Method for preparing endometrial organoid and uses thereof for treating uterine fibrosis and ameliorating aging

Abstract

A pharmaceutical composition according to an aspect can be applied to the prevention, treatment or the like of endometrial damage, Asherman's syndrome, or complications thereof by: reducing the level of collagen accumulation in the endometrium; increasing the expression level of VEGF; increasing the expression level of Ki67; increasing the expression level of Itgb3; increasing the expression level of Spp1; increasing the number of implantation sites; decreasing the expression levels of fibrosis-related factors; decreasing the expression levels of metabolism-related factors; increasing ATP production; and regulating the expression levels of mitochondrial function-related factors.

Description

Method for producing endometrial organoids and their use for treating uterine fibrosis and improving aging

[0001] The present invention relates to a method for producing endometrial organoids and their use in treating uterine fibrosis and improving aging.

[0002] The endometrium, a lining of the uterine cavity, plays a crucial role in embryo implantation. The endometrium secretes growth factors and cytokines, providing an environment conducive to embryo implantation, development, and pregnancy. Asherman's syndrome (AS) is a gynecological disorder characterized by intrauterine adhesions accompanied by severe fibrous lesions, leading to infertility and damage to the basilar artery. Severe postoperative trauma, primarily dilatation and curettage (D&C), or viral infections can disrupt the cyclical regeneration of the uterine functional layer, resulting in menstrual disorders such as amenorrhea or hypomenorrhea. A key feature of Asherman's syndrome is intrauterine adhesions, which reduce the volume of the uterine cavity. Methods used to treat endometrial damage, specifically endometrial adhesions, include hysteroscopic adhesiolysis, intrauterine balloon stents / Foley catheters to prevent re-adhesion, and postoperative adjuvant hormonal therapy. However, the treatment effect is minimal due to the high recurrence rate of intrauterine adhesions.

[0003] Against this backdrop, the present inventors confirmed the anti-endometrial effect of endometrial organoids or mitochondria isolated from endometrial organoids. Based on this, they developed a composition for the prevention or treatment of endometrial damage. Furthermore, they developed a composition capable of efficiently producing endometrial organoids, thereby developing an effective treatment method for Asherman syndrome or its complications.

[0004] One aspect is to provide a composition for promoting endometrial organoid culture, comprising a basic medium, antibiotics and growth factors.

[0005] Another aspect provides a method for producing an endometrial organoid, comprising the steps of: a) obtaining cells from normal endometrial tissue; b) obtaining a fraction enriched in epithelial cells from the isolated cells; and c) culturing the fraction in an organoid culture medium to obtain an endometrial organoid.

[0006] Another aspect provides a pharmaceutical composition comprising the endometrial organoid.

[0007] Another aspect provides a method for preventing or treating endometrial damage by injecting the pharmaceutical composition into a subject.

[0008] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein will be readily apparent and inferred by those skilled in the technical field of this application or similar technical fields, and therefore, their description will be omitted.

[0009] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0010] One aspect provides a composition for promoting organoid culture comprising a basic medium, antibiotics and growth factors.

[0011] The composition for promoting endometrial organoid culture may be provided, wherein the growth factor is at least one of resveratrol (RES), casin (Ca), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF1), and the growth factor may include at least one selected from the group consisting of resveratrol (RES) and casin (Ca). The same part as described above also applies to the composition for promoting endometrial organoid culture.

[0012] The composition for promoting endometrial organoid culture may include, but is not limited to, resveratrol at a concentration of 0.1 nM to 500 nM, 0.1 nM to 400 nM, 0.1 nM to 300 nM, 0.1 nM to 200 nM, 0.1 nM to 100 nM, 0.5 nM to 100 nM, or 1 nM to 100 nM.

[0013] The composition for promoting endometrial organoid culture may include, but is not limited to, a concentration of cassin of 0.1 nM to 50 nM, 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, or 1 nM to 10 nM.

[0014] Organoids cultured in media containing high concentrations of resveratrol, carcin, or resveratrol and carcin beyond the above range exhibit reduced proliferation capacity, making it difficult to produce organoids of an appropriate level.

[0015] The term "organoid culture" above encompasses any action that can generate or maintain organoids. For example, this may involve differentiating stem cells or cells isolated from a specific tissue into tissue or organ cells with a specific function, and / or causing the organoids to survive, grow, or proliferate.

[0016] The above "promoting organoid culture" may mean promoting the proliferation of cells that make up the organoid. The term "proliferation" refers to an increase in the number of cells and may be used in the same sense as "growth."

[0017] The above RES, PDGF, IGF1 or a combination thereof may be included at an appropriate concentration as long as it is suitable for the purpose of promoting the culture of the endometrial organoids.

[0018] The above RES may be comprised of 0.1 μM to 10 μM, 0.1 μM to 5 μM, 0.1 μM to 3 μM, 0.5 μM to 3 μM or 0.5 μM to 1.5 μM.

[0019] The PDGF may be contained in an amount of 0.1 ng / ml to 10 ng / ml, 0.1 ng / ml to 5 ng / ml, 0.1 ng / ml to 3 ng / ml, 0.5 ng / ml to 3 ng / ml, or 0.5 ng / ml to 1.5 ng / ml.

[0020] The above IGF1 may be included in an amount of 0.1 ng / ml to 10 ng / ml, 0.1 ng / ml to 5 ng / ml, 0.1 ng / ml to 3 ng / ml, 0.5 ng / ml to 3 ng / ml, or 0.5 ng / ml to 1.5 ng / ml.

[0021] If the concentration range is exceeded or below the above, adverse effects in vivo may occur or the organoid culture promotion effect may not be exhibited.

[0022] According to one embodiment, the composition for organoid culture was confirmed to significantly increase cell proliferation ability or organoid proliferation ability by including RES, PDGF, IGF1 or a combination thereof as a growth factor, thereby showing an effect of improving organoid culture efficiency.

[0023] The composition may be provided in the form of a medium composition to enhance the efficiency of organoid culture of stem cells. The RES, PDGF, IGF1, or a combination thereof may be added to a conventional medium used for organoid culture to achieve effects related to the enhancement of organoid culture. In one specific example, it was confirmed that cell proliferation capacity was enhanced by adding RES, PDGF, IGF1, or a combination thereof to the organoid culture medium.

[0024] The above medium composition may further include an antioxidant selected from the group consisting of selenium, ascorbic acid, vitamin E, catechin, lycopene, beta-carotene, coenzyme Q-10 (CoQ-10), T-BHQ, oltipraz, alder extract, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), insulin transferrin selenium (ITS), stem cell factor (SCF), WNT pathway activator, and combinations thereof.

[0025] In one specific example, the basic medium may be a basic medium selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), advanced DMEM, 80% knockout DMEM, MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax Medium, and Chang's Medium MesemCult-XF Medium, and in addition, any medium used for organoid culture in the art may be used without limitation.

[0026] In one specific example, the medium composition comprises Epidermal Growth Factor (EGF), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Fibroblast Growth Factor 10 (FGF10), Noggin (BMP inhibitor protein), R-spondin1 (Roof plate-specific spondin 1), R-spondin3 (Roof plate-specific spondin 3), Hepatocyte Growth Factor (HGF), Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF), Vascular Endothelial Growth Factor (VEGF), CHIR (CHIR99021, a GSK-3 inhibitor) and WNT3a. (Wingless-type MMTV integration site family, member 3a) may contain one or more growth factors selected from the group consisting of:

[0027] In one embodiment, the antibiotic may be primocin, penicillin, streptomycin, gentamycin, or a combination thereof.

[0028] Another aspect provides a method for producing an endometrial organoid, comprising the steps of: a) obtaining cells from normal endometrial tissue; b) obtaining a fraction enriched in epithelial cells from the isolated cells; and c) culturing the fraction in an organoid culture medium to obtain an endometrial organoid. The same parts as described above also apply to the method for producing an endometrial organoid.

[0029] The above endometrial organoid may be obtained from normal endometrial tissue of a human or mouse.

[0030] In one specific example, the endometrial organoids produced through the above production method may have CD326 expressed at 60% or higher, CD44 expressed at 60% or higher, and LGR5 expressed at 0.5% or higher.

[0031] Another aspect provides a method for isolating mitochondria from endometrial organoids prepared using the above method for preparing endometrial organoids. The same principles described above also apply to the method for isolating mitochondria.

[0032] The method for isolating the above mitochondria can be performed using a known method.

[0033] Another aspect provides a pharmaceutical composition comprising the endometrial organoid. The endometrial organoid may be derived from uterine tissue of a young individual, or may be a "normal" organoid with excellent function cultured using special additives. Furthermore, the term "comprising an endometrial organoid" may refer to a pharmaceutical composition for improving, alleviating, preventing, or treating endometrial damage, which comprises a normal endometrial organoid or mitochondria isolated from a normal endometrial organoid as an active ingredient.

[0034] The above normal endometrial organoids may express CD326 at 60% or more, CD44 at 60% or more, and LGR5 at 0.5% or more.

[0035] The term "organoid" above refers to a cell aggregate created by re-aggregating and recombining cells isolated from stem cells or organ-derived cells using a 3D culture method, and may include organoids or cell clusters formed from suspension cell cultures. The organoids may also be referred to as miniature organoids, organ analogs, or pseudo-organs. Specifically, the organoids include one or more cell types among the various types of cells that constitute an organ or tissue, and must be able to reproduce the shape and function of the tissue or organ.

[0036] The above normal endometrial organoid may be produced by a method comprising the steps of: a) obtaining cells from normal endometrial tissue; b) obtaining a fraction rich in epithelial cells from the isolated cells; and c) culturing the fraction in an organoid culture medium to obtain an endometrial organoid.

