Method for culturing and transplanting adrenal organoid

Abstract

The present invention relates to a composition for maturation of adrenal organoids, a method for maturation of adrenal organoids using same, and a method for transplantation of organoids prepared by the method. In the present invention, the structurally and functionally mature adrenal organoids prepared under optimized culture conditions using the composition for maturation of adrenal organoids containing adrenocortical hormone and cyclic adenosine monophosphate have been structurally and functionally improved, and it was confirmed that the adrenal organoid chip having a three-dimensional vascular network structure manufactured using the same actually replaces the function of the adrenal gland without side effects after transplantation into a mouse. Accordingly, the present invention can be advantageously used for transplantation or regenerative therapy, drug screening, drug responsiveness evaluation, and the like.

Description

Adrenal organoid culture and transplantation method

[0001] The present invention relates to an adrenal organoid maturation composition, a method for maturating adrenal organoids using the same, and a method for transplanting organoids produced by the method.

[0002] This invention is the result of the research project support below.

[0003]

[0004] [Research Project Number] RS-2021-NR059722

[0005] [Research Project Name] Research on the Production of Bio-Artificial Organs Using Organoid Module Integration and Alignment Technology

[0006] [Host] Yonsei University

[0007] [Research Period] March 1, 2024 - February 28, 2025

[0008]

[0009] [Research Project Number] 20024298

[0010] [Research Project Name] Development of High-Functional Decellularized Materials for the Fabrication and Transplantation of Long-Term Customized Biomimetic Organs

[0011] [Host Organization] SeratGen

[0012] [Research Period] January 1, 2024 - December 31, 2024

[0013]

[0014] [Research Project Number] KFRM 23A0203L1

[0015] [Research Project Name] Advancement of Nanovesicle Therapy Technology Combining Mesodermal and Endothelial Cell Characteristics Derived from the HiPSC-EC Differentiation Process in Patients with Peripheral Arterial Disease

[0016] [Host Organization] Yonsei University Industry-Academic Cooperation Foundation

[0017] [Research Period] January 1, 2024 - December 31, 2024

[0018]

[0019] [Research Project Number] RS-2023-00210680

[0020] [Research Project Title] Development of a 3D avatar system using human-derived adrenal cortex for biotransplantation.

[0021] [Host] Yonsei University

[0022] [Research Period] March 1, 2024 - February 28, 2025

[0023]

[0024] [Research Project Number] RS-2023-00248656

[0025] [Research Project Title] Development of a Diversified Aspirin Nanosystem Technology for the Treatment of Cardiovascular Disease

[0026] [Host] Yonsei University

[0027] [Research Period] March 1, 2024 - February 28, 2025

[0028]

[0029] The adrenal glands (adrenal glands, suprarenal glands) are small, triangular endocrine glands located one above each kidney. The inner part is composed of the medulla and the outer part is the cortex. In adults, the cortex accounts for almost 90% of the adrenal glands. The adrenal cortex, stimulated by adrenocorticotropic hormone secreted from the pituitary gland, produces steroid hormones such as cortisol, aldosterone, and androgens. The medulla is an endocrine organ composed of specialized nerve cells that produces so-called catecholamines such as epinephrine and norepinephrine. Cortisol, secreted from the adrenal cortex, is secreted in response to stress, and is closely related to carbohydrate metabolism by mobilizing energy stored in the body, especially glucose and amino acids, and is therefore also called a glucocorticoid. It also plays a major role in controlling the immune system, such as suppressing immune system cells such as lymphocytes. Aldosterone is called a mineralocorticoid, and when the body lacks fluid, the kidneys produce renin, which acts on angiotensinogen from the liver to produce angiotensin, which is then secreted in response to stimulation by the kidneys to promote salt reabsorption. This is called the renin-angiotensin-aldosterone system. Catecholamines released from the adrenal medulla are hormones that rapidly respond to stress, increasing blood pressure and heart rate to increase blood circulation, and mobilizing energy such as glucose and fatty acids stored in the liver and adipose tissue to supply it to necessary organs such as muscles. Epinephrine and norepinephrine are examples of this.

[0030] Adrenal diseases are divided into diseases of the adrenal medulla and diseases of the adrenal cortex. The only known disease of the adrenal medulla is pheochromocytoma, which causes excessive secretion of epinephrine and norepinephrine. Symptoms include periodic high blood pressure, palpitations, excessive sweating, severe hammer-like headaches, and anxiety. Nausea and vomiting may also occur, but these are usually treated with medication. Adrenal cortex diseases can be divided into hyperadrenocorticism, caused by excessive secretion of adrenal cortex hormones, and adrenal insufficiency, caused by undersecretion. Hyperadrenocorticism is a congenital condition in which both adrenal glands are enlarged, or it can be caused by acquired conditions such as adrenal tumors or hypertrophy. Acquired hyperadrenocorticism manifests as Cushing's syndrome or adrenogenital syndrome. Cushing's syndrome is characterized by weight gain, a rounded face like the moon, the absence of menstruation, and diseases such as high blood pressure, diabetes, and osteoporosis. Thin skin, easy bruising, general weakness, and sometimes mental disorders may also occur. Adrenogenital syndrome is characterized by masculinization in women, such as facial hair growth and muscle development, and sexual precocity in prepubescent men. Hypoadrenal insufficiency is a disease in which the adrenal glands produce insufficient hormones to meet the body's needs, causing complex symptoms such as general weakness, hypoglycemia, hyperpigmentation of the skin, and low blood pressure. It can be broadly divided into primary adrenal insufficiency and secondary adrenal insufficiency. Primary adrenal insufficiency, which manifests as Addison's disease, is caused by problems with the adrenal gland itself, and can also be caused by adrenal tuberculosis, autoimmune diseases, adrenal tumors, cancer that has spread from other organs, and, in rare cases, bleeding or congenital diseases.Secondary adrenal insufficiency is caused by a deficiency in the secretion of adrenocorticotropic hormone, which stimulates cortisol secretion in the brain, and can occur after a tumor in the pituitary gland / hypothalamus or surgery for a pituitary adenoma.

[0031] Currently, the treatment of adrenal insufficiency relies on supplemental administration of steroid hormones, but this requires repeated daily administration, which reduces the quality of life and carries the risk of causing side effects such as diabetes, osteoporosis, and fatty liver when administered for a long period of time.

[0032]

[0033] An object of the present invention is to provide an adrenal organoid maturation composition.

[0034] In addition, the present invention provides a method for producing an adrenal organoid with enhanced hormone secretion function.

[0035] In addition, the present invention provides an adrenal organoid with enhanced hormone secretion function.

[0036] In addition, the present invention provides a method for manufacturing an adrenal organoid chip.

[0037] In addition, the present invention provides an adrenal organoid chip.

[0038] In addition, the present invention provides a cell therapy agent for treating adrenal insufficiency.

[0039] In addition, the present invention provides a composition for transplantation.

[0040] In addition, the present invention provides a method for evaluating the effectiveness of a therapeutic agent for adrenal disease.

[0041] In addition, the present invention provides a pharmaceutical composition for preventing or treating adrenal diseases.

[0042] In addition, the present invention provides a method for screening for a preventive or therapeutic agent for adrenal disease.

[0043]

[0044] To achieve the above purpose, the present invention provides an adrenal organoid maturation composition comprising adrenocorticotropic hormone and cyclic adenosine monophosphate.

[0045] In addition, the present invention provides a method for producing an adrenal organoid with enhanced hormone secretion function using the above-described mature composition.

[0046] In addition, the present invention provides an adrenal organoid with enhanced hormone secretion function manufactured by the above method.

[0047] In addition, the present invention provides a method for manufacturing an adrenal organoid chip using the adrenal organoid.

