Method for manufacturing placenta-like organoids

Abstract

To provide a method for producing a placenta-like organoid that facilitates the establishment of the placenta-like organoid from chorionic cells collected from the placenta, which becomes unnecessary following delivery, in quicker and simpler manners than traditional methods.SOLUTION: A method for producing a placenta-like organoid includes a culture step for bringing human chorionic cells into contact with a medium containing a minimal essential medium, a medium supplement, and an extracellular matrix to culture the chorionic cells, where the medium supplement includes N2 supplement, B27 supplement minus vitamin A, primocin, N-acetyl-L-cysteine, human epidermal growth factor, CHIR99021, human R-spongin-1, human basic fibroblast growth factor, human hepatocyte growth factor, A83-01, and prostaglandin E2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a placenta-like organoid, a placenta-like organoid, and a method for evaluating a placenta-like organoid. [Background Art]

[0002] The placenta is an organ critical for fetal development in the uterus. The placenta connects the fetus to the maternal environment via the umbilical cord, exchanges gas, nutrients and excreta, and further supports the production of pregnancy-related hormones and the immune defense of the fetus. The placenta is composed of cells of the trophoblast lineage. Substances ingested by pregnant women are exposed to the fetus when they pass through the placenta. Whether a substance passes through the placenta greatly affects the developmental toxicity of the substance to the fetus. Therefore, development of an in vitro test system for evaluating the effect of a substance on human placental function and the placental permeability of the substance is required. However, when cells are induced to differentiate into trophectoderm and further into trophoblast cell lines via two-dimensional adherent culture, the production of human chorionic gonadotropin (abbreviated as hCG), a hormone produced during pregnancy, stops after about two weeks, making long-term culture difficult. As described above, the generation of human placental organoids is more difficult than that of other organs, and its development is the most delayed.

[0003] Patent Document 1 describes a method for producing a placenta-like organoid, which comprises suspension culturing pluripotent stem cells in the presence of bone morphogenetic protein 4 (BMP4).

[0004] Non-Patent Document 1 describes a method for establishing placenta-like organoids from villus cells. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Unexamined Patent Publication No. 2022-081375 [Non-patent literature]

[0006] [Non-Patent Document 1] Sheridan, MA, et al., “Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta,” Nature Protocols, October 2020, Vol. 15, pp. 3441-3463. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Although a human placenta model was finally reported in 2020 (Non-Patent Literature 1), the method for creating it proved to be extremely difficult in practice. The method for producing placenta-like organoids described in Patent Document 1 includes suspension culture of pluripotent stem cells in the presence of BMP4. Patent Document 1 states that ES cells (embryonic stem cells) or iPS cells (induced pluripotent stem cells) are preferred as the pluripotent stem cells. However, establishing ES cells requires destroying human embryos, which raises ethical issues, and iPS cells carry a risk of becoming cancerous, so the resulting placenta-like organoids may not be usable as mini-placentas. The placenta-like organoids obtained by the method described in Non-Patent Document 1 are not uniform in size during the chorionic cell treatment process, require multiple passages before establishment, and the appropriate cell concentration is unknown, making research applications difficult.

[0008] This invention has been made in view of the above circumstances, and aims to provide a method for producing placental-like organoids that can be established more easily and in a shorter period of time from chorionic cells collected from the placenta, which becomes unnecessary after childbirth. [Means for solving the problem]

[0009] The inventors of this invention discovered that placenta-like organoids can be established more easily and in a shorter time than conventional methods by culturing human chorionic cells in contact with a culture medium supplement containing specific components and a culture medium containing an extracellular matrix, and thus completed the present invention. The present invention includes the following embodiments.