[0037] The above endometrial organoid may be obtained from normal endometrial tissue of a human or mouse.

[0038] The above term 'epithelial cell-rich fraction' refers to a fraction with a high level of CD326 expression. In the initial cells obtained from the tissue, CD326 is maintained at approximately 10%, but as subculture progresses, it can be maintained close to 100%.

[0039] The step of obtaining cells of the above normal endometrial tissue may be a step of obtaining cells through a dissociation step, and the dissociation may be performed by mechanical dissociation, enzymatic dissociation, or a combination thereof.

[0040] In the case of the above mechanical dissociation, it means that the entire uterine tissue is finely divided into pieces using microscissors until it becomes a thin state without any large lumps.

[0041] In the case of the above enzymatic dissociation, it may mean a step of treating with TrypLE for about 10 minutes, then treating with collagenase solution for about 30 minutes, mixing by pipetting, removing tissue debris with a strainer of about 100 μm, and then recovering the cell concentrate from the remaining cell mixture using a centrifuge (about 800 rcf, about 3 min); then washing with serum-free medium about twice, preparing a mixture of cell concentrate: Matrigel (1:9 or 2:8), seeding it in a 48-well or 24-well plate, solidifying it in an incubator at about 37 C° for about 15 min, and processing the organoid culture.

[0042] The term "endometrial damage" as used herein means disruption of the normal integrity of all or part of the tissue structure of the endometrium, including damage caused by uterine surgery, tuberculosis infection, physical injury, or endometrial infection, and is intended to include "wounds," "lesions," "necrosis," and "ulcers." Damage to the endometrium may result in embryo implantation failure or abnormal embryo development, which may cause infertility.

[0043] The above prevention or treatment of endometrial damage may mean prevention or treatment of endometrial adhesions, Asherman syndrome or complications thereof.

[0044] As used herein, the term "Asherman's syndrome", also known as intrauterine adhesions (IUAs), occurs when the basal layer of the endometrium is detached and fails to regenerate normally, causing uterine adhesions. Asherman's syndrome primarily occurs in patients with a history of surgery such as endometrial curettage, cervical cone biopsy, or electrocautery, a history of pelvic inflammatory disease, or infection caused by an intrauterine device (IUD), and is characterized by endometrial fibrosis, damage to the cervix, destruction of the endometrium, and adhesions in the uterine cavity. In addition, Asherman's syndrome in the uterus may be accompanied by complications, and generally, decreased menstruation, amenorrhea, uterine pain, infertility, and subfertility may occur.

[0045] As used herein, the term "complication of Asherman syndrome" refers to a disease that may accompany Asherman syndrome or a general term for a disease that may increase the risk of Asherman syndrome. Specifically, it includes a disease or symptom that may accompany uterine adhesions or increase the risk of uterine adhesions. In one specific example, the complication of Asherman syndrome may be one or more selected from the group consisting of uterine adhesions, uterine fibroids, endometriosis, ectopic pregnancy miscarriage, ovarian cysts, menstrual disorders, infertility, sterility, pelvic adhesions, pelvic pain, and pelvic inflammatory disease, but is not limited thereto.

[0046] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0047] As used herein, the term "treatment" refers to any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms. Accordingly, the terms "treatment" and "prevention" are not intended to imply a cure or complete elimination of symptoms.

[0048] The pharmaceutical composition may be provided in the form of an injection. The composition in the form of an injection may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. Specifically, the composition may be provided in the form of an injection for easy topical administration. The active ingredient of the composition in the form of an injection may be dissolved in a pharmaceutically acceptable aqueous solution or may be frozen in a solution state.

[0049] The sterile composition for the above injection form can be prescribed according to the usual preparation practice using a vehicle such as distilled water for injection. As an aqueous solution for injection, there can be mentioned, for example, physiological saline, isotonic solution containing glucose or other auxiliary agent (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). As a suitable solubilizing agent, for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.), nonionic surfactant (polysorbate 80(TM), HCO-50, etc.) can be used in combination. As an oily liquid, there can be mentioned sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can also be used in combination as a solubilizing agent. Additionally, it may be combined with buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution may be conventionally filled into appropriate ampoules.

[0050] The above "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.

[0051] In one specific example, the normal endometrial organoid may express one or more factors selected from the group consisting of CD326, CD44, and LGR5.

[0052] The above CD326 may be expressed by 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more of the above normal endometrial organoids.

[0053] The above CD44 may be expressed by 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more of the above normal endometrial organoids.

[0054] The above LGR5 may be expressed in 0.5% or more, 1.0% or more, 1.3% or more, or 1.5% or more of the above normal endometrial organoids.

[0055] The above CD 326 is also known as 'Epitehlial cell adhesion molecule (EpCAM)' and is a well-known epithelial cell marker. In one specific example, the normal endometrial organoid may exhibit characteristics of endometrial epithelial cells by expressing CD326 by 80% or more. The CD 44 (cluster of differentiation 44) is a stem cell marker. In one specific example, the normal endometrial organoid may exhibit excellent cell regeneration ability by expressing CD44 by 80% or more. In addition, the normal endometrial organoid may exhibit characteristics of stem cells by expressing LGR5, a stem cell marker, by 1.5% or more.

[0056] In one embodiment, the pharmaceutical composition may exhibit any one or more of the following characteristics: a) decreased collagen accumulation level in the endometrium; b) increased VEGF expression level; c) increased Ki67 expression level; d) increased Itgb3 expression level; e) increased Spp1 expression level; f) increased number of implantation sites; g) decreased expression level of a fibrosis-related factor; h) decreased expression level of a metabolism-related factor; and i) increased ATP production. In one embodiment, the fibrosis-related factor may be Tgfb1, Timp1, or Col1a1. In one embodiment, the metabolism-related gene may be Hk2, a glycolysis-related marker, or Scd1 or Gpr84, a lipid metabolism-related marker. By exhibiting the above characteristics, the pharmaceutical composition may have an effect of improving, alleviating, preventing, or treating endometrial damage.

[0057] As used herein, the term "fibrosis" or "fibrosis" refers to the phenomenon of excessive formation of fibrous connective tissue in a tissue or organ during the process of damage recovery or remodeling, and may be used interchangeably with "fibrotic lesion" herein. This may be caused by excessive accumulation of extracellular matrix components such as collagen.

[0058] In one specific example, the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by improving or alleviating symptoms of endometrial fibrosis by reducing the level of collagen accumulation in the endometrium. Furthermore, by exhibiting such effects, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility.

[0059] The above-mentioned vascular endothelial growth factor (VEGF) is a factor related to angiogenesis and proliferative capacity. In one specific example, the pharmaceutical composition may exhibit an endometrial damage prevention or treatment effect by increasing the expression level of VEGF in endometrial cells. Furthermore, by exhibiting such effects, the pharmaceutical composition may exhibit an infertility or subfertility alleviation, improvement, prevention, or treatment effect.

[0060] The above Ki67, also known as Antigen Kiel 67 or MKI67 (marker of proliferation Kiel 67), is a protein encoded by the MKI67 gene and is a cell proliferation-related factor. In one specific example, the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by increasing the expression level of Ki67 in endometrial cells. In addition, by exhibiting such effects, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility.

[0061] The above Itgb3 (Integrin beta-3) or Spp1 (secreted phosphoprotein 1 or osteopontin) is a marker related to endometrial receptivity, and Spp1 increases the expression of extracellular matrix, adhesion molecules, and cytokines of the human uterus of the receptor, and is related to the pregnancy rate. In one specific example, the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by increasing the expression level of Itgb3 or Spp1 in the endometrium. In addition, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility by exhibiting such an effect.

[0062] In one specific example, the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by increasing the number of implantation sites within the endometrium. Furthermore, by exhibiting such effects, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility.

[0063] The above Tgfb1 (Transforming Growth Factor-beta 1), Timp1 (TIMP metallopeptidase inhibitor 1), or Col1a1 (Collagen, type I, alpha 1) are fibrosis-related factors. In one specific example, the pharmaceutical composition may exhibit an endometrial damage prevention or treatment effect by reducing the expression level of fibrosis-related factors, such as Tgfb1, Timp1, or Col1a1, in the endometrium. In addition, the pharmaceutical composition may exhibit such effects, thereby exhibiting an infertility or subfertility alleviation, improvement, prevention, or treatment effect.

[0064] The above Hk2 (Hexokinase 2) is a factor related to this action, and the above Scd1 (Stearyl-coenzyme A desaturase 1) or Gpr84 (G protein-coupled receptor 84) is a factor related to lipid metabolism. In one specific example, the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by reducing the expression level of Scd1 or Gpr84 in the endometrium. In addition, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility by exhibiting such an effect.

[0065] The pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by regulating the expression of mitochondrial function-related factors (Pgc1a, Nrf1, Mfn1, Fis1). In one embodiment, when AS-induced mice were treated with the pharmaceutical composition, it was confirmed that the levels of mitochondrial function-related factors changed by AS induction were regulated to the levels of normal (non-AS-induced) mice. Through this, it was confirmed that the pharmaceutical composition may exhibit an effect of preventing or treating endometrial damage by restoring mitochondrial function in the endometrium. In addition, by exhibiting such effects, the pharmaceutical composition may exhibit an effect of alleviating, improving, preventing, or treating infertility or subfertility.

