and weighing 200-220g as the research subjects, and use the mechanical injury method (endometrium scraping with a blade) to perform uterine injury.
  • the injury model was made, and the rats were sacrificed 2 weeks after the model was made.
  • the uterine tissue at the surgical site was cut out and fixed with 4% paraformaldehyde.
  • the abdominal cavity and uterus were surgically exposed, and the right uterus was selected as the experimental group, and the left uterus was used as the control group without any treatment.
  • the uterine and abdominal incisions were sutured and the abdominal incisions were disinfected.
  • the SD rat endometrial injury model was established through mechanical injury. Uterine tissue was taken for observation 2 weeks after the model was established, and the uterine sample photo of the SD rat shown in Figure 18 was obtained. Referring to Figure 18, there is a certain degree of adhesion between the damaged side of the uterus and the surrounding tissue. The damaged uterus is blocked, the distal uterus is enlarged and has uterine effusion, and the normal side of the uterus has a normal appearance and uniform thickness.
  • the difference between the construction method of the SD rat endometrial injury model with cell intervention and the above-mentioned SD rat endometrial injury model is that: when the injured uterus is sutured until the last stitch is left, different rats are treated with a syringe from Chitosan solution, a mixture containing chitosan and SSEA-1 + cells, a mixture containing chitosan and SUSD2 + cells, and a mixture containing chitosan and SSEA-1 + and SUSD2 + were injected into the distal end of the uterus.
  • the mixture of two types of cells formed a chitosan solution treatment group (abbreviated as CG group), SSEA-1 + cell treatment group (abbreviated as SSEA-1 + group), and SUSD2 + cell treatment group (abbreviated as SUSD2 + group) and combined group, the injection volume of each liquid was 0.8-1 ml.
  • liquid C The specific configuration of the chitosan solution (abbreviated as liquid C) is as follows: fully dissolve the chitosan powder in physiological saline at a concentration of 3% to make a clear and transparent mucus-like solution, and store it at 4°C for later use.
  • C-SSEA-1 + solution The specific configuration of the mixed solution containing chitosan and SSEA-1 + cells (abbreviated as C-SSEA-1 + solution) is: use C solution to resuspend the 1-P3-SSEA-1 + cells with a confluence of 90% , so that the cell concentration is 10 6 cells/ml.
  • C-SUSD2+ solution The specific configuration of the mixed solution containing chitosan and SUSD2 + cells (abbreviated as C-SUSD2+ solution) is as follows: use C solution to resuspend the 1-P3-SUSD2+ cells with a confluence of 90%, so that the cell concentration is 10 6 /ml.
  • C-SSEA-1 + -SUSD2 + solution The specific configuration of the mixed solution containing chitosan and SSEA-1 + and SUSD2 + cells (abbreviated as C-SSEA-1 + -SUSD2 + solution) is: use C solution to resuspend cells with a confluence of 90%. 1-P3-SUSD2 + and 1-P3-SSEA-1 + cells so that each cell concentration was 10 cells/ml.
  • Example 2-2 each uterine sample from the normal side, the CG group, the SSEA-1 + group, the SUSD2 + group and the combined group was paraffin embedded and sectioned, and then HE stained and Masson stained respectively, and the staining was completed. Afterwards, dehydration, transparency and neutral gum sealing are performed.
  • the above specific operation methods are routine technical means for those skilled in the art and will not be described again here.
  • Figures 19 to 23 are HE stained photos of uterine samples from the normal side of the uterus sample, the surgical side of the CG group, the surgical side of the SSEA-1 + group, the surgical side of the SUSD2 + group, and the combined group, respectively.
  • the magnification is 100 times.
  • the number of endometrial glands on the surgical side of each group was counted with reference to Figures 19 to 23. Please see Table 3 for specific data.
  • the number of endometrial glands on the surgical side of the SSEA-1 + group was not only significantly increased compared to the surgical side of the CG group (P ⁇ 0.05), but the structure of the endometrial glands on the surgical side was similar to that of normal tissue under microscope. Normal glandular structures can be seen as pointed by the arrows in Figure 21, indicating that SSEA-1 + cells have a better repair effect on the endometrium than SUSD2 + cells.
  • the number of endometrial glands on the surgical side of the combined group was significantly increased compared with the surgical side of the CG group (P ⁇ 0.05), and the structure of the endometrial glands on the surgical side was different from that of normal tissue. It looks similar when viewed under a microscope. Furthermore, the number of endometrial glands on the surgical side of the combined group had no statistical difference compared with the normal group (P>0.05), and was further increased compared with the surgical side of the SSEA-1 + group (P ⁇ 0.05). It is suggested that the combination of SSEA-1 + cells and SUSD2 + cells has a better repair effect on the endometrium than SSEA-1 + cells.
  • SUSD2 + cells have a limited repair effect on the endometrium; compared with SUSD2 + cells, SSEA-1 + cells have a stronger repair effect on the endometrium; and compared with SSEA-1 + cells , the combined use of SUSD2 + cells and SSEA-1 + cells further enhanced the repair effect on the endometrium.