[0048] In addition, the present invention provides an adrenal organoid chip manufactured by the above method.

[0049] In addition, the present invention provides a cell therapy for treating adrenal insufficiency comprising the adrenal organoid or adrenal organoid chip.

[0050] In addition, the present invention provides a transplant composition comprising the adrenal organoid or adrenal organoid chip.

[0051] In addition, the present invention provides a method for evaluating the effectiveness of an adrenal disease treatment agent, including a step of evaluating drug responsiveness after treating the adrenal organoid or adrenal organoid chip with a drug.

[0052] In addition, the present invention provides a pharmaceutical composition for preventing or treating adrenal disease, comprising the adrenal organoid, the adrenal organoid chip, or a culture medium thereof.

[0053] In addition, the present invention provides a method for screening for a preventive or therapeutic agent for adrenal disease, comprising a step of treating a test substance on the adrenal organoid or adrenal organoid chip.

[0054]

[0055] In the present invention, structurally and functionally mature adrenal organoids were produced under optimized culture conditions using an adrenal organoid maturation composition containing adrenocorticotropic hormone and cyclic adenosine monophosphate, and it was confirmed that the adrenal organoids produced in this way were structurally and functionally improved. In addition, it was confirmed that an adrenal organoid chip having a three-dimensional vascular network structure produced using the same replaced the function of the adrenal gland without side effects after transplantation into an actual mouse, and therefore, it can be usefully utilized for transplantation or regenerative treatment, drug screening, drug responsiveness evaluation, etc.

[0056]

[0057] Figure 1 is a diagram showing a method for producing adrenal organoids using human adrenal tissue and confirming the same:

[0058] a: Schematic diagram of a method for producing adrenal organoids of the present invention;

[0059] b to e: Formation of cell aggregates during 3 days of primary culture; and

[0060] f: Cell viability of the cell aggregate.

[0061] Figure 2 shows the analysis of adrenal organoids that were cultured for 7 days (Day 3+Day 7) using maturation medium after 3 days of primary culture:

[0062] a: Size of cell aggregate;

[0063] b: Expression of cells that make up the three layers of the adrenal cortex; and

[0064] c: Expression of steroidogenic enzyme genes.

[0065] Figure 3 is a diagram analyzing the additional culture period using the optimal maturation culture medium:

[0066] a: Morphology of adrenal organoids according to additional culture weeks (1 to 12 weeks) using maturation medium after 3 days of primary culture (Day 3); and

[0067] b: Hormone secretion of adrenal organoids according to additional culture weeks (1 to 12 weeks) using maturation medium after 3 days of primary culture (Day 3).

[0068] Figure 4 is a diagram analyzing the effect of adrenocorticotropic hormone (ACTH) and cyclic adenosine monophosphate (cAMP) contained in the maturation culture medium on enhancing the hormone secretion function of adrenal organoids:

[0069] MM: Adrenal organoids prepared by further culture in maturation medium containing ACTH and cAMP;

[0070] MM-A-c: Adrenal organoids prepared by further culture in maturation medium without ACTH and cAMP;

[0071] a: Morphology of adrenal organoids;

[0072] b: Size of adrenal organoids;

[0073] c: Expression of steroidogenic enzyme genes in adrenal organoids; and

[0074] d: Hormone secretion of adrenal organoids.

[0075] Figure 5 is a diagram analyzing the proteomic changes of adrenal organoids according to the inclusion of ACTH and cAMP in the maturation culture medium:

[0076] MM: Adrenal organoids prepared by further culture in maturation medium containing ACTH and cAMP;

[0077] MM-A-c: Adrenal organoids prepared by further culture in maturation medium without ACTH and cAMP;

[0078] a: Proteomic analysis of adrenal organoids;

[0079] b: Pearson correlation analysis for total protein expression;

[0080] c: number of adrenal expressed proteins; and

[0081] d: Gene ontology enrichment analysis.

[0082] Figure 6 is a diagram analyzing changes in the expression pattern of adrenal-specific proteins in adrenal organoids according to the inclusion of ACTH and cAMP in the maturation culture medium:

[0083] MM: Adrenal organoids prepared by further culture in maturation medium containing ACTH and cAMP;

[0084] MM-A-c: Adrenal organoids prepared by further culture in maturation medium without ACTH and cAMP;

[0085] Adrenal gland: Actual human adrenal tissue;

[0086] a: Number of proteins with increased adrenal-specific expression; and

[0087] b: Pearson correlation analysis.

[0088] Figure 7 is a diagram analyzing the expression patterns of individual proteins in adrenal organoids according to the presence or absence of ACTH and cAMP in the maturation culture medium:

[0089] MM: Adrenal organoids prepared by further culture in a maturation medium containing ACTH and cAMP; and

[0090] MM -A -c: Adrenal organoids prepared by further culture in maturation medium that does not contain ACTH and cAMP.

[0091] Figure 8 is a diagram analyzing differentially expressed proteins (DEPs) in adrenal organoids depending on the presence or absence of ACTH and cAMP in the maturation culture medium:

[0092] MM: Adrenal organoids prepared by further culture in maturation medium containing ACTH and cAMP;

[0093] MM-A-c: Adrenal organoids prepared by further culture in maturation medium without ACTH and cAMP;

[0094] a: Volcano plot of DEPs;

[0095] b: Gene ontology enrichment analysis of DEPs promoted in MM-A-c;

[0096] c: Gene ontology enrichment analysis of DEPs promoted in MM;

[0097] d: Scatter plot of GOBP enriched in MM; and

[0098] e: Protein-protein interaction network of proteins belonging to adrenal-related GOBPs among DEPs promoted in MM.

[0099] Figure 9 is a grouping network of significantly enriched reactome pathways for differentially expressed proteins (DEPs) in adrenal organoids depending on the presence or absence of ACTH and cAMP in the maturation medium:

[0100] MM: Adrenal organoids prepared by further culture in maturation medium containing ACTH and cAMP;

[0101] MM-A-c: Adrenal organoids prepared by further culture in maturation medium without ACTH and cAMP;

[0102] a: Grouping network of reactome pathways significantly enriched for DEPs promoted in MM; and

[0103] b: Grouping network of significantly enriched reactome pathways for DEPs enhanced in MM-A-c.

[0104] Figure 10 is a diagram showing the analysis of adrenal organoids formed from normal adrenal tissue isolated from donors with various adrenal diseases.

[0105] Figure 11 is a diagram showing the analysis of adrenal organoids using cells derived from frozen adrenal tissue:

[0106] Primary: Adrenal organoids manufactured directly using cells derived from adrenal tissue;

[0107] Thawed: Adrenal organoids prepared using cells isolated from adrenal tissue, frozen, and then later thawed;

[0108] a: Schematic diagram showing the process of manufacturing adrenal organoids using cells isolated from adrenal tissue, frozen, and then thawed;

[0109] b: morphology of adrenal organoids; and

[0110] c: Hormone secretion of adrenal organoids.

[0111] Figure 12 is a diagram showing the results of drug responsiveness evaluation using adrenal organoids manufactured by the method of the present invention:

[0112] a: Cortisol secretion in adrenal organoids treated with osilodrostat, a drug that inhibits the synthesis of adrenal hormones;

[0113] b: Cortisol secretion in adrenal organoids treated with metyrapone, a drug that inhibits the synthesis of adrenal hormones;

[0114] c: Cortisol secretion in adrenal organoids treated with ketoconazole, a drug that inhibits the synthesis of adrenal hormones; and

[0115] d: Aldosterone secretion in adrenal organoids treated with Baxdrostat, a drug that inhibits aldosterone synthase.