[0010] [1] A method for producing placenta-like organoids, comprising a culture step of culturing human chorionic cells in contact with a culture medium containing a basal medium, a culture medium supplement, and an extracellular matrix, A method for producing placenta-like organoids, wherein the culture medium supplement comprises N2 supplement, B27 supplement minus vitamin A, primosine, N-acetyl-L-cysteine, human epidermal growth factor, CHIR99021, human R-spongin-1, human basic fibroblast growth factor, human hepatocyte growth factor, A83-01, and prostaglandin E2. However, CHIR99021 refers to 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride (CAS registry number: 252917-06-9), and A83-01 refers to 3-(6-methyl-2-pyrimidinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (CAS registry number: 909910-43-6). [2] A method for producing a placenta-like organoid according to [1], wherein the extracellular matrix comprises a basement membrane extract obtained from a mouse Engelbreth-holmesworm tumor. [3] A method for producing placenta-like organoids according to [1] or [2], comprising a pre-culture step of culturing human chorionic cells in suspension in a culture medium containing a basal medium and the culture medium supplement before the culture step. [4] A method for producing placenta-like organoids according to [3], comprising a single-cell separation step after the pre-culture step and before the culture step, in which aggregates of human chorionic cells are separated into single cells using a cell strainer. [5] A method for producing a placenta-like organoid according to any one of [1] to [4], wherein the basal medium is Dulbecco's modified Eagle medium. [6] A placenta-like organoid manufactured by the method for manufacturing a placenta-like organoid described in any one of items [1] to [5]. A method for evaluating placental organoids described in [7] [6], A method for evaluating placental-like organoids, including a combination of a detection method specific to syncytiotrophoblast cells and a detection method specific to cellular trophoblast cells. [8] [6] A method for evaluating placental organoids described above, A method for evaluating placenta-like organoids, comprising contacting the placenta-like organoids with a test substance and performing metabolome analysis. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a method for producing placental-like organoids that can be established more easily and in a shorter period of time from chorionic cells collected from the placenta, which becomes unnecessary after childbirth. [Brief explanation of the drawing]

[0012] [Figure 1] These are the results of light microscopy observations of cultured cells after the first passage. A: day 0 of passage; B: day 3 of passage; C: day 6 of passage; D: day 10 of passage. [Figure 2] These are fluorescence images of chorionic tissue (9 weeks of gestation). A (top left), Syndecan staining; B (bottom left), ITGA6 staining; C (top right), DAPI staining; D (bottom right), fully composite image. [Figure 3] These are fluorescence images of the fabricated placenta-like organoids. A (top left), Syndecan staining; B (bottom left), ITGA6 staining; C (top right), DAPI staining; D (bottom right), fully composite image. [Figure 4] This is the result of FACS analysis. A: All cells; B: ITGA6 stained cells (52.44%). [Figure 5]The results of FACS analysis. A, whole cells; B, Syndecan-stained cells (39.04%). [Figure 6] The results of metabolome analysis. Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments of the method for producing a placenta-like organoid, the placenta-like organoid, and the method for evaluating a placenta-like organoid of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below, and various modifications can be made without departing from the gist of the present invention.

[0014] Method for Producing Placenta-like Organoid Culture Step The method for producing a placenta-like organoid of the present embodiment comprises a culture step of contacting and culturing human villus cells with a medium containing a basal medium, a medium supplement, and an extracellular matrix. Examples of the basal medium include basal media conventionally used for cell culture, such as Dulbecco's modified Eagle medium. Culture is preferably performed using a CO2 incubator at 37°C with a 5% CO2 concentration. The culture time is preferably 10 to 14 days. By culturing the cells in an extracellular matrix, a three-dimensional structure can be formed.

[0015] Preculture Step The method for producing a placenta-like organoid of the present embodiment preferably includes, before the culture step, a preculture step of performing suspension culture of human villus cells in a culture solution containing a basal medium and a medium supplement. The method for producing a placenta-like organoid of the present embodiment preferably includes, after the preculture step and before the culture step, a step of separating aggregates of human villus cells into uniform cell clumps with a cell strainer. The cell strainer is not particularly limited. The capacity of the cell strainer is limited to a maximum of 6 mL, so it is more preferably 1 to 5 mL, even more preferably 2 to 4 mL, and particularly preferably about 3 mL. The pore size of the cell strainer is not particularly limited, but is preferably 70 to 100 μm.

[0016] <Extracellular matrix> The extracellular matrix preferably contains a basement membrane extract extracted from a mouse Engelbreth-Holm-Swarm tumor, and more preferably Corning Matrigel basement membrane matrix (manufactured by Corning) or an equivalent product. Corning Matrigel basement membrane matrix is ​​a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is rich in ECM proteins including laminin (main component), collagen IV, heparin sulfate proteoglycan, entactin / nidogen, and numerous growth factors.

[0017] <Culture medium supplements> One embodiment of the culture medium supplement is a culture medium supplement containing N2 supplement, B27 supplement minus vitamin A, primosine, N-acetyl-L-cysteine, human epidermal growth factor, CHIR99021, human R-spongin-1, human basic fibroblast growth factor, human hepatocyte growth factor, A83-01, and prostaglandin E2.