[0066] The pharmaceutical composition comprising the endometrial organoid may exhibit an effect of preventing or treating endometrial damage in a subject by transferring mitochondria within the endometrial organoid into the endometrium of the subject. In one embodiment, when the endometrial organoid was treated in AS-induced mice, the migration of mitochondria within the organoid into the endometrium was confirmed. In addition, when mitochondria isolated from the endometrial organoid were treated in the endometrium of AS-induced mice, the expression levels of Timp1 and Col1a1 were reduced to a level similar to that observed when the endometrial organoid was treated, and the expression levels of Hk2, Scd1, and Gpr84 were reduced. Through this, it was confirmed that the endometrial organoid according to one embodiment can exhibit an effect of preventing or treating endometrial damage by transferring mitochondria.

[0067] Therefore, it was confirmed that a pharmaceutical composition comprising mitochondria isolated from a normal endometrial organoid or an endometrial organoid according to one embodiment can exhibit the following properties for a damaged endometrium: a) a decrease in the level of collagen accumulation in the endometrium; b) an increase in the level of VEGF expression; c) an increase in the level of Ki67 expression; d) an increase in the level of Itgb3 expression; e) an increase in the level of Spp1 expression; f) an increase in the number of implantation sites; g) a decrease in the level of expression of a fibrosis-related factor; h) a decrease in the level of expression of a metabolism-related factor; i) an increase in ATP production; and k) a regulation of the level of expression of a mitochondrial function-related factor, thereby exhibiting an effect of preventing or treating endometrial damage. In addition, it was confirmed that the endometrial organoid can exhibit the above-mentioned properties by transferring mitochondria in the organoid to the endometrium, thereby exhibiting an effect of preventing or treating endometrial damage.

[0068] The pharmaceutical composition may comprise a pharmaceutically effective amount of the endometrial organoid or mitochondria isolated from the endometrial organoid; and / or a pharmaceutically acceptable carrier.

[0069] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0070] Mitochondria isolated from the endometrial organoid or endometrial organoid; And the pharmaceutically acceptable weight ratio between carriers may be, for example, 500:1 to 1:500, and as an example, the weight ratio may be 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or It can be 2:1 or 1:2, but is not limited thereto.

[0071] The above pharmaceutical composition may additionally include, but is not limited to, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above ingredients.

[0072] The above pharmaceutical composition can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, transdermal administration, etc., but is not limited thereto.

[0073] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 ug (micrograms, 0.001 to 1000 ug, 0.01 to 1000 ug, 0.1 to 1000 ug, or 1.0 to 1000 ug) per day, and may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity.

[0074] The above pharmaceutical composition can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0075] The above formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally contain a dispersing agent and / or a stabilizer.

[0076]

[0077] Another aspect is to provide a cell therapy comprising the endometrial organoids. The same principles described above apply to the cell therapy.

[0078] The term "cell therapy" as used herein refers to a medicine used for the purposes of treatment, diagnosis, and prevention from cells and tissues isolated, cultured, and specially prepared from humans, and refers to a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise changing the biological characteristics of living autologous, allogenic, or xenogenic cells in vitro to restore the function of cells or tissues.

[0079] The dosage of the above cell therapy composition may vary depending on the age, weight, sex, dosage form, health status, and disease severity of the individual, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the typical dosage of the cell therapy composition for humans is 10 4 10 inland 10 It can be cells / body, preferably 10 6 ~10 8Cells / body, can be administered once or in multiple doses. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0080] The term "administration" as used herein means introducing the cell therapy composition into a patient by any appropriate method, and the cell therapy composition may be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. Examples of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0081] The above cell therapy composition may be administered in a pharmaceutically effective amount. As used herein, the term "effective amount" means the amount necessary to delay or completely stop the onset or progression of a specific disease to be treated. The appropriate total daily usage amount may be determined by the treating physician within the scope of sound medical judgment. For the purpose of the above cell therapy composition, the specific therapeutically effective amount for a specific patient may be applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the duration of treatment, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0082]

[0083] Another aspect provides a method for improving, alleviating, preventing, or treating endometrial damage by injecting a pharmaceutical composition comprising normal endometrial organoids or mitochondria isolated from normal endometrial organoids into a subject. The same principles as described above also apply to the method for improving, alleviating, preventing, or treating endometrial damage.

[0084]

[0085] Another aspect provides a cell therapy composition comprising organoids cultured in a culture medium containing the composition for promoting organoid culture. In one specific example, the cell therapy composition may be a cell therapy comprising, as an active ingredient, organoids treated with RES, PDGF, IGF1, or a combination thereof.

[0086]

[0087] By treating endometrial organoids with the above RES, PDGF, IGF1, or a combination thereof, the proliferative capacity of the cells constituting the organoids can be increased. Therefore, organoids obtained through a method of treating RES, PDGF, IGF1, or a combination thereof have significantly increased cell proliferative capacity, thereby activating the organoids to a state that is efficient and suitable for transplantation, or modifying their characteristics to suit the human body, and thus can be included as an effective ingredient in cell therapy.

[0088] Another aspect provides a method for enhancing organoid culture efficiency, comprising culturing cells in the above-described composition for enhancing organoid culture. The same portions described above also apply to the method for enhancing organoid culture efficiency.

[0089] Another aspect provides organoids obtained through the aforementioned method for enhancing organoid culture efficiency. The same portions described above also apply to organoids obtained through the aforementioned method for enhancing organoid culture efficiency.

[0090] According to the pharmaceutical composition according to one aspect, it can be applied to the prevention or treatment of endometrial damage, Asherman syndrome or complications thereof, etc. by reducing the level of collagen accumulation in the endometrium; increasing the level of VEGF expression; increasing the level of Ki67 expression; increasing the level of Itgb3 expression; increasing the level of Spp1 expression; increasing the number of implantation sites; reducing the level of expression of fibrosis-related factors; reducing the level of expression of metabolism-related factors; increasing ATP production; and regulating the level of expression of factors related to mitochondrial function.

[0091] According to the composition for promoting organoid culture according to the aspect, efficient organoid production is possible by increasing cell proliferation ability.

[0092] Figure 1 shows the results of collagen volume fraction (A), α-SMA DAB intensity level (B), and Ki67+ fluorescence intensity (C) in endometrial tissues derived from normal (n=1) and AS patients (n=1), and mouse endometrial tissues derived from normal (n=3) and AS-induced uteri (n=3).

[0093] Figure 2 shows the results of qRT-PCR analysis of mRNA expression of Tgfb1 (A), Timp1 (B), and Col1a1 (C) in AS-induced mouse uterus on the indicated days (days 7, 14, and 21) compared to normal mouse uterus (total number of mice used = 48, 3 mice per group, triplicates).

[0094] Figure 3 is an image of the uterus showing the implantation site on day 14 of pregnancy in normal and AS-induced mice (n=6).

[0095] Figure 4 shows the results of the number of implantation sites (A), fetal weight (B), and placental weight (C) of normal and AS-induced mice.

[0096] Figures 5a and 5b show the results of measuring the expression levels of CD326 and CD44 in human endometrial organoids according to one embodiment. Figure 5c shows the results of additionally confirming the expression level of LGR5, and Figure 5d shows the results of confirming the human endometrial expression factors MUC1, CDH1, ERα, and Ki67 through fluorescent staining.

[0097] Figure 6 shows the results of immunohistochemical analysis to determine the expression levels of h-MITO and STEM121 according to human endometrial organoid treatment in AS-induced endometrium according to one embodiment (total number of mice used = 4; 2 mice per group; duplicates).

[0098] Figure 7 is a DIC and GFP fluorescence image of a mouse endometrial organoid used for transplantation according to one embodiment.

[0099] Figures 8a and 8b show the results of measuring the expression levels of CD326 and CD44 in mouse endometrial organoids used for transplantation according to one embodiment.

[0100] Figure 9 shows GFP according to one embodiment in AS-induced endometrium. + The results of measuring GFP +ve fluorescence intensity according to mouse endometrial organoid treatment (total number of mice used = 8, 4 mice per group, quadruplicate).

[0101] Figure 10 shows the results of measuring the levels of collagen accumulation (A), Col1a1 expression (B), VEGF expression (C), and Ki67 expression (D) in mice following treatment with human endometrial organoids according to one embodiment in AS-induced mouse endometrium (total number of mice used = 8, 2 per group, triplicate).

[0102] Figure 11 shows the results of qRT-PCR analysis to determine the expression levels of uterine receptivity-related markers (Itgb3 and Spo1) according to the treatment of human endometrial organoids according to one embodiment in normal and AS-induced mouse endometrium.

[0103] Figure 12 is an image (A) and a graph (B) showing the number of implantation sites according to whether human endometrial organoids were treated in an AS-induced mouse uterus according to one embodiment.

[0104] Figure 13 shows the results of qRT-PCR analysis to determine the expression levels of mitochondrial function-related markers (Pgc1a, Nrf1, Mfn1, and Fis1) according to the treatment of GFP mouse-derived endometrial organoids according to one embodiment in normal and AS-induced endometrium. (Total number of mice used = 54, 3 per group (normal / non-transplanted, transplanted) per day; triplicates) (****; p<0.001 normal endometrium vs. non-transplanted endometrium, ####; p<0.001, ###; p<0.005, ##; p<0.01 and #; p<0.05 non-transplanted endometrium vs. organoid-transplanted endometrium, ††††; p<0.001, †††; p<0.005, 쪌; p<0.05 normal endometrium vs. organoid-transplanted endometrium).

[0105] Figure 14 is a schematic diagram showing the structure of an endometrium-on-a-chip used in one embodiment.

[0106] Figure 15 shows the results of measuring the Mitotracker fluorescence intensity (A) and the number of DAPI+ and Mitotracker+ cells in the stromal channel according to whether Tgf-beta was treated after treating the organoid according to one embodiment of the endometrium-on-chip.

[0107] Figure 16 shows the results of confirming the number of GFP+ TOM20+ cells depending on whether or not organoids were processed according to one embodiment.