[0116] Figure 13 is a diagram evaluating the effects of hormones secreted by adrenal organoids produced by the method of the present invention on other organs:

[0117] Dexa: Hepatocytes treated with dexamethasone;

[0118] Dexa + RU486: Hepatocytes treated with dexamethasone and RU486, a glucocorticoid receptor antagonist;

[0119] CM (Cortisol): Hepatocytes treated with a culture medium (final concentration of cortisol: 2 μM) cultured with adrenal organoids prepared by the method of the present invention; and

[0120] CM (Cortisol) + RU486: Culture medium in which adrenal organoids prepared by the method of the present invention were cultured (final concentration of cortisol: 2 μM) and hepatocytes treated with RU486, an antagonist of the glucocorticoid receptor.

[0121] Figure 14 is a diagram showing the process of manufacturing an adrenal organoid chip having a three-dimensional vascular microchannel network structure (a) and the experimental schedule and mouse groups using the same (b).

[0122] Figure 15 is a diagram analyzing the three-dimensional microchannel network structure (a) and the diameter of the microchannel (b) within the adrenal organoid chip.

[0123] Figure 16 is a diagram showing the transplantation of the adrenal organoid chip of the present invention into an adrenal-removed mouse and its confirmation:

[0124] a: Location of the chip confirmed at 1 and 7 weeks after adrenal organoid chip transplantation;

[0125] b: 3D vascular network structure analyzed 2 weeks after adrenal organoid chip implantation; and

[0126] c: Survival rate of adrenal organoids in the adrenal organoid chip harvested 11 weeks after adrenal organoid chip transplantation.

[0127] Figure 17 is a diagram analyzing the survival rate of adrenal-removed mice transplanted with the adrenal organoid chip of the present invention:

[0128] Normal: Normal mouse control group;

[0129] Adx: bilateral adrenalectomized mouse group; and

[0130] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed.

[0131] Figure 18 is a diagram analyzing changes in the concentration of cortisol and corticosterone in the plasma of adrenal-removed mice implanted with the adrenal organoid chip of the present invention:

[0132] 3 week: 3rd week after transplant;

[0133] 5 week: 5 weeks after transplant;

[0134] Normal: Normal mouse control group;

[0135] Adx: bilateral adrenalectomized mouse group;

[0136] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed; and

[0137] Adx+H: A group of mice administered corticosterone to mice with bilateral adrenal removal.

[0138] Figure 19 is a diagram analyzing the change in the concentration of aldosterone in the plasma of an adrenal-removed mouse transplanted with the adrenal organoid chip of the present invention:

[0139] 3 week: 3rd week after transplant;

[0140] 5 week: 5 weeks after transplant;

[0141] Normal: Normal mouse control group;

[0142] Adx: bilateral adrenalectomized mouse group;

[0143] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed; and

[0144] Adx+H: A group of mice administered corticosterone to mice with bilateral adrenal removal.

[0145] Figure 20 is a diagram analyzing the change in the concentration of adrenocorticotropic hormone in the plasma of an adrenal-removed mouse transplanted with the adrenal organoid chip of the present invention:

[0146] 3 week: 3rd week after transplant;

[0147] 5 week: 5 weeks after transplant;

[0148] Normal: Normal mouse control group;

[0149] Adx: bilateral adrenalectomized mouse group;

[0150] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed; and

[0151] Adx+H: A group of mice administered corticosterone to mice with bilateral adrenal removal.

[0152] Figure 21 is a diagram analyzing the wound healing effect of adrenal-removed mice implanted with the adrenal organoid chip of the present invention:

[0153] Normal: Normal mouse control group;

[0154] Adx: bilateral adrenalectomized mouse group;

[0155] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed;

[0156] a: wound area;

[0157] b: H&E and MT staining of skin tissue; and

[0158] c: Immunostaining of Involucrin and CD31.

[0159] Figure 22 is a diagram showing the analysis of blood sugar (a), heart rate (b), and potassium ion concentration (c) of adrenal-removed mice implanted with the adrenal organoid chip of the present invention:

[0160] Normal: Normal mouse control group;

[0161] Adx: bilateral adrenalectomized mouse group; and

[0162] Adx+chip: A group of mice in which adrenal organoid chips were transplanted into mice with both adrenal glands removed.

[0163] Figure 23 is a diagram analyzing whether side effects due to excessive cortisol occur in adrenal-removed mice implanted with the adrenal organoid chip of the present invention:

[0164] a: Bone density analysis photograph of the femur; and

[0165] b: Quantitative graph of bone density of the femur.

[0166]

[0167] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0168] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0169] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0170]

[0171] In one aspect, the present invention relates to an adrenal organoid maturation composition comprising adrenocorticotropic hormone (ACTH) and cyclic adenosine monophosphate (cAMP).

[0172] In one embodiment, the composition can increase the expression of CYP17A1, CYP11B2, CYP11A1, HSD3B2 or CYP21A2 in adrenal organoids.

[0173] In one embodiment, the composition can increase hormone secretion of adrenal organoids.

[0174] In one embodiment, the composition can increase secretion of cortisol, corticosterone or aldosterone from adrenal organoids.

[0175] In one aspect, the present invention relates to a method for producing adrenal organoids with enhanced hormone secretion function, comprising the steps of first culturing adrenal tissue-derived cells to form cell aggregates; and second culturing them in a culture medium containing the adrenal organoid maturation composition of claim 1.

[0176] In one embodiment, the primary culture in the method may be performed for 2 to 4 days, and the secondary culture may be performed for 5 to 9 days.

[0177] In one embodiment, the adrenal tissue-derived cells may be cells that have been frozen and then thawed.

[0178] In one embodiment, the method can mature adrenal organoids in secondary culture, wherein the maturation can be structural or functional, and the functional maturation can be enhancement of the secretory function of hormones.

[0179] In one embodiment, the adrenal tissue-derived cells can be labeled, and can be labeled with one or more selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a luminescent substance, and a fluorescent substance, and the fluorescent substance can be a fluorescent substance of the Cy (cyanine) series, Rhodamine series, Alexa series, BODIPY series, or ROX series, and rhodamine, Cyanine 3, Cyanine 5, pyrene, Cyanine 2, green fluorescent protein (GFP;Green Fluorescent Protein (GFAP), red fluorescent protein (RFP), Calcein, fluorescein isothiocyanate (FITC), Alexa 488, 6-carboxy-fluorescein (FAM), 2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein (HEX), 2',7'-dichloro-6-carboxy-4,7-dichlorofluorescein (TET), Fluorescein Chlorotriazinyl, Fluorescein, Oregon Green, Magnesium Green, Calcium Green, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), Tetramethylrhodamine, Tetramethyl-rhodamine isothiocyanate (TRITC), Levoxytetramethyl rhodamine (TAMRA), Rhodamine Phalloidin, Pyronin Y, Lissamine, X-rhodamine (ROX), Calcium Crimson, Texas Red, Nile Red, Malachite green, Thiadicarbocyanine, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), or To1-biotin.;

[0180] In one embodiment, the secondary culture in the method can enhance structural maturation or functional maturation of the adrenal organoids.

[0181] The term "organoid" used in the present invention refers to a "mini-organ" that is made to perform minimal functions using cells. It is characterized by its three-dimensional structure, allowing for the creation of an environment similar to an actual organ in the laboratory. In other words, an "organoid" refers to a three-dimensional in vitro organ-like model that reproduces the structure and function of an organ in the body by being a collection of cells that have self-organized and differentiated into functional cells in three dimensions. The origin of the cells that constitute the organoid is not limited. The organoid can have an environment that allows the cells to interact with the surrounding environment during the cell growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoid of the present invention can almost perfectly mimic the organs that actually interact in the body, making it an excellent model for observing the development of treatment methods for diseases, etc.