[0018] N2 supplement is a supplement that enables serum-free culture of various cells, including nerve cells. N2 supplement allows cells that previously required serum to be cultured to be cultured using known factors. N2 supplement contains five factors: insulin, transferrin, ethanolamine, 2-mercaptoethanol, and sodium selenite. It may also contain oleic acid in addition to these five factors. Commercially available N2 supplements such as Gibco N-2 Supplement (100x) (Thermo Fisher Scientific, product number: 17502048), NDiff Neuro-2 Medium Supplement (200x) (Merck, product number: SCM012), N2 Supplement [containing transferrin (holo)] (x100) (Fujifilm Wako Pure Chemical Industries, product number: 141-08941), or N2 Supplement [containing transferrin (apo)] (x100) (Fujifilm Wako Pure Chemical Industries, product number: 141-09041) can be used. The concentration of N2 supplement in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 0.7 to 1.3 times, more preferably 0.8 to 1.2 times, and most preferably 1 times, relative to a 100x concentration of Gibco N-2 Supplement (100x).

[0019] B27 Supplement Minus Vitamin A is a supplement that contains 20 factors including insulin, but with vitamin A removed. Commercially available products such as Gibco B-27 Supplement (50x), minus vitamin A (Thermo Fisher Scientific, product number: 12587010), B-27 Supplement (50x), minus Vitamin A (Tribioscience, product number: TBS8080), or NeuroCult SM1 Without Vitamin A (50x) (STEMCELL Technologies, product number: 05731) can be used as B27 supplements without vitamin A. The concentration of B27 supplement minus vitamin A in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 0.7 to 1.3 times, more preferably 0.8 to 1.2 times, and even more preferably 1 times, relative to 50 times Gibco B-27 Supplement (50x), minus vitamin A.

[0020] Primosin is an antimicrobial agent against mycoplasma, bacteria, and fungi (yeast, mold). Mycoplasma are bacteria, but they lack a cell wall and have plasticity in cell shape, allowing them to pass through a 0.22 μm filter. Therefore, even with sterilization by filtration, it is difficult to avoid contamination by mycoplasma in culture media used for cell culture. Also, because they lack a cell wall, β-lactam (penicillin, cephalosporin) antimicrobial agents that target cell wall synthesis enzymes are ineffective. Primosin contains four types of antimicrobial agents, three of which inhibit DNA and protein synthesis in Gram-positive bacteria, Gram-negative bacteria, and mycoplasma. The fourth type of antimicrobial agent interferes with ion exchange across the cell membrane of fungi (yeast, mold). As primosine, commercially available products such as Primocin (manufactured by InvivoGen, product number: ant-pm-1, product concentration: 50 mg / mL) can be used. The concentration of primosin in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 70 to 130 μg / mL, more preferably 90 to 110 μg / mL, and even more preferably 100 μg / mL.

[0021] N-acetyl-L-cysteine ​​(abbreviated as NAC, CAS registry number: 616-91-1) is an antioxidant. It increases the accumulation of free radical scavengers within cells. It is known to prevent apoptosis in nerve cells. The concentration of N-acetyl-L-cysteine ​​in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 1.0 to 1.5 mM, more preferably 1.1 to 1.4 mM, and even more preferably 1.25 mM.

[0022] Human epidermal growth factor (hEGF, CAS registry number: 62229-50-9) is a protein with a molecular weight of 6045, consisting of 53 amino acid residues and 3 intramolecular disulfide bonds. It binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface and plays a crucial role in regulating cell growth and proliferation. Recombinant hEGF may also be used. As hEGF, commercially available products such as Epidermal Growth Factor (EGF), Human, Recombinant, Animal-Free (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 059-07873), rhEGF (Human, Recombinant) (manufactured by Tokyo Chemical Industry Co., Ltd., product number: R0262), or EGF, Human, Recombinant, E. coli (manufactured by Merck, product number: 324831) can be used. The concentration of hEGF in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 75 to 125 ng / mL, more preferably 90 to 110 ng / mL, and even more preferably 100 ng / mL.