[0108] Figure 17 shows the results of TMRE (red) immunofluorescence analysis of mouse endometrial organoids treated with FCCP, taxol, and rotenone compared to the untreated normal group. Nuclei were stained with DAPI (blue).

[0109] Figure 18 shows the results of flow cytometry analysis of TMRE, CD326, CD44, and Ki67 in mouse endometrial organoids (FCCP, taxol, and rotenone treated groups, and untreated control group).

[0110] Figure 19 shows the results of immunohistochemical analysis of MT and α-SMA in mouse endometrial organoids (Taxol or rotenone treated and untreated groups).

[0111] Figure 20 shows the results of qRT-PCR analysis of the expression levels of Tnf-a (E), Tgf-β (F), and Col1a1 (G) in mouse endometrial organoids (taxol or rotenone treated and untreated groups).

[0112] Figure 21 shows the results of confirming the expression levels of Timp1, Col1a1, Hk2, Scd1, and Gpr84 through qRT-PCR after co-culturing mitochondria isolated from normal mouse endometrial organoids with mESCs according to one embodiment.

[0113] Figure 22 shows the results of confirming the expression levels of fibrosis-related markers (Timp1, Col1a1) and metabolism-related markers (Hk2, Scd1, Gpr84) through qRT-PCR after transplanting mitochondria isolated from mouse endometrial organoids and normal mouse endometrial organoids according to one embodiment into an AS-induced mouse model.

[0114] Figure 23 shows the results of confirming the growth rate when the endometrial organoids derived from the endometrial tissue of a 20-year-old patient were cultured for 5 to 7 passages with different substances added to the culture medium according to one embodiment.

[0115] Figure 24 shows the results of confirming the growth rate when the endometrial organoids derived from the endometrial tissue of a 20-year-old patient were cultured for 6 to 7 passages with different substances added to the culture medium according to one embodiment.

[0116] Figure 25 shows the results of confirming the growth rate when the endometrial organoids derived from the endometrial tissue of a 36-year-old patient were cultured for 6 to 7 passages with different substances added to the culture medium according to one embodiment.

[0117] Figure 26 shows the results of culture morphology and proliferation rate when endometrial organoids derived from aged mouse uterine tissue were cultured in a medium containing resveratrol and carcin according to one embodiment.

[0118] Figure 27 shows the results of culture morphology and proliferation rate when endometrial organoids derived from mouse uterine tissue that were induced to age by injecting doxorubicin into a medium containing resveratrol and carcin according to one embodiment.

[0119] Figure 28 shows the results showing the degree of proliferation of endometrial organoids derived from uterine tissue according to the concentration of resveratrol or carcin according to one embodiment.

[0120] Figure 29 shows the results of confirming the growth rate according to the composition of the culture medium of endometrial organoids derived from the endometrial tissue of a 20-year-old young patient and a 36-year-old aged patient according to one embodiment.

[0121] Figure 30 shows the results of confirming the expression levels of E-cadherin, an endometrial epithelial cell marker, and Ki67, a cell proliferation marker, in endometrial organoids derived from uterine tissue, with or without the addition of resveratrol or carcin according to one embodiment.

[0122] Figure 31a shows the results of examining the expression level of a senescence marker (p16) in endometrial organoids derived from uterine tissue, with or without the addition of resveratrol or carcin, according to one embodiment.

[0123] Figure 31b shows the results of examining the expression level of a stem cell potential marker (LGR5) in endometrial organoids derived from uterine tissue, with or without the addition of resveratrol or carcin, according to one embodiment.

[0124] Figure 32 shows the results of examining the oxygen consumption of mitochondria in endometrial organoids derived from aging-induced mouse uterus by varying the presence or absence of resveratrol or carcin in the culture medium according to one embodiment.

[0125] Figure 33 shows the results of transplanting endometrial organoids derived from uterine tissue of an aging-induced mouse according to one embodiment into an aging-induced mouse, and then collecting the mouse uterine tissue to check the expression level of aging markers.

[0126] Figure 34 shows the results of confirming the movement of endometrial organoid-derived mitochondria in an in vitro environment after injecting endometrial organoids onto the upper part of the stromal cell layer of a uterine chip using uterine tissue-derived stromal cells.

[0127] Figure 35 shows the results of confirming the movement of endometrial organoid-derived mitochondria in an in vitro environment after injecting endometrial organoids cultured by adding resveratrol or carcin to the upper part of the stromal cell layer of a uterine chip utilizing uterine tissue-derived stromal cells.

[0128] Figure 36 shows the results of confirming the mitochondrial mobility of endometrial organoids derived from aging-induced mouse uterus cultured in a culture medium supplemented with resveratrol or carcin, as determined by the level of COX2 expression.

[0129] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0130] Example 1. Preparation of reagents and experiments

[0131] 1-1. Preparation of human samples

[0132] Endometrial tissue from normal or AS patients was obtained after diagnostic hysteroscopy from the CHA Fertility Center Bundang. Samples were obtained for research purposes with the patients' informed consent. This study was approved by the Institutional Review Board (IRB) of CHA Bundang Hospital (Approval No. 2020-10-007).

[0133]

[0134] 1-2. Preparation for animal testing

[0135] All animal experiments were conducted in accordance with the animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC, Approval Nos. 200193, 210055, 220011, LMO Approval No. LMI 22-852) of CHA University. C57BL / 6 female mice (6–8 weeks old), male mice (7–9 weeks old), BALB / c-nude female mice (7 weeks old), and male mice (7 weeks old) were purchased from Orientbio (Korea). C57BL / 6-Tg (CAG-EGFP strain #:003291) mice (male, 7 weeks old) were provided by Jackson Laboratory (USA). Genotype analyses for line maintenance were performed by tail cutting using a gDNA extraction kit (Bioneer, Korea). Mice were housed in an SPF environment under standard temperature, humidity, and light-controlled conditions (12 h light / 12 h dark).

[0136]

[0137] 1-3. Creation of an Asherman syndrome mouse model

[0138] Female mice were anesthetized with an intraperitoneal injection of 2,2,2-tribromoethanol (Avertin) using a 26G 1 / 2 syringe needle tip bent 2 mm at a 45° angle and inserted into the uterine horn. The endometrium was scraped 20 times in four directions to roughen and bleed the uterine wall, while leaving the perimetrium intact. Mice were sacrificed on days 2, 7, 14, and 21 postoperatively, and the uteri were harvested for model validation.

[0139]

[0140] 1-4. Generation of human endometrial organoids

[0141] Normal endometrial tissue was finely chopped with fine scissors until there were no visible lumps, and cells were isolated by incubating with 50 ug / ml Liberase (Roche, Swiss) in HBSS (Gibco, USA) + 0.5% BSA at 37 °C for 30 min. The enzyme solution was then filtered through a 100 uM strainer, and the epithelial cells filtered out on top were washed with PBS. The epithelial-enriched fraction remaining at the bottom was obtained by centrifugation at 800 rpm for 3 min. The separated epithelial fraction was suspended in DMEM / F12 medium and mixed with Matrigel (Corning, USA) at a ratio of 1:10 (v / v), then mixed with 6 mg / ml TeloCol-6 (Advanced BioMatrix, USA), and a neutralizing solution (Advanced BioMatrix, USA) was added at a ratio of 1:1 (v / v), and finally seeded in a 48-well culture plate. After solidification, the cell droplets were treated with organoid culture medium. The culture medium was changed every 2 days, and passages were performed every 6–7 days.

[0142]

[0143] 1-5. Generation of mouse endometrial organoids

[0144] Mouse endometrial tissues were mechanically and enzymatically dissociated for 10 minutes using TrypLE Express (Gibco, USA) and incubated for 1 hour at 37*?*C with an enzyme solution containing 1.25 IU / ml Dispase II (Sigma, USA) and 0.4 mg / ml Collagenase V (Sigma, USA). After incubation, the solution was filtered through cell strainers to remove the stromal fraction, and the cell strainers were washed with fresh PBS to collect the epithelial fraction. The separated epithelial fraction was suspended in DMEM / F12 medium, mixed with Matrigel (Corning, USA) at a ratio of 1:10 (v / v), and gently seeded into 48-well culture plates. The medium used was DMEM / F12 (Gibco, USA) with 1 (Biogems, USA), -10 μM A83-01 (Peprotech, USA), 1 mM Nicotinamide (Biogems, USA), 50 ng / mL recombinant human EGF (Peprotech, USA), 100 ng / mL recombinant human Noggin (Peprotech, USA), 500 ng / mL recombinant human R-spondin1 (Peprotech, USA), 100 ng / mL recombinant human FGF-10 (Peprotech, USA), and 50 ng / mL recombinant human HGF1 (Peprotech, USA) were treated to prepare the medium.

[0145] Afterwards, the above cells were treated with organoid culture medium and cultured at 37°C in 5% CO2.

[0146]

[0147] 1-6. Cell culture

[0148] Mouse endometrial stromal cells (mESC) were obtained from the dissociated stromal fraction. The filtered enzyme and stromal cell mixture (filtrate) was treated at 1500 rpm for 5 minutes. After removing the supernatant, the pellet was resuspended in DMEM / F12 medium supplemented with 20% FBS (Gibco, USA), 1% L-glutamine (Gibco, USA), and 1% penicillin-streptomycin (Gibco, USA). The culture medium was changed daily, and cells were subcultured when confluent using 0.25% trypsin-EDTA (GenDEPOT, USA). Cells from the first to fourth passages were used in the experiment.

[0149]

[0150] 1-7. Intrauterine transplantation of organoids and isolated mitochondria into AS mice.