  • Figures 24 to 28 are Masson stained photos of uterine samples from the normal side, the surgical side of the CG group, the surgical side of the SSEA-1 + group, the surgical side of the SUSD2 + group and the combined group, with a magnification of 100 times.
  • the proportion of fibrosis area on the surgical side of the SUSD2 + group was second only to the surgical side of the CG group, and was reduced compared with the surgical side of the CG group (P ⁇ 0.05), suggesting that the surgical side samples of the SUSD2 + group The degree of fibrosis was lower than that on the surgical side in the CG group, and SUSD2 + cells had the effect of inhibiting fibrosis.
  • the proportion of fibrosis area on the surgical side of the SSEA-1 + group was significantly reduced (P ⁇ 0.05), indicating that the degree of fibrosis in the surgical side samples of the SSEA-1 + group was lower than that of the surgical side.
  • SSEA-1 + cells have a stronger inhibitory effect on fibrosis than SUSD2 + cells.
  • the proportion of fibrosis area on the surgical side of the combined group was significantly reduced compared with the surgical side of the CG group and the surgical side of the SUSD2 + group (P ⁇ 0.05). Furthermore, the proportion of fibrosis area on the surgical side of the combined group was further significantly reduced compared with the surgical side of the SSEA-1 + group (P ⁇ 0.05), and there was no statistical difference compared with the normal group (P>0.05), suggesting that The degree of fibrosis in the samples from the surgical side of the combined group was further lower than that of the surgical side of the SSEA-1 + group. Compared with SSEA-1 + cells, the combination of SSEA-1 + cells and SUSD2 + cells had a stronger inhibitory effect on fibrosis.
  • SUSD2 + cells have the inhibitory effect on fibrosis; compared with SUSD2 + cells, SSEA-1 + cells have a stronger inhibitory effect on fibrosis; and compared with SSEA-1 + cells, SUSD2 + cells and The combined use of SSEA-1 + cells further enhanced the inhibitory effect on fibrosis.
  • test group normal group CG group SSEA-1 + group SUSD2 + group joint group TGF- ⁇ 1 1.012 ⁇ 0.168 9.71 ⁇ 1.263 1.935 ⁇ 1.506 3.13 ⁇ 1.183 0.896 ⁇ 0.216 ⁇ -catenin 1.008 ⁇ 0.129 2.807 ⁇ 0.472 1.349 ⁇ 0.108 2.63 ⁇ 0.717 0.859 ⁇ 0.099 COLA-1 1.016 ⁇ 0.189 6.351 ⁇ 0.988 1.059 ⁇ 0.144 4.071 ⁇ 0.575 1.15 ⁇ 0.115 ⁇ SMA 1.054 ⁇ 0.405 23.515 ⁇ 4.376 1.593 ⁇ 0.307 9.84 ⁇ 2.721 1.766 ⁇ 0.364
  • TGF- ⁇ 1, ⁇ -catenin, COLA-1 and ⁇ SMA were significantly reduced in the SSEA-1 + group (P ⁇ 0.05). More importantly, TGF- ⁇ 1, ⁇ -catenin, COLA-1 and ⁇ SMA in the SSEA-1 + group not only had no statistically significant differences compared with the normal group (P>0.05), but also had significant differences compared with SUSD2 + decreased (P ⁇ 0.05), indicating that SSEA-1 + cells inhibited the fibrotic response to a higher degree than SUSD2 + cells.
  • TGF- ⁇ 1, ⁇ -catenin, COLA-1 and ⁇ SMA in the combination group were significantly lower than those in the CG group (P ⁇ 0.05), but there was no statistical significance compared with the normal group. Difference (P>0.05), significantly lower than SUSD2 + (P ⁇ 0.05). Furthermore, the expression of TGF- ⁇ 1 and ⁇ -catenin in the combination group was further reduced compared with the SSEA-1 + group, suggesting that the combination of SSEA-1 + cells and SUSD2 + cells has a better response to fibrosis than SSEA-1 + cells. The degree of inhibition is higher.
  • SSEA-1 + cells can significantly reduce the expression of fibrosis-related factors and inhibit the fibrosis response to a high degree; the combination of SUSD2 + cells and SSEA-1 + cells can It further reduces the expression of fibrosis-related factors, thereby exerting a higher inhibitory effect on the fibrotic response.
  • the internal standard used in the qPCR test was ⁇ -actin, and each primer was from Shanghai Sangon Bioengineering Co., Ltd.

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Abstract

L'invention concerne une préparation associée à une réparation endométriale, une cellule endométriale, son procédé de préparation et son utilisation. La préparation associée à une réparation endométriale comprend un milieu de culture conditionnel d'une cellule dérivée de l'endomètre ou d'une cellule dérivée de l'endomètre. La cellule dérivée de l'endomètre présente les caractéristiques d'une cellule précurseur et exprime positivement un antigène embryonnaire spécifique au stade. La culture In vitro et la culture de différenciation de cellules de type précurseur épithélial endométrial avec des effets favorisant la réparation sur une lésion endométriale sont obtenues avec succès, afin que la source cellulaire de cellules souches endométriales soit étendue. Un système de co-culture in vitro ayant un effet positif sur la réparation des lésions de l'endomètre est également construit en se basant sur le milieu de culture conditionnel des cellules précurseurs de l'épithélium endométrial, ce qui revêt une grande importance pour la recherche sur la réparation physiopathologique de l'endomètre et le traitement des adhérences intra-utérines.
PCT/CN2022/091967 2022-05-10 2022-05-10 Préparation associée à une réparation endométriale, cellule endométriale, son procédé de préparation et son utilisation WO2023216104A1 (fr)

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