[0182] The cell types used in the present invention can be cultured by any method known in the art. Methods for culturing cells and tissues are well known in the art and are described, for example, in Cell & Tissue Culture: Laboratory Procedures; Freshney (1987), and Culture of Animal Cells: A Manual of Basic Techniques, the contents of which are incorporated herein by reference. General mammalian cell culture techniques, cell lines, and cell culture systems that can be used in conjunction with the present invention are also described in Doyle, A., Griffiths, JB, Newell, DG, (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley (1998), the contents of which are incorporated herein by reference.

[0183] In one aspect, the present invention relates to an adrenal organoid with enhanced hormone secretion function produced by the method of the present invention.

[0184] In one embodiment, the adrenal organoids may have enhanced structural maturation or functional maturation.

[0185] In one embodiment, the adrenal organoid may have increased secretion of cortisol, corticosterone, or aldosterone.

[0186] In one embodiment, the adrenal organoid may have increased expression of CYP17A1, CYP11B2, CYP11A1, HSD3B2, or CYP21A2.

[0187] In one aspect, the present invention relates to a method for manufacturing an adrenal organoid chip, comprising: forming a fiber structure using a temperature-responsive polymer; injecting a solution containing the adrenal organoid of the present invention and a biocompatible polymer into the fiber structure to gel it; and removing the fiber structure.

[0188] In one embodiment, the thermoresponsive polymer is poly(N-isopropyl acrylamide) [poly(N-isopropylacrylamide), pNIPAM], poly(N-isopropyl acrylamide-co-allylamine) [poly(N-isopropyl acrylamide-co-allylamine), poly(NIPAM-co-AA)], poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl methacrylate) [poly(Nisopropylacrylamide-co-2-(dimethylamino)ethyl methacrylate), poly(NIPAM-co-DMAEMA)], poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate), poly(NIPAM-co-DMAEA)], poly(N-isopropyl acrylamide-co-acrylic acid) [poly(N-isopropyl acrylamide-co-acrylic acid), poly(NIPAM-co-AAc)], poly(N-isopropyl acrylamide-co-methacrylic acid), poly(NIPAM-co-MAAc)], poly(N,N-diethylacrylamide)], poly(N-vinylcaprolactam)], poly(ethylene glycol)], poly(ethylene glycol-b-propylene glycol-b-ethylene glycol)], or polyvinyl methyl ether.

[0189] In one embodiment, the biocompatible polymer is selected from the group consisting of cellulose, pectin, chondroitin sulfate, decellularized extracellular matrix, fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone) (PCL), polyanhydride, polyorthoester, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), It may be poly-N-isopropylacrylamide (PNIPAAm), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, a copolymer thereof, or a mixture thereof.

[0190] In one embodiment, gelation of a solution containing the adrenal organoid and biocompatible polymer of the present invention may be performed by an enzymatic reaction, and for this purpose, an enzyme such as Microbial transglutaminase (mTG) may be additionally included.

[0191] In one embodiment, the fiber structure formed from the heat-sensitive polymer can be removed by perfusing with a solution at a temperature at which the heat-sensitive polymer becomes soln, thereby forming a microchannel structure inside the biocompatible polymer including the gelled adrenal organoid of the present invention, thereby manufacturing an adrenal organoid chip having a three-dimensional vascular microchannel network structure.

[0192] In one embodiment, the adrenal organoid chip may have a three-dimensional vascular microchannel network structure, and the diameter of the microchannel may be 5 to 50 μm.

[0193] In the present invention, the term "biocompatible polymer" refers to a polymer that is slowly and spontaneously decomposed in a living body after a certain period of time, and has one or more of the following properties: biodegradability, blood compatibility, anti-calcification properties, cellular nutritional properties, and intercellular matrix formation ability.

[0194] In one aspect, the present invention relates to an adrenal organoid chip manufactured by the method of the present invention.

[0195] In one embodiment, the adrenal organoid chip may have a three-dimensional vascular microchannel network structure.

[0196] In one aspect, the present invention relates to a cell therapy for treating adrenal insufficiency comprising the adrenal organoid or adrenal organoid chip of the present invention.

[0197] In the present invention, the term "cell therapy agent" refers to a medicine used for the purposes of treatment, diagnosis, and prevention by isolating, culturing, and manufacturing cells and tissues from humans through special processing, and refers to a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the function of cells or tissues.

[0198] The cell therapy agent according to the present invention can be injected into the body of a subject, for example, using the clinical method announced by Lindvall et al. (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka (Pittsburgh, 1998). The preparation may contain, in addition to the active ingredient, the mature organoid, a pharmaceutically acceptable conventional carrier, and in the case of an injection, a preservative, an analgesic, a solubilizer, or a stabilizer, and in the case of a preparation for topical administration, a base, an excipient, a lubricant, or a preservative, etc.

[0199] The cell therapy agent according to the present invention can be manufactured in a unit dose form or manufactured by inserting it into 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. The pharmaceutically acceptable carrier included in the cell therapy agent of the present invention is one that is commonly used in formulation, and includes, but is 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, or mineral oil. In addition to the above components, the cell therapy agent of the present invention may additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0200] The cell therapy agent according to the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally, or topically. The appropriate dosage of the cell therapy agent according to the present invention can vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity.

[0201] As used herein, the terms "administering," "introducing," and "transplanting" are used interchangeably and may refer to the placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site. Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location within a viable subject.

[0202] In the above method, the drug can be administered to the lesion site requiring organoid transplantation. Endoscopic equipment can be used for administration, but is not limited thereto. For example, administration via endoscope is a typical example, and surgical administration to the adrenal gland is also possible. For example, in cases where part or all of the adrenal tissue has been lost, the adrenal organoid or adrenal organoid chip of the present invention can be transplanted to replace the damaged tissue.

[0203] In one aspect, the present invention relates to a transplant composition comprising the adrenal organoid or adrenal organoid chip of the present invention.

[0204] In one embodiment, the composition may be a composition for adrenal transplantation.

[0205] In one aspect, the present invention relates to a method for evaluating the effectiveness of a therapeutic agent for adrenal disease, comprising the step of evaluating drug responsiveness after treating the drug to an adrenal organoid or adrenal organoid chip of the present invention.

[0206] In one embodiment, drug responsiveness can be assessed by examining in vivo kinetics or in vitro cellular functionality, wherein the in vivo kinetics can be metabolism, absorption, membrane permeability, drug interaction, induction of drug metabolizing enzymes, or induction of drug transporters, and the in vitro cellular functionality can be hormone secretion.

[0207] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating adrenal disease, comprising the adrenal organoid, adrenal organoid chip, or culture medium thereof of the present invention.

[0208] In one embodiment, the adrenal disease may be an adrenal cortical disease or an adrenal medullary disease, and the adrenal cortical disease may be hyperadrenocorticism or adrenal insufficiency.

[0209] In one embodiment, the hyperadrenalism may be Cushing's syndrome or adrenogenital syndrome, the hypoadrenalism may be primary hypoadrenalism or secondary hypoadrenalism, and the primary hypoadrenalism may be Addison's disease.

[0210] In one embodiment, the culture medium of the adrenal organoid or adrenal organoid chip may be a culture obtained during or after culturing the adrenal organoid or adrenal organoid chip, a supernatant thereof, a filtrate thereof, a fraction thereof, a concentrate of the supernatant thereof, a lyophilized product thereof, or a supercritically dried product thereof.

[0211] In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of adrenal disease by administering a pharmaceutical composition according to the present invention.

[0212] The term "treatment" as used herein refers to any action that improves or beneficially alters the symptoms of adrenal disease through administration of the composition of the present invention. Those skilled in the art to which the present invention pertains will be able to accurately determine the criteria for diseases for which the composition of the present invention is effective and determine the degree of improvement, enhancement, and treatment by referencing materials provided by the Korean Medical Association and other sources.