[0151] Two days after AS modeling, female mice were anesthetized and a small abdominal incision was made to access the uterine cavity. Organoids (1x10 6 Cells) were slowly transferred into a small niche created in the uterus using a 31-gauge syringe. All mice were sacrificed on days 7, 14, and 21 after transplantation for further analysis. Mitochondria were isolated using a mitochondrial isolation kit (Thermo, USA) according to the manufacturer's protocol.

[0152]

[0153] 1-8. Histology and immunohistochemistry

[0154] Tissues and organoids were fixed with 4% paraformaldehyde and embedded in paraffin, respectively. Paraffin-embedded sections (5 μm) were deparaffinized with Histoclear (National Diagnostics, USA), rehydrated through a descending ethanol gradient (100–70), and stained with H&E and Masson's trichrome staining kit (Agilent, USA). For immunohistochemistry, antigen retrieval was performed in a microwave for 30 min in sodium citrate buffer (pH 6). Samples were blocked overnight at 4°C with primary antibodies against α-SMA (Abcam, UK), MUC1 (Abcam, UK), STEM121 (Takara, Japan), h-MITO (Novus, USA), COL1A1 (Santacruz, USA), and VEGF (Invitrogen, USA), and then incubated with biotinylated goat anti-rabbit or mouse secondary antibodies (Vector laboratories, USA) for 1 h at room temperature. The localization of each protein was confirmed by diaminobenzidine tetrachloride reaction (Vector laboratories, USA).

[0155]

[0156] 1-9. Immunofluorescence and microscopy

[0157] CD31 (Abcam, UK), Ki67 (BD Biosciences, USA), E-CADHERIN (Santacruz, USA), CK14 (Abcam, UK), ER (GeneTex, USA), TOM20 (Abcam, UK), and GFP (Santacruz, USA) were detected in fixed and paraffin-embedded endometrial tissues or organoid sections using immunofluorescence. Finally, nuclei were stained with DAPI (Sigma, USA). Images were observed with a Mica microhub and Thunder imager (Leica Microsystems, Germany) and analyzed with LAS X software (Leica Microsystems, Germany).

[0158]

[0159] 1-10. Quantitative RT-PCR-based mRNA expression analysis

[0160] The whole uterus was homogenized with Labozol (Cosmo, Korea), and complementary DNA for the target gene was synthesized using a cDNA synthesis kit (Takara, Japan). RT-qPCR was performed on the cDNA using SYBR Green (Enzynomics, Korea) to assess the expression of the gene of interest. Experimental gene expression data were normalized to the housekeeping genes Rpl7 and Actb. The primer sequence pairs used in this experiment are listed in Table 1.

[0161]

[0162] 1-11. Flow cytometry

[0163] Cultured organoids were harvested using cold PBS and dissociated into single cells by incubation with TrypLE Express. Single-cell suspensions were washed with FACS buffer (5% BSA in 0.1 X PBS) and stained with anti-CD326 (APC / human; Miltenyi, Germany), anti-CD326 (APC / mouse; Miltenyi, Germany), anti-CD44 (APC / mouse; Miltenyi, Germany), and anti-rat IgG2b (APC; Miltenyi, Germany), anti-mouse IgG1 (PE; Miltenyi, Germany), anti-human IgG1 (APC; Miltenyi, Germany), and anti-human IgG1 (PE; Miltenyi, Germany) for 30 min. In addition, staining for LGR5 was also performed. Cells were then washed three times and finally resuspended in 400 μl of FACS buffer. All samples were tested on a Cyto-FLEX-Analyzer (Beckman Coulter, USA) and data were analyzed using CytExpert software (Beckman Coulter, USA).

[0164]

[0165] 1-12. Fertility Assessment

[0166] AS-induced mice were mated overnight with males of the same strain at a 1:1 ratio. The following morning, vaginal plugs were observed, defining day 1 of pregnancy. Uterines were harvested on day 14 of pregnancy, and the total number of implantation sites, fetal and placental weights were recorded. Transplanted mice were mated 21 days after transplantation, and fertility was assessed using the same method as described above.

[0167]

[0168] 1-13. Loading my hydrogel and cells onto the endometrium-on-a-chip

[0169] A microfluidic device that reproduces the endometrial structure was designed and fabricated. HUVECs and mESCs (5 x 10 ) were cultured in a fibrin gel solution containing 2.5 mg / mL fibrinogen (Sigma, USA) and 0.15 U / mL aprotinin (Sigma, USA). 6 cells / ml) were mixed and rapidly loaded into each channel. The device was cultured at 37°C with 5% CO2. To reproduce AS conditions, the device was treated with EBM basal medium (Lonza, Switzerland) with or without 5 ng / ml TGF-β (R&D Systems, USA) for 24 h. The upper reservoir of the device was filled with EGM2 medium (Lonza, Switzerland), and the lower reservoir was gently aspirated to wet the hydrophobic medium channel. The next day, mouse endometrial organoids stained with Mitotracker (Invitrogen, USA) were placed in the stromal channel as an epithelial layer (6 × 10 6 cells / mL). The device was rotated 90º for 30 min with the matrix channel facing downward to evenly distribute the organoid layer on the gel surface. The matrix channel was filled with organoid culture medium, and the endothelial channel was filled with EGM2 medium supplemented with 1 nM sphingosine 1-phosphate (Sigma, USA), 100 ng / ml VEGF-A (R&D Systems, USA), 50 ng / ml VEGF-C (R&D Systems, USA), and 50 ng / ml bFGF (R&D Systems, USA). The device was then fixed at 0 h, 1 h, and 24 h after organoid loading. Images were observed with a Zeiss LSM880 confocal microscope (Carl Zeiss, Germany) and further analyzed with ZEN black software (Carl Zeiss, Germany).

[0170]

[0171] 1-14. Transmigration Analysis

[0172] To identify the transferred mitochondria, CRL-4003 (2 x 10 5 cells / ml) were seeded in a 24-well plate and cultured at 37°C for 24 hours. Afterwards, the cultured cells were seeded in the upper compartment of a transwell (Corning, USA) to determine the mouse endometrial organoid untreated group and mouse endometrial organoid (1.5 x 10 6 cells / ml) were maintained as a treatment group for 48 hours.

[0173]

[0174] 1-15. Digital RT-PCR analysis

[0175] RT-dPCR analysis was performed to detect small copy number changes. Two micrograms of cDNA, 400 nm of each primer, and 13.3 μl of 4x EG PCR master mix (Qiagen, Germany) were mixed in a final volume of 40 μl. The mixture was prepared in a Nanoplate 26k 24-well (Qiagen, Germany) and subdivided into 26,000 partitions. The experiment was performed using a Qiacuity One 5plex (Qiagen, Germany). The fluorescence signal of each sample was detected and calculated by automatically setting the threshold compared to the non-template control (NTC / negative control).

[0176]

[0177] 1-16. Mitochondrial membrane potential assessment

[0178] To induce resident mitochondrial dysfunction in mouse endometrial organoids, FCCP (Abcam, USA), Taxol (Selleckchem, USA), and Rotenone (Sigma, USA) were applied. Organoids (1x10 6(cells / well) were treated with FCCP (25 μM, 24 h), Taxol (10 nM, 24 h), and Rotenone (100 nM, 48 h). After treatment, the organoids were dissociated into single cells and then incubated with TMRE (Abcam, USA) at a final concentration of 250 nM for 30 min. Active mitochondria were detected using a Cyto-FLEX-Analyzer at 488 nm for excitation and 575 nm for emission.

[0179]

[0180] 1-17. Co-culture of endometrial organoid-derived mitochondria and mESCs

[0181] To investigate the recovery of mitochondrial-mediated metabolism in AS state, mouse endometrial stromal cells (mESCs (5x10 4 A group was prepared by treating mESCs with 5 ng / ㎖ of TGF-β for 24 hours and a group was prepared by not treating them. Afterwards, mitochondria isolated from mouse endometrial organoids were added to the mESCs and maintained for 48 hours.

[0182]

[0183] 1-18. Measuring ATP levels

[0184] ATP levels were measured using a luminescent ATP detection assay kit (Abcam, USA) according to the manufacturer's instructions. Briefly, co-cultured samples were gently collected and centrifuged. Purified islets were lysed, and a luciferin-luciferase solution was added. After incubation, intracellular ATP concentrations were measured using a Spectramax ID5 (Molecular Devices, USA).

[0185]

[0186] 1-19. Statistical Analysis

[0187] Comparison groups were analyzed using unpaired t-tests for parametric distributions. For multiple comparisons, either a regular one-way ANOVA analysis using Dunnett's multiple comparisons test or a two-way ANOVA analysis using Tukey's multiple comparisons test was used. In all cases, a P value less than 0.05 was considered statistically significant (P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****)).

[0188] Example 1. Preparation of Asherman's syndrome (AS) mouse model

[0189] In this example, in order to confirm the effect of the composition according to one embodiment on alleviating or improving endometrial damage, an animal model was prepared as described in the above 'Preparation of an Asherman's syndrome mouse model', and its characteristics were compared with those of human AS patients.

[0190]

[0191] 1-1. Comparison of Characteristics of Mouse AS Models and Human AS Patients

[0192] Specifically, endometrial tissues were obtained from normal or AS patients, and biopsies were performed to assess subfertility complaints. The AS patient was a 38-year-old nulligravid woman with a history of abortion (one abortion) and was diagnosed with RIF (five failed implantations). Hysterosalpingography (HSG) and transvaginal ultrasound (TVS) images did not reveal congenital uterine anomalies or tubal ligation, but hysteroscopy images revealed severe intrauterine adhesions in the uterine lumen. To mimic the endometrial condition of these AS patients in mice, an experimentally induced AS mouse model was established by inducing physical trauma through repeated mechanical curettage of the mouse uterine horns. To verify successful modeling, mouse uterine tissues obtained 2, 7, 14, and 21 days after AS induction were compared with endometrial tissue from AS patients. To control for issues related to variable estrous cycles, one side of the uterine cavity was induced with AS, while the other side served as a control.