[0023] CHIR99021 [IUPAC name: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, CAS registry number: 252917-06-9] is an inhibitor of glycogen synthase kinase 3 (GSK3, GSK3α, and GSK3β). 50 The values ​​are 10 and 6.7 nM, respectively. For CHIR99021, commercially available products such as CHIR99021 (Cayman Chemical Corporation, product number: 13122), Laduviglusib (MedExpress, product number: HY-10182), and Laduviglusib (CHIR-99021) (Selleck Chemicals, product number: S1263) can be used. The concentration of CHIR99021 in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 2.7 to 3.3 μM, more preferably 2.85 to 3.15 μM, and even more preferably 3 μM.

[0024] Human R-spongin-1 (abbreviated as Rspo-1) is a protein belonging to the Rspo family (Rspo1-4) of Wnt modulators. Rspo-1 is expressed in specific sites of central nervous system development, as well as in the adrenal glands, ovaries, testes, thyroid gland, and trachea. Rspo interacts with the Frizzled / LRP6 receptor complex in the Wnt / β-catenin signaling pathway. For Rspo-1, commercially available products such as R-spondin-1, human, recombinant (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 181-02801), recombinant human R-spondin 1 protein (manufactured by Qkine, Inc., product number: Qk006-0100), or Recombinant Human R-Spondin 1, Carrier-free (manufactured by R&D Systems, Inc., product number: 4645-RS-025 / CF) can be used. The concentration of Rspo-1 in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 70 to 90 ng / mL, more preferably 75 to 85 ng / mL, and even more preferably 80 ng / mL.

[0025] Human basic fibroblast growth factor (bFGF, FGF-basic, FGF2, CAS registry number: 106096-93-9) is a growth factor that exhibits multifaceted effects, and in addition to angiogenesis, its involvement in the development of the nervous system, lungs, muscles, bones, and skin has been suggested. bFGF is an important component of the culture medium for human embryonic stem cells. FGF2 is required to maintain cells in an undifferentiated state. As bFGF, commercially available products such as fibroblast growth factor (basic) (FGF-basic / bFGF / FGF2), human, recombinant, animal-free (154aa) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 060-05383), Recombinant Human bFGF (manufactured by Hygieia Biosciences, Inc., product number: 16000101), or Fibroblast Growth Factor basic, human recombinant, animal-free (manufactured by Merck, product number: GF003AF-100UG) can be used. The concentration of bFGF in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 7 to 13 ng / mL, more preferably 8.5 to 11.5 ng / mL, and even more preferably 10 ng / mL.

[0026] Human hepatocyte growth factor (hHGF) is a cytokine that has been purified as a factor that strongly promotes the proliferation of primary cultured hepatocytes. As a growth factor, hHGF is large and has a heterodimer structure in which a heavy chain with a molecular weight of approximately 60,000 and a light chain with a molecular weight of approximately 35,000 are linked by disulfide bonds. As hHGF, commercially available products such as Human HGF recombinant protein (Proteintech, product number: HZ-1084) or human hepatocyte growth factor (Merck, product number: H5791) can be used. The concentration of hHGF in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 7 to 13 ng / mL, more preferably 8.5 to 11.5 mg / mL, and even more preferably 10 mg / mL.

[0027] A83-01 (also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, CAS registry number: 909910-43-6) is a selective inhibitor of TGF-β type I / activin receptor-like kinase (ALK5), type I activin / nodal receptor-like kinase (ALK4), and type I nodal receptor-like kinase (ALK7). It inhibits Smad2 / 3 phosphorylation and TGF-β-induced mesenchymal cell transition. For A83-01, commercially available products such as CultureSure A-83-01 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 035-24113), A83-01 (manufactured by Merck Ltd., product number: SML0788), or A83-01 (manufactured by R&D Systems, Ltd., product number: 2939 / 10) can be used. The concentration of A83-01 in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 400 to 600 nM, more preferably 450 to 550 nM, and even more preferably 500 nM.