[0213] The term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage for use in humans should be determined as an appropriate amount by taking both safety and efficacy into consideration. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0214] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, the type and severity of the adrenal disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0215] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0216] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and examples thereof include compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0217] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0218] Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0219] In one aspect, the present invention relates to a method for screening for an agent for preventing or treating adrenal disease, comprising a step of treating a test substance to an adrenal organoid or adrenal organoid chip of the present invention.

[0220]

[0221] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0222]

[0223] Example 1. Production of adrenal organoids with enhanced hormone secretion function.

[0224] 1-1. Basic cultivation and analysis

[0225] Cells were isolated from normal human adrenal tissue obtained incidentally during adrenalectomy by treating it with collagenase, dispensed into 300 μm diameter microwells, and cultured for 3 days to form cell aggregates. Microscopic observation revealed that after 3 days of culture, the cells gradually clumped together and cell aggregates of similar size were evenly formed (Figs. 1b to e). Analysis of cell viability using Live / Dead staining also confirmed that there was no problem with the viability of the cells in the cell aggregates (Fig. 1f).

[0226]

[0227] 1-2. Analysis of the effect of additional culture and hormone secretion enhancement using maturation medium

[0228] To determine whether mature adrenal organoids with enhanced hormone secretion function could be produced by further culturing the formed cell aggregates using a maturation medium, the cell aggregates formed for 3 days in Example 1-1 (Day 3) were further suspended and cultured in a maturation medium (MM) containing adrenocorticotropic hormone (ACTH) and cyclic adenosine monophosphate (cAMP) for 7 days (Day 3+7) to produce human adrenal organoids (Fig. 1a). Afterwards, the manufactured adrenal organoids were observed under a microscope, and immunofluorescence staining was performed for CYP17A1, a marker protein mainly expressed in the zona fasciculata (zF) and zona reticularis (zR), CYB5, a marker protein mainly expressed in the zona reticularis, and CYP11B2, a marker protein mainly expressed in the zona glomerulosa (zG), to confirm the expression of cells constituting the three layers of the adrenal cortex: the zona globularis, zona fasciculata, and zona reticularis. In addition, the gene expression of other steroidogenic enzymes involved in the biosynthesis of adrenal cortical hormones was analyzed by quantitative PCR.

[0229] Microscopic observations revealed that the size of the cell aggregates increased during the 7-day additional culture period (Fig. 2a), and immunofluorescence staining analysis revealed that the expression of markers specific to each layer of the adrenal cortex increased after 7 days of additional culture compared to the cell aggregates on Day 3 (Fig. 2b). In addition, the expression of CYP11A1, HSD3B2, and CYP21A2, which are genes for steroidogenic enzymes, was found to increase by additional culture (Fig. 2c). Through this, it was confirmed that the formation of structurally mature adrenal organoids with enhanced hormone secretion function was possible through additional culture using the maturation culture medium of the present invention.

[0230]

[0231] 1-3. Analysis of the effects according to the additional incubation period

[0232] In order to compare the hormone secretion enhancement effect of adrenal organoids according to the additional culture period of the present invention, cell aggregates were formed for 3 days, and then cultured for 1 to 12 weeks using a maturation culture medium, and the secretion of cortisol and aldosterone of adrenal organoids was confirmed.

[0233] As a result, adrenal organoids were found to survive and maintain their morphology even after 12 weeks of culture (Fig. 3a). However, the amount of hormone secreted sufficiently in the first week began to decrease from the second week of culture, and by the 12th week, it was found to have decreased by approximately 50% compared to the first week (Fig. 3b). Therefore, the subsequent maturation culture period of adrenal organoids was optimized to 7 days.

[0234]

[0235] Example 2. Analysis of the effect of mature culture solution

[0236] 2-1. Analysis of morphology and hormone secretion of adrenal organoids

[0237] To confirm the effect of ACTH and cAMP included in the maturation medium, the cell aggregates formed by primary culture for 3 days were further cultured for 7 days using the maturation medium supplemented with ACTH and cAMP. The morphology and hormone secretion levels of adrenal organoids (MM) were compared with those cultured for 7 days without the addition of ACTH and cAMP (MM -ACTH -cAMP).

[0238] As a result, compared to adrenal organoids cultured further using a maturation medium containing ACTH and cAMP, adrenal organoids cultured further without ACTH and cAMP showed a reduced size and a shrunken morphology (Figs. 4a and b), and the expression of steroidogenic enzyme genes CYP11A1, HSD3B2, and CYP21A2, as well as the secretion of cortisol and aldosterone, were significantly reduced (Figs. 4c and d). Through this, it was confirmed that the maturation medium containing ACTH and cAMP enhances the hormone secretion function of adrenal organoids.

[0239]

[0240] 2-2. Proteomic analysis of adrenal organoids

[0241] To further investigate the effects of ACTH and cAMP contained in the maturation medium on adrenal organoids, adrenal organoids formed by primary culture for 3 days were cultured for an additional 7 days using the maturation medium supplemented with ACTH and cAMP (MM) and adrenal organoids cultured for an additional 7 days without the addition of ACTH and cAMP (MM -ACTH -cAMP) were disrupted and subjected to proteomic analysis (Fig. 5a).

[0242] Pearson correlation analysis of total protein expression revealed similar expression patterns within the same group, but differences between the MM and MM -ACTH -cAMP groups (Fig. 5b). In addition, when the number of adrenal gland-expressed proteins was confirmed based on the Human Protein Atlas database, 1,980 proteins were commonly included, 120 proteins were present only in the MM group, and 80 proteins were present only in the MM -ACTH -cAMP group, confirming that there were differences in the types of proteins included in the two groups (Fig. 5c). In addition, Gene ontology enrichment analysis was performed on the 120 proteins present only in the MM group, and the 10 most significant Gene ontology biological processes (GOBPs) were identified based on the false discovery rate (FDR) value. As a result, GOBP terms related to adrenal steroid hormone metabolic processes, such as Sterol metabolic process and Steroid metabolic process, were enriched (Fig. 5d).

[0243]

[0244] 2-3. Analysis of protein expression patterns in adrenal organoids

[0245] To determine whether there were differences in adrenal-specific protein expression patterns in adrenal organoids prepared with or without ACTH and cAMP in the maturation medium, MM and MM -ACTH -cAMP (MM -A -c) were additionally analyzed for proteins known to be expressed more than four times higher in the adrenal gland than in other tissues (Adrenal gland-elevated proteins) based on the Human Protein Atlas database. In addition, Pearson correlation analysis was performed on the expression of proteins known to be highly expressed in the adrenal gland, and the expression of actual human adrenal gland tissue was compared using the Human Proteome Map database.

[0246] As a result, 43 proteins known to be highly expressed in the adrenal gland were common to both groups, 6 proteins were present only in the MM group, and 5 proteins were present only in the MM -ACTH -cAMP group (Fig. 6a), confirming that there were differences in the types of proteins expressed in the two groups. In addition, the Pearson correlation analysis results showed that the organoids of the MM group showed a high correlation with human adrenal tissue with a Pearson correlation coefficient of 0.73-0.74, but the organoids of the MM -ACTH -cAMP group showed a relatively low correlation with human adrenal tissue with a Pearson correlation coefficient of 0.52-0.59 (Fig. 6b). Through this, it was confirmed that the protein expression pattern of the adrenal organoids manufactured using the maturation culture medium containing ACTH and cAMP of the present invention was more similar to that of actual human adrenal tissue.