[0193] As a result, as shown in Fig. 1, both human AS patients and AS-induced mouse models exhibited increased collagen accumulation compared to normal tissues, and activated fibroblasts in the endometrium showed highly expressed blue staining for Masson's trichrome (MT) and α-SMA. In addition, the number of Ki67-positive cells was drastically reduced (Fig. 1). These results confirmed that the AS-induced mouse model according to one embodiment exhibited characteristics similar to those of human AS patients, excellently reproducing the AS condition of human patients.

[0194]

[0195] 1-2. Characteristics Evaluation of the Mouse AS Model

[0196] In addition, to confirm AS induction in a mouse AS mouse model that reproduces the characteristics of human AS patients as described above, the expression levels of AS-related factors in the endometrium were measured.

[0197] Specifically, the expression levels of Tgfb1, Timp1, and Col1a1, which are molecular markers of fibrosis, were measured, and it was confirmed that their levels increased after AS induction (Fig. 2). In addition, it was observed that the number of embryo implantation sites recorded on day 14 of pregnancy was significantly reduced compared to the control mice (p=0.0040; number of mice=6; number of implantation sites=44). Although the weight of the fetuses was observed to be similar in the control and AS-induced endometria (excluding embryo resorption), morphologically delayed fetuses, placentas, and resorption sites were observed in the AS mice (Figs. 3 and 4). These characteristics are observed in AS patients, and these results confirmed that the mouse AS model manufactured according to one embodiment can well reproduce the AS condition.

[0198]

[0199] Example 2. Production and transplantation of endometrial organoids

[0200] In this example, after manufacturing an endometrial organoid according to one embodiment, the following experiment was performed to confirm whether successful engraftment was achieved by treating the endometrial organoid with an AS-induced mouse model.

[0201]

[0202] 2-1. Manufacturing of endometrial organoids

[0203] Human endometrial organoids were prepared by biopsy of endometrial tissue from four patients diagnosed with normal endometrial status admitted to the Infertility Center of Bundang CHA Hospital. The normal endometrial tissue was mechanically and enzymatically dissociated and filtered to include the epithelial cell-rich fraction. The obtained isolates were then embedded with collagen droplets in endometrial organoid culture medium to induce self-organization into organoid-like structures. The organoids prepared in this manner expanded well and were passaged up to six times, demonstrating a 3.10-fold increase in cell number. Furthermore, the organoids expressed CD326 (EpCAM) (a luminal and glandular epithelial marker) and CD44 (a stem cell marker) (Fig. 5a), and the number of positively stained cells increased with increasing passage number (CD326, 5.00-fold; CD44, 2.25-fold).

[0204]

[0205] 2-2. Confirmation of transplantation of endometrial organoids

[0206] To confirm whether the organoids manufactured in this way successfully engrafted into the endometrium, human endometrial-derived organoids (1x10) manufactured as in Example 2-1 were 6 Cells) were transplanted into one side of the AS endometrial cavity of AS-induced BALB / c-nude mice, and the contralateral side without transplantation was used as a control. The organoids used for transplantation were well expanded, showing 84.84% (±11.16) of CD326-positive staining and 67.6% (±9.40) of CD44-positive staining (Figs. 5a and 5b). In addition, when the expression level of LGR5, a stem cell marker, was additionally checked, as shown in Fig. 5c, LGR5 expression was also observed, confirming that they also had characteristics of stem cells.

[0207] In addition, when the expression of MUC1, CDH1, ERα, and Ki67 markers, which are characteristic expression factors of the endometrium, was observed in organoids by immunofluorescence staining, it was shown that the expression was maintained in the organoids or rather increased, as shown in Fig. 5d.

[0208] For more efficient transplantation, the organoids were mixed with Matrigel at a 1:1 ratio 2 days after AS induction and transplanted into a mouse AS model. Twenty-one days after transplantation, DAB intensities of human mitochondria (h-MITO) and cytoplasm (STEM121) were measured, confirming successful engraftment of the human endometrial organoids into the recipient mouse endometrium (Fig. 6).

[0209] In addition, when mouse endometrial organoids were prepared from the uterine tissue of C57BL / 6-Tg(CAG-EGFP) 1Osb / J mice, cell expandability was observed to increase 6.7-fold up to passage 15, and most cells expressed CD326 (99%) and CD44 (97%) (Fig. 8a), confirming that epithelial progenitor cells were enriched during the culture period. In addition, immunofluorescence analysis of mouse endometrial organoids (Fig. 8b) showing 94.3% (±3.7) of CD326 and 70.6% (±14.4) of CD44 positivity showed a significantly higher GFP-positive signal level in the endometrium compared to the non-transplantation group (NT), confirming successful transplantation of the organoids (Fig. 9).

[0210]

[0211] Example 3. Confirmation of the effect of endometrial organoids on improving endometrial damage.

[0212] In this example, to confirm the endometrial damage improvement effect of the endometrial organoid according to one example, the following experiment was performed.

[0213]

[0214] 3-1. Confirmation of the effect of endometrial organoids on improving AS symptoms.

[0215] Specifically, human endometrial organoids were transplanted into one side of the endometrial cavity of AS-induced BALB / c-nude mice, and the uterus of the mice was removed on day 21 after transplantation to perform immunohistochemical analysis.

[0216] As a result, we confirmed that the excessive collagen accumulation level of AS-induced mouse endometrium was restored to the normal level through transplantation of human endometrial organoids (accumulated collagen volume: 0.49-fold, COL1A1: 0.52-fold) (Fig. 10, A and B). In addition, we confirmed that the reduced expression levels of vascular endothelial growth factor (VEGF) and Ki67 in AS-induced mouse endometrium were restored to the levels observed in normal endometrium (VEGF: 1.64-fold, Ki67: 1.68-fold) (Fig. 10, C and D). In addition, we confirmed that the mRNA expression levels of endometrial receptivity-related markers (Itgb3, Spp1) were also significantly increased to levels similar to those detected in normal endometrium (Itgb3, 34.15-fold; Spp1, 53.02-fold) (Fig. 11). In addition, we observed a significant increase in the number of implantation sites in AS-derived uteri transplanted with human endometrial organoids compared to AS-derived uteri not transplanted (Fig. 12).

[0217] Furthermore, transplantation of mouse endometrial organoids, similar to transplantation of human endometrial organoids, showed a significant decrease in collagen accumulation and recovery of fibrotic lesions. In addition, revascularization and re-proliferation of the endometrium were confirmed by an increase in CD31 and Ki67 expression. In addition, a significant increase in the total number of implantation sites (p=0.0075) was confirmed compared to the non-transplanted uterus.

[0218] In summary, the above results show that treating endometrial organoids with damaged endometrium resulted in a decrease in increased collagen deposition, an increase in vascular endothelial growth factor expression, an increase in Ki67 expression, an increase in endometrial receptivity-related gene expression, and an increase in the number of implantation sites. Through these results, it was confirmed that treating endometrial organoids according to one embodiment can improve the endometrial environment damaged by AS into an environment suitable for embryo implantation and pregnancy, thereby showing an effect of improving AS symptoms.

[0219] 3-2. Confirmation of the effect of endometrial organoids on improving mitochondrial function in the endometrium.

[0220] In this example, in order to confirm that the endometrial-derived organoid according to one embodiment can exhibit an effect of improving AS symptoms by improving mitochondrial metabolic activity in the endometrium, the following experiment was performed.

[0221] Specifically, GFP-mouse endometrial-derived organoids were transplanted into AS-induced recipient endometrium, and changes in the expression levels of mitochondrial function-related genes (including Pgc1a, Nrf1, Mfn1, and Fis1) were compared with those of normal endometrium or non-transplanted AS-induced endometrium on days 7, 14, and 21 after transplantation.

[0222] As a result, as shown in Fig. 13, a significantly higher level of Pgc1α was detected in the non-implant AS-induced endometrium on day 7 compared to the normal endometrium (3.17-fold), and then decreased back to the level of the normal endometrium. In general, Pgc1α, a master regulator of mitochondrial biogenesis, tends to decrease in a fibrogenic state. Based on this background, the results of Fig. 13 may suggest a selective mechanism that preferentially responds to acute damage induced by physical trauma rather than dynamic changes in mitochondrial biogenesis in response to fibrosis progression in the early stage of AS induction (day 7), and it was confirmed that Pgc1α was down-regulated to respond to the chronic state as the inflammatory response persisted thereafter. In comparison, when endometrial organoids were transplanted, we found that Pgc1a expression in the recipient endometrium decreased (0.15-fold) initially (7 days after transplantation), but increased after 14 and 21 days after transplantation (day 14, 2.88-fold; day 21, 2.60-fold), confirming that the cellular activity of AS endometrium was restored to normal through endometrial organoid transplantation. In addition, Nrf1, a key transcription factor regulating mitochondrial function, was rapidly increased in AS endometrial organoid-transplanted endometrial endometrium on day 7 after transplantation, and almost no difference in Nrf1 expression was observed in all groups on days 14 and 21. Similarly, Mfn1, a representative marker of mitochondrial fusion, also showed a similar expression pattern, whereas Fis1, a representative marker of mitochondrial fission, showed an exactly opposite expression pattern. Specifically, in AS endometrium on day 7, we observed an exceptional increase (4.72-fold) in the mRNA levels of Mfn1 and a decrease (0.67-fold) in Fis1 compared to normal endometrium, and the expression of Mfn1 and Fis1 showed an opposite trend after endometrial organoid transplantation (Mfn1, 0.61-fold; Fis1, 2.29 times) (Fig. 13). In mammals, the number of mitochondria is controlled by removing dysfunctional mitochondria through mitophagy through the dynamic balance between fission and fusion. In light of this, the above results suggest that inducing AS through repetitive mechanical trauma upsets the mitochondrial function of the recipient endometrium, and subsequent endometrial organoid transplantation restores the balance of mitochondrial fission and fusion, thereby removing dysfunctional mitochondria and restoring them to normal.