[0028] Prostaglandin E2 (abbreviated as PGE2, CAS registry number: 363-24-6) is a type of physiologically active prostaglandin that is involved in fever, bone resorption by osteoclasts, and childbirth via PGE receptors. The nomenclature of PGE2 is common to all prostaglandins; it is designated "E" because it has an oxo group at position 9 and a hydroxyl group at position 11 of the five-membered ring portion, and a hydroxyl group at position 15 of the side chain portion, and "2" because it has double bonds at positions 5 and 13. As PGE2, commercially available products such as prostaglandin E2 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 165-10813), prostaglandin E2 (manufactured by Nacalai Tesque, Ltd., product number: 29334-34), or prostaglandin E2 (manufactured by Tokyo Chemical Industry Co., Ltd., product number: P1884) can be used. The concentration of PGE2 in the culture medium supplement used in the method for producing placenta-like organoids of this embodiment is not particularly limited, but the final concentration in the culture medium to which the culture medium supplement used in the method for producing placenta-like organoids of this embodiment has been added is preferably 0.7 to 1.3 μM, more preferably 0.85 to 1.15 μM, and even more preferably 1.0 μM.

[0029] The culture medium supplement used in the method for producing placenta-like organoids of this embodiment preferably contains 0.7-1.3 × concentration N2 supplement, 0.7-1.3 × concentration B27 supplement minus vitamin A, 70-130 μg / mL primosine, 1.0-1.5 mM N-acetyl-L-cysteine, 75-125 ng / mL human epidermal growth factor, 2.7-3.3 μM CHIR99021, 70-90 ng / mL human R-spongin-1, 7-13 ng / mL human basic fibroblast growth factor, 50-60 ng / mL human hepatocyte growth factor, 400-600 nM A83-01, and 0.7-1.3 μM prostaglandin E2 in the respective concentration ratios. However, the concentration of the N2 supplement in the culture medium supplement is based on the concentration of Gibco N-2 Supplement (100x) (manufactured by Thermo Fisher Scientific, product number: 17502048), and the concentration of the B27 supplement minus vitamin A in the culture medium supplement is based on the concentration of Gibco B-27 Supplement (50x), minus vitamin A (manufactured by Thermo Fisher Scientific, product number: 12587010) at 50x concentration.

[0030] The culture medium supplement used in the method for producing placenta-like organoids of this embodiment may contain components other than those described above, as long as they do not interfere with the effects of the present invention.

[0031] The culture medium supplement used in the method for producing placenta-like organoids according to this embodiment can be prepared by dissolving or dispersing each component in water.

[0032] In the method for producing placenta-like organoids according to this embodiment, purified water is preferred for preparing the culture medium supplement. For example, distilled water, RO water (water filtered using an RO membrane (reverse osmosis membrane)), ion-exchanged water (water from which ionic impurities have been removed using an ion-exchange resin), Elix water (water purified using an RO membrane and an EDI membrane (continuous ion exchange membrane)) can be used.

[0033] The pH of the culture medium supplement used in the method for producing placenta-like organoids according to this embodiment is not particularly limited, but is preferably 6.5 to 7.5, more preferably 6.8 to 7.4, and even more preferably 7.1 to 7.3. The pH is a measured value obtained using a pH meter.

[0034] The culture medium supplement used in the method for producing placenta-like organoids according to this embodiment may be provided in the form of a concentrated liquid and diluted before use.

[0035] [Placenta-like organoid] The placenta-like organoid of the present invention is a placenta-like organoid manufactured by the method for manufacturing the placenta-like organoid of the present invention described above. The placenta-like organoid of the present invention has a two-layer structure of cellular trophoblast cells (VCTs) and syncytiotrophoblast cells (SCTs), similar to the chorionic tissue of the human placenta. However, unlike the chorionic tissue of the human placenta, the cellular trophoblast cells (VCTs) are on the outside and the syncytiotrophoblast cells (SCTs) are on the inside. The interstitium inside is constructed of a sparse matrix structure and capillaries. When the organoid is established, it inverts; that is, the basement membrane (BM) outside the outer VCTs is in contact with Matrigel.

[0036] [Methods for evaluating placental-like organoids] One embodiment of the method for evaluating placenta-like organoids of the present invention includes combining a detection method specific to syncytiotrophoblast cells and a detection method specific to cytotrophoblast cells. One specific method for detecting syncytiotrophoblastic cells is Sydecan staining. One example of a method for specifically detecting cytotrophoblast cells is ITGA6 staining.

[0037] Another embodiment of the method for evaluating placenta-like organoids of the present invention includes contacting the placenta-like organoids of the present invention with a test substance and performing metabolome analysis. The method of metabolome analysis is not particularly limited. [Examples]

[0038] The present invention will be described more specifically below with reference to examples. However, the present invention is not limited to the examples described later.