[0247]

[0248] 2-4. Analysis of protein expression levels related to hormone synthesis and secretion in adrenal organoids

[0249] To analyze the expression patterns of individual proteins in adrenal organoids MM and MM -ACTH -cAMP (MM -A -c), the relative expression levels of each protein belonging to Steroid biosynthetic process, C21-steroid hormone metabolic process, and Cortisol synthesis and secretion, which are GOBPs related to adrenal hormone synthesis / secretion function, were displayed as a heatmap. As a result, it was confirmed that the expression of two adrenal-related GOBPs and proteins belonging to one KEGG pathway were generally high in adrenal organoids cultured additionally in a maturation medium containing ACTH and cAMP (Fig. 7).

[0250]

[0251] 2-5. DEPs analysis of adrenal organoids

[0252] Differentially expressed proteins (DEPs) were selected and analyzed between the adrenal organoid MM group and the MM -ACTH -cAMP group based on the criteria of Fold change > 2 and p-value < 0.05. 155 DEPs that were increased in the MM group and 271 DEPs that were increased in the MM -ACTH -cAMP group were displayed as Volcano plots (Fig. 8a). In addition, as a result of performing Gene ontology enrichment analysis on the DEPs that were enhanced in each group, the MM -ACTH -cAMP group was found to be enriched in GOBP terms related to energy production and cellular respiration as a whole, such as Generation of precursor metabolites and energy, Energy derivation by oxidation of organic compounds, Cellular respiration, and ATP biosynthetic process (Fig. 8b), whereas the MM group was found to be enriched in GOBP terms related to the metabolic / biosynthetic processes of adrenal steroid hormones, such as Cholesterol metabolic process, Sterol metabolic process, Steroid metabolic process, Cholesterol biosynthetic process, Sterol biosynthetic process, and Steroid biosynthetic process (Fig. 8c).In addition, using REVIGO, the core GOBP terms among the significantly enriched GOBPs were displayed in a scatter plot according to semantic similarity, and the results showed that Lipid metabolic process, Cholesterol homeostasis, Sterol metabolic process, and Steroid metabolic process, etc. were identified (Fig. 8d). In addition, among the DEPs (155) that were enhanced in the MM group, proteins belonging to the adrenal-related GOBPs, Lipid metabolic process, Steroid metabolic process, and Cholesterol metabolic process, were selected and the protein-protein interaction network was displayed. As a result, it was found that many proteins commonly belonged to the three GOBPs, and it was confirmed that the proteins formed close interactions with each other (Fig. 8e).

[0253]

[0254] 2-6. Reactome pathway analysis of adrenal organoids

[0255] The significantly enriched reactome pathways for the 155 DEPs enhanced in the MM group were grouped according to their relevance and displayed as a network, and were represented by cholesterol biosynthesis, metabolism of amino acids and derivatives, etc. (Fig. 9a). On the other hand, the 271 DEPs enhanced in the MM -ACTH -cAMP group were represented by respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins, mRNA splicing, etc. (Fig. 9b).

[0256]

[0257] In summary, the results of the proteomic analysis of the adrenal organoids of Examples 2-2 to 2-6 above showed that the protein expression pattern of the adrenal organoids produced when ACTH and cAMP were added to the maturation medium was more similar to that of actual human adrenal tissue, and it was confirmed that the expression of proteins related to adrenal steroid hormone synthesis / secretion was generally high. In addition, the results of the analysis of differentially expressed proteins also confirmed that GOBPs and pathways functionally related to adrenal steroid hormone metabolism were enriched. This suggests that the hormone secretion function of adrenal organoids can be enhanced by adding ACTH and cAMP to the maturation medium.

[0258]

[0259] Example 3. Applicability analysis of the adrenal organoid production method

[0260] 3-1. Production of adrenal organoids using various adrenal tissues

[0261] In order to confirm whether the optimized culture medium composition and culture period conditions of the method for producing adrenal organoids of the present invention using adrenal tissues obtained incidentally during adrenalectomy without a separate additional process for tissue acquisition can be applied to form adrenal organoids regardless of the type of adrenal disease in patients undergoing adrenalectomy, normal adrenal tissues (Table 1) obtained incidentally after adrenalectomy from patients who underwent adrenalectomy due to various adrenal diseases and scheduled for disposal were received and adrenal organoids were produced using the method of the present invention (culture for 3 days and then cultured for an additional 7 days in a maturation medium).

[0262] Donor No.AgeSexPreoperative diagnosisPathologyORG1#148MaleAdrenal incidentalomaCushing's syndromeGangliogliomaORG2#254FemaleAdrenal incidentalomaLymphangiomatous cystORG3#354MalePrimary hyperaldosteronismAdrenal cortical adenomaORG4#466FemaleAdrenal incidentalomaPrimary hyperaldosteronismAdrenal cortical adenoma

[0263] As in Example 1, cells were seeded into 300 μm diameter microwells and cultured for 3 days to form cell aggregates, and then further cultured for 7 days using a maturation medium containing ACTH and cAMP to produce adrenal organoids (Fig. 11a). The morphology and hormone secretion amount of adrenal organoids produced from cryopreserved cells (Thawed) and adrenal organoids produced directly without cell freezing (Primary) were analyzed.

[0264] As a result, the organoids (Thawed) produced from cells thawed after freezing were well formed in a similar shape to the organoids (Primary) produced directly without the freezing process (Fig. 11b), and the secretion amounts of cortisol and aldosterone were also maintained at similar levels (Fig. 11c).

[0265]

[0266] 3-3. Evaluation of drug responsiveness of adrenal organoids

[0267] In order to confirm the drug responsiveness of the adrenal organoids manufactured by the method of the present invention, the adrenal organoids were treated with various concentrations of Osilodrostat, Metyrapone, and Ketoconazole, which are drugs known to treat Cushing's syndrome by inhibiting the synthesis of adrenal hormones, and Baxdrostat, a drug that inhibits aldosterone synthase and reduces aldosterone production in the body, for 3 days, and the degree of hormone secretion was analyzed.

[0268] As a result, adrenal organoids showed a concentration-dependent decrease in cortisol secretion when treated with osilodrostat, metyrapone, and ketoconazole (Figs. 12a to c), and aldosterone secretion was significantly reduced by treatment with vaxdrostat (Fig. 12d). Thus, it was confirmed that the adrenal organoids produced by the method of the present invention are responsive to cortisol or aldosterone secretion inhibitors, and thus can be used for evaluating drug responsiveness.

[0269]

[0270] Example 4. Functional evaluation of adrenal organoid secreting hormones

[0271] In order to confirm whether the hormone secreted from the adrenal organoid manufactured by the method of the present invention affects other organs as in the actual body, the cortisol concentration of the culture medium (conditioned media, CM) in which the adrenal organoid was cultured was measured, and the liver cells, HepG2, were treated with cortisol to a final concentration of 2 μM and incubated for 48 hours, and the expression of G6PC and PEPCK1, genes related to gluconeogenesis, was confirmed by quantitative PCR analysis. At this time, a group (Dexa) treated with dexamethasone, a synthetic glucocorticoid, was added as a positive control group, and a group (RU486) treated with RU486, a glucocorticoid receptor antagonist, was added to confirm the effect of cortisol among various components in the culture medium.

[0272] As a result, the expression of G6PC and PEPCK1 was significantly increased in the group treated with the conditioned media (CM) cultured with adrenal organoids, similar to the group treated with dexamethasone, and the effect was reduced when treated with RU486 (Fig. 13), confirming that cortisol contained in the conditioned media (CM) cultured with adrenal organoids promotes gluconeogenesis in liver cells.

[0273]

[0274] Example 5. Fabrication of an adrenal organoid chip with a three-dimensional vascular microchannel network structure.