[0223]

[0224] 3-3. Confirmation of transfer of organoid-derived mitochondria into endometrial organoids

[0225] In addition, to determine whether the above-described effects in the transplanted endometrium were induced by the transfer of mitochondria derived from endometrial organoids, the following experiments were performed.

[0226] Specifically, to recapitulate the stratified structure of the endometrium, a 3D microfluidic system was fabricated that mimics the vascularized endometrial stromal layer, including stroma (mouse endometrial stromal cells, mESCs) and endothelial channels (human umbilical vein endothelial cells) (Fig. 14). To mimic AS-induced fibrosis, mESCs were treated with TGF-β for 24 h, and increased COL1A1 expression was observed. Mitotracker-stained organoids were then loaded onto the stromal channels to recapitulate in vivo endometrial organoid transplantation (Fig. 14). Mitotracker fluorescence intensity was measured using Image J to detect organoid-derived mitochondrial movement. The movement of endometrial organoid-derived mitochondria was observed in the stromal channel 1 hour after endometrial organoid loading, and after 24 hours, significantly more active migration was observed in the TGF-β-treated mESC layer, with a 2.18-fold higher Mitotracker fluorescence intensity compared to TGF-β-untreated mESCs (Fig. 15A). Furthermore, a greater number of Mitotracker-stained mitochondria were detected in TGF-β-treated stromal cells, indicating that the transferred mitochondria preferentially migrated toward damaged cells over normal cells (Fig. 15B). Furthermore, strong co-localization of GFP and TOM20 signals in GFP mouse-derived endometrial organoid-derived mitochondria confirmed successful transplantation into the recipient endometrium 21 days after organoid transplantation (Fig. 16).

[0227] In addition, to confirm the migration of endometrial organoid-derived mitochondria, a transwell migration assay was performed by treating mouse endometrial organoids and human endometrial stromal cells (CRL-4003) to the upper and lower chambers, respectively. After 48 hours of co-culture, the levels of mouse nuclear DNA (mNuDNA) and mitochondrial DNA (mMtDNA) within the stromal layer were measured using digital polymerase chain reaction (PCR).

[0228] As a result, we observed a significant increase in mMtDNA levels in the lower chamber (1.27x10 4 (pear), mitochondria from co-cultured endometrial organoids were confirmed to migrate to the stromal layer of the lower chamber. Furthermore, no difference in mNuDNA levels was observed between single-cultured and co-cultured mouse endometrial organoid stromal cells, confirming mitochondrial transfer rather than organoid itself.

[0229] Through this, it was confirmed that a pharmaceutical composition including an endometrial organoid according to one embodiment can exhibit an effect on the endometrium by transferring mitochondria in the organoid into the endometrium of the subject.

[0230]

[0231] 3-4. Confirmation of the effect of transfer of mitochondria derived from endometrial organoids.

[0232] To determine whether mitochondria transferred from transplanted endometrial organoids to AS-induced endometrium mediate anti-fibrotic effects in the endometrium of recipient mice, experiments were performed as follows.

[0233] Specifically, after treating endometrial organoids with mitochondrial function inhibitors Taxol and Rotenone, the inhibition of organoid-derived mitochondrial function was confirmed by measuring the penetration status of tetramethyl rhodamine ethyl ester (TMRE) through immunofluorescence and flow cytometry.

[0234] As a result, a decrease in TMRE expression was observed in inhibitor-treated endometrial organoids, which showed TMRE levels similar to those observed in FCCP (positive control, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone))-treated organoids (Fig. 17), confirming that mitochondrial function in endometrial-derived organoids was suppressed by taxol and rotenone treatment. In addition, CD326, CD44, and Ki67 levels were observed to be maintained after inhibitor treatment, confirming that only mitochondrial function, not other important features of the organoids, was suppressed (Fig. 18).

[0235] Next, we transplanted endometrial organoids with dysfunctional mitochondria into AS-induced endometrial cavities and compared their therapeutic effects with those of organoids with functional mitochondria. Immunohistochemical analysis of MT and α-SMA revealed that the endometrium transplanted with Taxol- or rotenone-treated endometrial organoids did not recover the endometrial state, exhibited dense collagen deposition throughout the endometrium, and exhibited significantly higher expression levels of Tnfa, Col1a1, and Tgfb1 compared to the control (inhibitor-free) endometrial organoid-transplanted endometrium (Figure 20), which was similar to that observed in non-transplanted AS endometrium. This indicates that endometrial organoids with mitochondrial dysfunction attenuate the regenerative therapeutic effects of organoid transplantation.

[0236] These results led us to further investigate the specific role of transplanted endometrial organoid-derived mitochondria in the recovery of AS-induced endometrial fibrosis. Mitochondria isolated from endometrial organoids were co-cultured with mESCs pre-treated with Tgf-β in vitro for 48 h. As a result, compared to the group not co-cultured with mitochondria, the increase in Timp1 and Col1a1 expression levels induced by Tgf-β treatment was significantly reduced in the co-cultured cells. Furthermore, the sudden increase in Hk2 (an important factor in glycolysis) induced by Tgf-β treatment was stabilized, and the rapid increase in the expression levels of Scd1 and Gpr84 (lipid metabolism markers) induced by Tgf-β treatment was restored to the levels observed in normal mESCs (non-Tgf-β-pretreated), with no significant change in Scd1, and ATP production levels were also restored (Fig. 21). To further verify these results in vivo, mitochondria isolated from mouse endometrial organoids were administered into the AS-damaged uterine cavity, and maternal endometrial organoids were administered into the contralateral uterine cavity as a control (Fig. 22). Comparing the organoid-derived mitochondria-transplanted endometrium with the maternal organoid-transplanted endometrium, the mRNA expression levels of Timp1 and Col1a1 were similar in both groups, which were significantly reduced compared to the non-transplanted AS-induced endometrium (Fig. 23). In addition, administration of organoid-derived mitochondria or organoids resulted in a significant decrease in the levels of glycolytic markers (Hk2) and lipid metabolism markers (Scd1 and Gpr84), confirming that the changes in mitochondrial metabolism were balanced (Fig. 23).

[0237] Through the above results, it was confirmed that a pharmaceutical composition containing the endometrial organoids can exhibit an endometrial damage prevention or treatment effect by transferring mitochondria within the endometrial organoids into the endometrium. Furthermore, it was confirmed that mitochondria isolated from the endometrial organoids can exhibit an endometrial damage prevention or treatment effect.

[0238]

[0239] Example 4. Production of organoids and confirmation of organoid cell viability according to treatment with a composition for promoting organoid culture.

[0240]

[0241] 4-1. Organoid production

[0242] 1) Isolation of cells from human endometrial tissue

[0243] 2) The separated cells are constructed in three dimensions using a Matrigel scaffold.

[0244] 3) Culturing human endometrial organoids by adding organoid culture medium

[0245]

[0246] 4-2. Preparation of a composition for promoting organoid production

[0247] In this example, in order to confirm the efficiency of promoting the proliferation of organoid cells by treating the composition for promoting organoid culture according to one embodiment, an organoid culture composition having a composition as shown in Table 2 below was prepared. In addition, the organoid culture medium prepared above was used as the basic medium (Control) for the organoid culture. 1% P / S (Invitrogen, USA) was added as an antibiotic. The medium was treated in an amount of 5 to 10 times the volume of the cell and ECM mixture and cultured.

[0248] Control Example 4-1 Example 4-2 Example 4-3 Example 4-4 Example 4-5 Basic medium OOOOOO Resveratrol (RES) - 1 nM - 1 nM 1 nM Platelet-derived growth factor (PDGF) - 1 ng / ml - 1 ng / ml Insulin-like growth factor 1 (IGF1) - 1 ng / ml -

[0249] Example 5. Confirmation of the effect of organoid cell proliferation according to the composition for promoting organoid culture.

[0250] In this example, the following experiment was performed to confirm the efficiency of organoid cell proliferation according to the treatment of the composition for promoting organoid culture according to one example.

[0251] Specifically, the three-dimensional cell aggregate according to Example 1 was treated with the composition according to Example 2. To confirm cell viability, after treatment with TrypLE for 15 min, Matrigel was removed, washed twice with cold PBS, and then the organoids were finely fragmented by pipetting 10 to 30 times. Dead cells were stained with tryphan blue, and the number of cells was counted using a hematocytometer.

[0252] As a result, as shown in FIGS. 23 to 25, it was confirmed that cell proliferation was significantly increased by treatment with the composition for promoting organoid culture according to one embodiment. In particular, as shown in FIG. 23, when a composition containing PDGF or RES was treated to organoids derived from the endometrial tissue of a 20-year-old patient, it was confirmed that the cell proliferation ability was significantly increased with excellent efficiency. In addition, as shown in FIG. 24, when a composition containing RES alone or a composition containing RES and PDGF together was treated to organoids derived from the endometrial tissue of a 20-year-old patient, it was confirmed that the cell proliferation ability was significantly increased with excellent efficiency. In addition, as shown in FIG. 25, when a composition containing RES alone or a composition additionally containing PDGF or IGF1 was treated to organoids derived from the endometrial tissue of a 36-year-old patient, it was confirmed that the cell proliferation ability was significantly increased with excellent efficiency.