[0039] [Manufacturing Example 1] Preparation of Culture Medium Supplements <Material> N2 Supplement Gibco N-2 Supplement (100x) (Manufactured by Thermo Fisher Scientific, Product Number: 17502048) • B27 Supplement Minus Vitamin A Gibco B-27 Supplement (50x), minus vitamin A (manufactured by Thermo Fisher Scientific, product number: 12587010) Primocin Primocin (manufactured by InvivoGen, product number: ant-pm-1, product concentration: 50 mg / mL) • N-acetyl-L-cysteine N-acetyl-L-cysteine ​​(manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 015-05132) • Human epidermal growth factor Epidermal growth factor (EGF), human, recombinant, animal-derived free (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 059-07873) ·CHIR99021 CHIR99021 (Manufactured by Cayman Chemical Corporation, Product Number: 13122) Human R-spongin-1 R-Spongin-1, Human, Recombinant (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Product Number: 181-02801) Human basic fibroblast growth factor Fibroblast Growth Factor (Basic) (FGF-basic / bFGF / FGF2), Human, Recombinant, Animal-derived Free (154aa) (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Product Number: 060-05383) Human hepatocyte growth factor Hepatocyte growth factor, human (Merck, product number: H5791) ·A83-01 CultureSure A-83-01 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 035-24113) • Prostaglandin E2 Prostaglandin E2 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 165-10813) ·water Ion-exchanged water

[0040] <Preparation method> Each component was dissolved in water to the following concentrations to prepare a 10-fold concentrated solution. N2 Supplement 10x Concentration B27 Supplement Minus Vitamin A 10x Concentration Primosin 1 mg / mL N-acetyl-L-cysteine ​​12.5 mM Human epidermal growth factor 1 μg / mL CHIR99021 30μM Human R-spongin-1 800 ng / mL Human basic fibroblast growth factor 100 ng / mL Human hepatocyte growth factor 500 ng / mL A83-01 5μM Prostaglandin E 210 μM

[0041] [Example 1] Production of placenta-like organoids <Material> Basic culture medium Advanced DMEM / F12 (Gibco) Culture medium supplement prepared in Manufacturing Example 1 Washing culture medium: Dulbecco's modified Eagle medium (manufactured by Nacalai Tesque) Digestive culture medium 0.17% collagenase B (manufactured by Roche) ROCK Inhibitor (Y-27632) (Manufactured by Merck, Product Number: SCM075) Matrigel Corning Matrigel Basement Membrane Matrix (manufactured by Corning) Cell strainer (Corning Corporation, volume: 3 mL, pore size: 70 μm or 100 μm)

[0042] <Instructions> Placenta-like organoids were produced from human chorionic cells using the following procedure. (1) Human chorionic cells (approximately 1 g) obtained from childbirth, etc., were finely chopped. (2) Place the culture medium (2) with approximately 10 mL of washing medium into a 15 mL tube. (3) After the tissue settled naturally, the supernatant was removed. (4) The mixture was pipetted again with approximately 10 mL of the washing culture medium (1 × Dulbecco's modified Eagle medium). (5) Repeat (3). (6) 4 mL of digestive culture medium and 4 μL of 10 mM ROCK inhibitor (1000-fold dilution) were added. (7) Shake at 37°C for 30 minutes. Adjust the cell aggregates to a uniform size using a cell strainer. (8) After the tissue settled naturally, the supernatant was transferred to another 15 mL tube (on ice). (9) Repeat steps (6) to (7) for the remaining pellets. (10) The entire volume was poured into the 15 mL tube on the ice mentioned earlier. (11) Centrifuge at 4°C for 5 minutes (at 200 rpm). (12) Discard the supernatant, embed the pellet in Matrigel (swelled with 1x Dulbecco's modified Eagle medium + medium supplement), and seed approximately 6 drops per well (20 μL / drop) onto a 24-well plate. (13) 1 mL / well of culture medium (1 × Dulbecco's modified Eagle medium + medium supplement) was added. (14) The cells were cultured in a CO2 incubator (37°C). Matrigel composition per well: 90 μL Matrigel, 30 μL culture medium (1 × Dulbecco's modified Eagle medium + medium supplement), 1 μL 10 mM ROCK inhibitor

[0043] <Optical observation of cells> Cultured cells after the first passage were photographed with an optical microscope. The photographing results are shown in Figure 1. A to D in Figure 1 represent day 0, day 3, day 6, and day 10 after passage, respectively.