[0275] 5-1. Adrenal organoid chip manufacturing

[0276] Using the adrenal organoids produced by the method of the present invention, an adrenal organoid chip having a three-dimensional vascular microchannel network structure was fabricated (Fig. 14a). Specifically, a fiber-shaped chip was prepared by injecting heat-sensitive poly(N-isopropyl acrylamide) (PNIPAM) into a polydimethylsiloxane (PDMS) mold using a cotton candy machine (top of Fig. 14a). Microbial transglutaminase (mTG) was dissolved at 10% (w / v) in phosphate-buffered saline (PBS), sterilized using a 0.2 μm filter, and gelatin powder was dissolved at 5.5% (w / v) in phosphate-buffered saline (PBS) at 37°C for 2 hours, and then sterilized using a vacuum filter. The prepared microvial transglutaminase and gelatin solution were mixed in a volume ratio of 1:9, and then injected into a chip having a PNIPAM fiber structure together with the adrenal organoid manufactured by the method of the present invention, and gelation was induced through an enzymatic reaction at 37°C for 1 hour, and then perfused with a phosphate buffer solution below 32°C to remove the PNIPAM fiber (bottom of Fig. 14a), thereby manufacturing an adrenal organoid chip having a three-dimensional vascular microchannel network structure.

[0277]

[0278] 5-2. Morphological analysis of the adrenal organoid chip

[0279] In order to confirm the three-dimensional microchannel network structure within the adrenal organoid chip manufactured in Example 5-1, perfusion staining was performed with microbeads labeled with a fluorescent dye, and as a result, a three-dimensional microchannel network structure was found to have been formed within the adrenal organoid chip (Fig. 15a), and the diameter of the microchannel was observed to be within 20 μm (Fig. 15b).

[0280]

[0281] Example 6. Analysis of the transplantation effect of the adrenal organoid chip.

[0282] 6-1. Transplantation of adrenal organoid chips

[0283] In order to confirm the therapeutic effect of the adrenal organoid chip having the three-dimensional vascular microchannel network structure manufactured in Example 5 above, both adrenal glands of nude mice were resected, and one adrenal organoid chip was transplanted into each location, for a total of two chips per mouse (200 adrenal organoids per mouse), and the subsequent experiments were performed (Fig. 14a bottom and Fig. 14b).

[0284]

[0285] 6-2. Analysis of transplanted adrenal organoid chips

[0286] Adrenal organoid chips were implanted and examined one and seven weeks later, demonstrating that the chips were well maintained over the kidney tissue, the initial implantation site (Fig. 16a). Furthermore, perfusion staining performed two weeks after implantation of the adrenal organoid chips confirmed the formation of a three-dimensional vascular network structure capable of perfusing fluid within the chip (Fig. 16b). Furthermore, live / dead staining of the adrenal organoid chips harvested 11 weeks after implantation confirmed that the adrenal organoids maintained a high viability rate even 11 weeks after implantation (Fig. 16c).

[0287]

[0288] 6-3. Analysis of Mice Transplanted with Adrenal Organoid Chips

[0289] 6-3-1. Survival rate analysis

[0290] To confirm the transplantation effect of the adrenal organoid chip, the survival rate of mice transplanted with the adrenal organoid chip was analyzed. As a result, the survival rate of mice with bilateral adrenal removal (Adx) decreased rapidly after surgery, but mice with adrenal organoid chips transplanted immediately after bilateral adrenal removal (Adx+chip) showed the same survival rate as normal mice (Normal) (Fig. 17).

[0291]

[0292] 6-3-2. Hormone Analysis

[0293] The mice were divided into a normal mouse group (Normal), a bilateral adrenal-removed mouse group (Adx), a mouse group in which adrenal organoid chips were transplanted after bilateral adrenal removal (Adx+chip), and a mouse group in which corticosterone dissolved in saline at a concentration of 3 μg / ml was supplied instead of drinking water from the day after bilateral adrenal removal (Adx+H). Then, blood was collected from the mice at 3 and 5 weeks after adrenal organoid chip transplantation, and plasma was separated. The concentrations of cortisol, a hormone secreted from the adrenal cortex, corticosterone, another major hormone secreted from the adrenal cortex, and adrenocorticotropic hormone secreted from the anterior pituitary when cortisol is not sufficiently secreted were measured using an enzyme-linked immunosorbent assay (ELISA) (Fig. 14b). For reference, both cortisol and corticosterone are hormones secreted from the adrenal cortex and play the same role in the body, but due to differences in enzymes between species, cortisol is primarily produced and used in humans, and corticosterone is primarily produced and used in rodents.

[0294] As a result, both cortisol and corticosterone levels, which were significantly reduced in the bilateral adrenal removal mouse group (Adx), were significantly recovered in the adrenal organoid chip-implanted mouse group (Adx+chip), showing hormone levels almost similar to those of the normal mouse group (Normal) (Fig. 18). In addition, the level of aldosterone, another major hormone secreted from the adrenal cortex, was significantly reduced in the bilateral adrenal removal mouse group (Adx) and the mouse group administered only corticosterone in drinking water (Adx+H), while it was recovered to a level similar to normal in the adrenal organoid chip-implanted mouse group (Adx+chip) (Fig. 19). In addition, the concentration of ACTH, which was significantly increased in the bilateral adrenal removal mouse group (Adx), was found to be secreted at a level similar to that of the normal mouse group (Normal) in the adrenal organoid chip-implanted mouse group (Adx+chip) (Fig. 20). It was confirmed that the above results were maintained up to 5 weeks after transplantation.

[0295] As described above, when the adrenal organoid chip of the present invention was transplanted after removal of the adrenal gland, the concentration of adrenal cortex hormones (cortisol, corticosterone, and aldosterone) and the adrenocorticotropic hormone associated therewith were restored to a level similar to normal, and thus it was confirmed that the adrenal organoid chip of the present invention is useful as a cell therapy agent.

[0296]

[0297] 6-3-3. Wound healing analysis

[0298] One week after adrenal gland removal and adrenal organoid chip transplantation, a full-thickness wound with a diameter of 8 mm was induced on the back skin of the mouse. Three weeks after transplantation, skin tissue was collected and the degree of wound healing was confirmed through quantitative analysis of wound area, histological analysis (H&E and MT staining), and immunostaining analysis (Involucrin and CD31) (Fig. 14b).

[0299] Quantitative analysis of wound area showed that the group of mice implanted with adrenal organoid chips (Adx+chip) showed wound healing similar to that of the normal mouse group (Normal), whereas the group of mice with bilateral adrenal removal (Adx) showed slower wound healing (Fig. 21a). In addition, H&E (hematoxylin & eosin) and MT (Masson's trichrome) staining of the skin tissue of the back of the mouse showed that the group of mice implanted with adrenal organoid chips (Adx+chip) showed wound healing similar to that of the normal mouse group (Normal) (Fig. 21b). In addition, the epidermis formation was evaluated by Involucrin immunostaining of mouse skin tissues, and the degree of neovascularization was analyzed by CD31 immunostaining. As a result, unlike the bilateral adrenal removal mouse group (Adx), the mouse group implanted with the adrenal organoid chip (Adx+chip) showed epidermis and blood vessels formed at a level similar to that of the normal mouse group (Normal) (Fig. 21c).

[0300]

[0301] 6-3-4. Analysis of body function recovery

[0302] At 3 weeks after adrenal gland removal and adrenal organoid chip transplantation, the recovery of various functions related to adrenal hormones in the body was evaluated in mice. Specifically, since cortisol secreted from the adrenal gland plays a role in regulating sugar metabolism in the body, the concentration of glucose in the mouse plasma was measured, and since heart rate can be controlled, the heart rate was analyzed. Since aldosterone secreted from the adrenal gland is known to regulate electrolyte homeostasis in the body, and hyperkalemia is induced when aldosterone is insufficient, the potassium (K) in the mouse plasma was measured. + ) concentration was analyzed.