[0253] Through these results, it was confirmed that the composition according to one embodiment can significantly increase the production efficiency of human endometrial-derived organoids.

[0254]

[0255] Example 6. Confirmation of organoid function according to organoid culture composition

[0256] 6-1. Confirmation of organoid proliferation ability according to the presence and concentration of additives

[0257] In this example, experiments were conducted to determine the effect of enhancing the functionality of organoids by treating them with a composition for promoting organoid culture according to one embodiment. Specifically, as described in the above example, advanced DMEM or DMEM was used for culture, and antibiotics such as primocin and penicillin-streptomycin were additionally added.

[0258] Additives to enhance function were selected from the group consisting of resveratrol (RES), casin (Ca), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF1), and finally, resveratrol and casin were selected to conduct the experiment.

[0259] As a result, as shown in Fig. 26, when resveratrol and carcin were added separately or together and cultured, the proliferation level was confirmed to be significantly increased compared to the control group (existing culture medium without additives) compared to endometrial organoids cultured in a culture medium that did not contain the substances. In addition, when endometrial organoids derived from mice that were induced to age by injection of doxorubicin were cultured by adding resveratrol and carcin separately or together, the proliferation level was also significantly increased (Fig. 27).

[0260] These results confirmed that endometrial organoids cultured in a culture medium supplemented with one or more additives, particularly resveratrol and carcin, showed excellent proliferation effects even in a damaged or aged endometrial environment with poor endometrial function.

[0261] In addition, when these effects were performed by culturing with different concentrations of resveratrol and carcin, it was confirmed that the proliferation ability significantly increased even at low doses, where the resveratrol concentration was set at 1 nM to 100 nM and the carcin concentration was set at 1 nM to 10 nM. However, it was confirmed that the proliferation ability actually decreased under high dose conditions, where resveratrol was 10 uM or higher and carcin was 100 nM or higher (Fig. 28).

[0262]

[0263] 6-2. Confirmation of the effect of improving the proliferation and function of organoids according to the presence or absence of additives.

[0264] To confirm the effect of enhancing organoid proliferation capacity according to the additives identified in Example 6-1 above, experiments were conducted on endometrial organoids derived from the uterine tissue of a 20-year-old young patient and a 36-year-old old patient, respectively. Specifically, during the process of culturing endometrial organoids, proliferation capacity was confirmed by treating one or more additives selected from the group consisting of RES, IGF1, and PDGF.

[0265] As a result, as shown in Figure 29, it was confirmed that the proliferative capacity of endometrial organoids derived from uterine tissue of an old patient was restored to a level similar to that of organoids prepared from tissue derived from a young patient.

[0266] To further confirm these results, organoids prepared from mouse uterine tissue induced to age through doxorubicin injection were cultured with resveratrol (R) and Ca, either individually or in combination. The cultured organoids exhibited significantly enhanced epithelial cell characteristics and cell proliferation potential compared to the control group (Fig. 30). Furthermore, senescence markers were significantly reduced (Fig. 31a), and stem cell potential was significantly increased (Fig. 31b).

[0267] Furthermore, when resveratrol and carcin were added individually or together, mitochondrial oxygen consumption was significantly increased, as shown in Figure 32. Therefore, it was confirmed that the addition of these substances can also enhance mitochondrial function within organoids.

[0268]

[0269] Example 7. Confirmation of the effects of culture medium additives on organoids through in vivo transplantation of organoids.

[0270] We conducted experiments to determine whether endometrial organoids exhibited superior function and activity after in vivo transplantation. To this end, endometrial organoids derived from young mouse uterine tissue or mouse uterine tissue induced to age by doxorubicin injection were transplanted into the uterine cavity of mice induced to age by doxorubicin injection. Furthermore, endometrial organoids cultured with resveratrol and carcin, either individually or together, were transplanted into the uterine cavity to determine the expression levels of senescence markers.

[0271]

[0272] When comparing the expression of senescence markers in uterine tissues of senescent mice transplanted with endometrial organoids, as shown in Figure 33, the experimental group transplanted with senescent endometrial organoids cultured by treating resveratrol and carcin, either individually or together, showed a decrease in the expression of senescence markers, similar to when young mouse endometrial organoids were transplanted. On the other hand, when endometrial organoids derived from senescent mice were injected into the endometrium of senescent mice, the expression of senescence markers increased compared to the non-transplantation group. Through these effects, it was confirmed that aging can also be improved when organoids are cultured by treating them with resveratrol and carcin.

[0273]

[0274] Example 8. Confirmation of the effect of organoids according to culture medium additives.

[0275] 8-1. In vitro uterine chip transplantation experiment

[0276] After setting up a uterine chip using uterine tissue-derived stromal cells, endometrial organoids were injected on top of the stromal cell layer and the endometrial organoids were transplanted into the uterine chip in vitro.

[0277] When mitochondrial movement was confirmed in endometrial organoids transplanted onto the uterine chip, as shown in Figure 34, mitochondrial movement was confirmed. Furthermore, mitochondrial mobility was also confirmed in endometrial organoids cultured with resveratrol and carcin, either individually or together. As a result, as shown in Figure 35, it was confirmed that the mitochondrial mobility of organoids cultured with the above additives significantly increased. Specifically, endometrial organoids derived from uterine tissue of aged mice also showed improved mitochondrial mobility to the level of organoids derived from uterine tissue of young, normal mice.

[0278]

[0279] 8-2. In vivo intrauterine transplantation experiment

[0280] Similar effects to Example 8-1 were observed when endometrial organoids were transplanted into the uterine cavity of aged mice. Specifically, endometrial organoid-derived mitochondria were found in the uterine tissue of the transplanted mice. Furthermore, it was confirmed that the amount of mitochondria detected was significantly higher when endometrial organoids cultured in a medium supplemented with resveratrol and carcin, either individually or together, were transplanted.

[0281] Through the above results, it was confirmed that the endometrial organoids of one type function properly even through in vivo transplantation, and that this effect was enhanced through culture using a culture medium containing the above additives, and among them, it was confirmed that the effect was significantly increased when resveratrol and carcin were added.

[0282]

[0283] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for promoting endometrial organoid culture, comprising a basic medium, antibiotics, and growth factors.

2. In claim 1, The composition comprises Epidermal Growth Factor (EGF), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Fibroblast Growth Factor 10 (FGF10), Noggin (BMP inhibitor protein), R-spondin1 (Roof plate-specific spondin 1), R-spondin3 (Roof plate-specific spondin 3), Hepatocyte Growth Factor (HGF), Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF), Vascular Endothelial Growth Factor (VEGF), CHIR (CHIR99021, a GSK-3 inhibitor) and WNT3a (Wingless-type A composition for promoting endometrial organoid culture, comprising at least one growth factor selected from the group consisting of MMTV integration site family, member 3a).

3. In claim 1, A composition for promoting endometrial organoid culture, wherein the composition further comprises at least one selected from the group consisting of resveratrol (RES), casin (Ca), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF1).

4. In claim 3, The composition above is a composition for promoting endometrial organoid culture, comprising resveratrol (RES) and casin (Ca).

5. In claim 4, The composition is a composition for promoting endometrial organoid culture, comprising resveratrol at a concentration of 0.1 nM to 500 nM.

6. In claim 4, The composition is a composition for promoting endometrial organoid culture, comprising carcin at a concentration of 0.1 nM to 50 nM.

7. In claim 1, the composition for promoting endometrial organoid culture increases the cell proliferation capacity of organoids derived from the endometrium of a human patient. 8.a) A step of obtaining cells from normal endometrial tissue; b) obtaining a fraction rich in epithelial cells from the separated cells; and c) A method for producing an endometrial organoid, comprising the step of culturing the fraction with an organoid culture promoting composition according to any one of claims 1 to 5 to obtain an endometrial organoid.

9. In claim 8, A method for producing an endometrial organoid, wherein the step of obtaining cells from the above tissue is obtained through mechanical dissociation, enzymatic dissociation, or a combination thereof.

10. In claim 8, A method for producing an endometrial organoid, wherein the method further comprises a step of attaching the endometrial organoid obtained in step c) to a seed on a three-dimensional scaffold.

11. A normal endometrial organoid manufactured by the manufacturing method of claim 8.

12. A pharmaceutical composition for preventing or treating endometrial damage, comprising the normal endometrial organoid of claim 11, wherein the normal endometrial organoid expresses CD326 at 60% or more, CD44 at 60% or more, and LGR5 at 0.5% or more.

13. In claim 12, A pharmaceutical composition, wherein the above normal endometrial organoid is derived from the endometrium of a human or mouse.

14. In claim 12, A pharmaceutical composition for preventing or treating endometrial damage, comprising the normal endometrial organoid or mitochondria isolated from the normal endometrial organoid as an active ingredient.

15. In claim 14, The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of at least one selected from endometrial damage, Asherman syndrome and complications thereof.

16. In claim 15, the pharmaceutical composition exhibits at least one of the following characteristics: a) Reduced levels of collagen accumulation within the endometrium; b) Increased VEGF expression levels; c) Increased Ki67 expression level; d) Increased Itgb3 expression level; e) Increased Spp1 expression level; f) Increased number of implantation sites; g) Reduced expression levels of fibrosis-related factors; h) reduction in the level of expression of metabolic-related factors; and i) Increased ATP production.

17. A pharmaceutical composition according to claim 16, wherein the fibrosis-related factor is at least one of Tgfb1, Timp1, and Col1a1.

18. A pharmaceutical composition according to claim 17, wherein the metabolic related factor is at least one of Hk2, Scd1, and Gpr84.

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