[0044] <Immunostaining> Chorionic villus tissue (9 weeks gestation) and the prepared placental-like organoids were stained with Syndecan selective for syncytiotrophoblast cells, ITGA6 selective for cytotrophoblast cells, and DAPI selective for nuclei, and then photographed. Figure 2 shows fluorescence observation images of the chorionic villus tissue, and Figure 3 shows fluorescence observation images of the prepared placental-like organoids. A (upper left): Syndecan staining B (lower left): ITGA6 staining C (upper right): DAPI staining D (lower right): Overlaid composite image

[0045] <Analysis of cell distribution by FACS> The fractionation ratio was confirmed by FACS analysis. The results are shown in Figure 4 and Figure 5. A in Figure 4 represents all cells, and B represents ITGA6-stained cells (52.44%). A in Figure 5 represents all cells, and B represents Syndecan-stained cells (39.04%).

[0046] <Metabolome analysis> Prepared placental-like organoids at the same concentration were seeded in one Matrigel using 48-well plates, and metabolome analysis was performed on primary metabolites distributed in the supernatant in a time-dependent manner under hypoxic conditions. At time points (0, 1, 3, 6, 24 hours), 200 µL of supernatant was collected for analysis. As shown in Figure 6, OPLS-DA analysis revealed clear clustering for each time point, and the cells grew uniformly (N=3).

Industrial Applicability

[0047] By using placenta-like organoids (mini-placentas), which are human placenta models produced using the method for producing placenta-like organoids of the present invention, it is expected that placenta research will advance dramatically as a living tissue that has been largely unexplored until now. It is expected to become one of the powerful placenta research tools for the future. Specific application examples include toxicity testing of the placenta during pregnancy in humans, elucidation of pathological mechanisms through medical models of miscarriage and premature birth, and elucidation of the implantation mechanism of fertilized eggs in the field of infertility, contributing to a wide range of medical advancements and drug discovery for disease treatment.

Claims

1. A method for producing placenta-like organoids, comprising a culture step of culturing human chorionic cells in contact with a culture medium containing a basal medium, a culture medium supplement, and an extracellular matrix, The culture medium supplement comprises N2 supplement, B27 supplement minus vitamin A, primosine, N-acetyl-L-cysteine, human epidermal growth factor, CHIR99021, human R-spongin-1, human basic fibroblast growth factor, human hepatocyte growth factor, A83-01, and prostaglandin E2. The aforementioned medium is at the final concentration. 0.7-1.3x concentration N2 supplement, 0.7-1.3x concentration B27 supplement minus vitamin A 70-130 μg / mL Primosin 1.0–1.5 mM N-acetyl-L-cysteine, 75-125 ng / mL Human epidermal growth factor, 2.7-3.3μM CHIR99021, 70-90 ng / mL Human R-Spongin-1 7-13 ng / mL Human basic fibroblast growth factor, 50-60 ng / mL human hepatocyte growth factor, 400-600 nM A83-01, and A method for producing placenta-like organoids containing 0.7–1.3 μM prostaglandin E2. However, CHIR99021 means 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride (CAS registration number: 252917-06-9), and A83-01 means 3-(6-methyl-2-pyrimidinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (CAS registration number: 909910-43-6). Furthermore, the concentration of the N2 supplement in the culture medium supplement is based on the concentration of Gibco N-2 Supplement (100x) (manufactured by Thermo Fisher Scientific, product number: 17502048), and the concentration of the B27 supplement minus vitamin A in the culture medium supplement is based on the concentration of Gibco B-27 Supplement (50x), minus vitamin A (manufactured by Thermo Fisher Scientific, product number: 12587010) at 50x concentration.

2. A method for producing a placenta-like organoid according to claim 1, wherein the extracellular matrix comprises a basement membrane extract extracted from a mouse Engelbrethholmsworm tumor.

3. A method for producing placenta-like organoids according to claim 1, comprising a pre-culture step of suspension culturing human chorionic cells in a culture medium containing a basal medium and the culture medium supplement, prior to the culture step.

4. The method for producing a placenta-like organoid according to claim 1, wherein the basal culture medium is Dulbecco's modified Eagle medium.

5. The method for producing a placenta-like organoid according to claim 1, wherein the human chorionic cells are human chorionic cells collected from a placenta obtained during childbirth.

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