[0303] As a result of measuring the concentration of glucose in the plasma, the blood sugar level decreased in the group of mice with both adrenal glands removed (Adx), whereas the blood sugar level in the group of mice with the adrenal organoid chip implanted (Adx+chip) was similar to that in the normal mouse group (Normal) (Fig. 22a). In addition, the heart rate analysis result showed that the heart rate decreased in the group of mice with both adrenal glands removed (Adx), whereas the heart rate in the group of mice with the adrenal organoid chip implanted (Adx+chip) was similar to that in the normal mouse group (Normal) (Fig. 22b). In addition, the potassium (K) in the plasma of the mice + ) As a result of concentration analysis, the plasma potassium concentration, which was significantly increased in the bilateral adrenal removal mouse group (Adx), was found to be similar to that in the normal mouse group (Normal) in the mouse group implanted with the adrenal organoid chip (Adx+chip) (Fig. 22c). Through this, it was confirmed that various functions in the body regulated by adrenal hormones, which are abnormal when the adrenal glands are removed, are restored to a similar level to normal when the adrenal organoid chip of the present invention is implanted.

[0304]

[0305] 6-3-5. Side Effect Analysis

[0306] Since it is known that osteoporosis can occur as a side effect when cortisol is excessive in the body, in order to confirm whether there are any side effects due to the transplantation of the adrenal organoid chip of the present invention, femurs were obtained from each group of mice and bone mineral density was analyzed using micro-CT. As a result, it was found that there was no significant difference between the normal mouse group (Normal) and the mouse group transplanted with the adrenal organoid chip (Adx+chip) (Fig. 23). Through this, it was confirmed that the adrenal organoid of the present invention does not have the side effect of inducing osteoporosis.

[0307]

[0308] The present invention relates to an adrenal organoid maturation composition, a method for maturating adrenal organoids using the same, and a method for transplanting organoids produced by the method. In the present invention, structurally and functionally mature adrenal organoids produced under optimized culture conditions using the adrenal organoid maturation composition comprising adrenocorticotropic hormone and cyclic adenosine monophosphate are structurally and functionally improved, and it was confirmed that an adrenal organoid chip having a three-dimensional vascular network structure produced using the same replaces the function of the adrenal gland without side effects after transplantation into an actual mouse. According to the present invention, the adrenal organoid maturation composition comprising adrenocorticotropic hormone and cyclic adenosine monophosphate can be usefully utilized for adrenal organoid transplantation or regenerative therapy, drug screening, drug responsiveness evaluation, etc., and through this, low-cost, high-functionality medical products can be produced, which can be effectively used for the prevention or treatment of diseases.

[0309] Therefore, the adrenal organoid maturation composition according to one embodiment of the present invention can be seen as having industrial applicability.

Claims

1. An adrenal organoid maturation composition comprising adrenocorticotropic hormone (ACTH) and cyclic adenosine monophosphate (cAMP).

2. An adrenal organoid maturation composition according to claim 1, which increases the expression of CYP17A1, CYP11B2, CYP11A1, HSD3B2 or CYP21A2 in an adrenal organoid.

3. An adrenal organoid maturation composition that increases hormone secretion of adrenal organoids according to claim 1.

4. An adrenal organoid maturation composition according to claim 1, which increases the secretion of cortisol, corticosterone or aldosterone of the adrenal organoid. 5.1) A step of forming cell aggregates by primary culturing cells derived from adrenal tissue; and 2) A method for producing an adrenal organoid with enhanced hormone secretion function, comprising a step of secondary culturing in a culture medium containing the adrenal organoid maturation composition of claim 1.

6. A method for producing an adrenal organoid with enhanced hormone secretion function, wherein the primary culture is performed for 2 to 4 days in paragraph 5.

7. A method for producing an adrenal organoid with enhanced hormone secretion function, wherein the secondary culture is performed for 5 to 9 days in paragraph 5.

8. An adrenal organoid with enhanced hormone secretion function manufactured by the method of Article 5.

9. An adrenal organoid having increased secretion of cortisol, corticosterone or aldosterone in accordance with claim 8.

10. An adrenal organoid having increased expression of CYP17A1, CYP11B2, CYP11A1, HSD3B2 or CYP21A2 according to claim 8. 11.1) A step of forming a fiber structure using a temperature-responsive polymer; 2) a step of injecting a solution containing the adrenal organoid and biocompatible polymer of clause 8 into a fiber structure to gel it; and 3) A method for manufacturing an adrenal organoid chip, comprising the step of removing a fibrous structure.

12. In the 11th paragraph, the thermoresponsive polymer is poly(N-isopropyl acrylamide) [poly(N-isopropylacrylamide), pNIPAM], poly(N-isopropyl acrylamide-co-allylamine) [poly(N-isopropyl acrylamide-co-allylamine), poly(NIPAM-co-AA)], poly(N-isopropylacrylamide-co-2-(dimethylamino)ethyl methacrylate) [poly(Nisopropylacrylamide-co-2-(dimethylamino)ethyl methacrylate), poly(NIPAM-co-DMAEMA)], poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate) [poly(N-isopropyl acrylamide-co-2-(dimethylamino)ethyl acrylate), poly(NIPAM-co-DMAEA)], poly(N-isopropyl A method for manufacturing an adrenal organoid chip, wherein the adrenal organoid chip is poly(N-isopropyl acrylamide-co-acrylic acid), poly(NIPAM-co-AAc), poly(N-isopropyl acrylamide-co-methacrylic acid), poly(NIPAM-co-MAAc), poly(N,N-diethylacrylamide), poly(N-vinylcaprolactam), poly(ethylene glycol), poly(ethylene glycol-b-propylene glycol-b-ethylene glycol), or polyvinyl methyl ether.

13. In the 11th paragraph, the biocompatible polymer is selected from the group consisting of cellulose, pectin, chondroitin sulfate, decellularized extracellular matrix, fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone) (PCL), polyanhydride, polyorthoester, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), A method for manufacturing an adrenal organoid chip, comprising: poly-N-isopropylacrylamide (PNIPAAm), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, a copolymer thereof, or a mixture thereof.

14. A method for manufacturing an adrenal organoid chip, wherein the chip is gelled by an enzymatic reaction in claim 11.

15. A method for manufacturing an adrenal organoid chip, wherein the fiber structure is removed by perfusing a solution having a temperature at which a heat-sensitive polymer becomes sol in the 11th paragraph.

16. An adrenal organoid chip manufactured by the method of Article 11.

17. An adrenal organoid chip having a three-dimensional vascular microchannel network structure in claim 16.

18. A cell therapy for treating adrenal insufficiency comprising the adrenal organoid of clause 8 or the adrenal organoid chip of clause 16.

19. A composition for transplantation comprising the adrenal organoid of clause 8 or the adrenal organoid chip of clause 16.

20. A method for evaluating the effectiveness of an adrenal disease treatment agent, comprising a step of evaluating drug responsiveness after treating the adrenal organoid of clause 8 or the adrenal organoid chip of clause 16 with a drug.

21. A pharmaceutical composition for preventing or treating adrenal disease, comprising the adrenal organoid of clause 8, the adrenal organoid chip of clause 16, or a culture medium thereof.

22. A pharmaceutical composition for preventing or treating adrenal disease, wherein the adrenal disease in clause 21 is an adrenal cortex disease or an adrenal medulla disease.

23. A pharmaceutical composition for preventing or treating adrenal disease, wherein the adrenal cortex disease in clause 22 is hyperadrenocorticism or adrenal insufficiency.

24. A method for screening for the prevention or treatment of adrenal disease, comprising a step of treating a test substance on the adrenal organoid of clause 8 or the adrenal organoid chip of clause 16.

Images