An adrenal cortex fascicular ribbon organoid and a preparation method and application thereof

Abstract

The application relates to the technical field of organoid culture, in particular to an adrenal cortex ribbon zone organoid and a preparation method and application thereof. The preparation method of the adrenal cortex ribbon zone organoid comprises the following steps: placing adrenal primary cells in a mixed solution of a culture medium and Matrigel for seeding and solidification; and placing the adrenal primary cells in an adrenal cortex organoid culture medium for culture. The adrenal cortex organoid culture medium comprises the following components on the basis of a basic culture medium: nicotinamide 4-6 mM, adrenocorticotropic hormone 10-30 nM, epidermal growth factor 40-60 ng / mL, basic fibroblast growth factor 30-50 ng / mL and forskolin 10-30 muM. The adrenal cortex organoid is successfully constructed in Matrigel through a specific culture medium, the adrenal cortex organoid has complete structure, high cell survival rate and high stability, and has important value for adrenal cortex related treatment target screening and drug development and the like.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to an adrenal cortex zona fasciculata organoid, its preparation method, and its application. Background Technology

[0002] Secondary hypertension has a high prevalence among hypertensive patients, and adrenal disease is one of its core causes. This is particularly pronounced in patients with refractory hypertension, where secondary hypertension caused by adrenal disease accounts for a significant proportion. Common clinical pathological types include primary aldosteronism, Cushing's syndrome, pheochromocytoma and related paragangliomas, and congenital adrenal hyperplasia. These diseases are often accompanied by excessive hormone secretion, causing severe and persistent damage to target organs such as the cardiovascular system and kidneys, and even endangering life. With the widespread use of imaging technology, the detection rate of adrenal incidental tumors has been increasing year by year, but the differentiation between benign and malignant tumors, the elucidation of molecular mechanisms, and the development of individualized treatment strategies for these diseases still face significant challenges.

[0003] Currently, treatment options for adrenal diseases and related hormone-secreting tumors are relatively limited, and patients often face problems such as hormonal imbalances, decreased quality of life, and difficulty in restoring adrenal function after surgery. In the fields of drug development and pathological research, existing evaluation systems mainly rely on specific cell lines, animal models, and two-dimensional culture of primary cells. However, cell lines often fail to simulate the heterogeneity of patient tumors; animal models cannot be fully equivalent to human physiological processes due to species differences; and traditional two-dimensional culture systems, lacking three-dimensional spatial structure, cannot reproduce the complex tumor microenvironment in vivo, leading to the gradual loss of original gene expression and physiological functions in cells. Therefore, establishing a reliable model system that more closely reflects the clinical pathological characteristics of humans has become an urgent need in this research field.

[0004] Organoid technology, as an emerging biomedical approach, involves the three-dimensional culture of tissue stem cells in vitro to create miniature tissues that are highly similar to the source organs in terms of spatial structure, gene expression, and physiological function. Compared to traditional culture methods, organoids, as organic combinations of different types of functional cells, can more realistically simulate the living space and growth state of cells in vivo, demonstrating significant application value in drug screening, disease modeling, and tissue engineering. Although preliminary explorations have emerged in the industry regarding adrenal cortex organoids, significant limitations remain in the stability of the culture system, the integrity of structure and function, and the complexity of cell composition, which greatly restricts their widespread application in basic research and clinical translation. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides an adrenal cortical zona fasciculata organoid, its preparation method, and its application.

[0006] In a first aspect, the present invention provides an adrenal cortex organoid culture medium, wherein the adrenal cortex organoid culture medium, based on a basal culture medium, comprises: Nicotinamide 4-6 mM, adrenocorticotropic hormone 10-30 nM, epidermal growth factor 40-60 ng / mL, basic fibroblast growth factor 30-50 ng / mL, and forsocrine 10-30 μM.

[0007] During the research process, this invention explored various formulations of adrenocortical zona fasciculata organoid culture media containing different growth factors or cell signaling molecules. For example, when using Wnt3a, RSPO1, and Noggin, regardless of the optimized dosage, the organoid culture success rate was extremely low after long-term culture, and no obvious three-dimensional structure was observed. This invention further employed a combination of growth factors and cell signaling molecules without adrenocorticotropic hormone, basic fibroblast growth factor, epidermal growth factor, and forscolin. The results showed that forscolin was an essential component for organoid formation and growth in the constructed organoid culture system. In the forscolin-free conditioned culture system, organoids could not form or proliferate; while in the culture system with added forscolin, organoid structural formation and development were significantly enhanced, effectively promoting functional maturation and hormone secretion capacity. After repeated exploration, the finally constructed adrenocortical organoids showed an unexpectedly high degree of similarity to primary tissues in morphology, structure, and physiological characteristics, and also exhibited high consistency in hormone secretion and protein expression. In addition, this adrenocortical organoid also showed high passage stability, with no substantial changes in morphology and structure after multiple passages.

[0008] The amounts of each component in the culture medium described in this invention are based on the basal culture medium.

[0009] Furthermore, the adrenal cortex organoid culture medium, in addition to the basal culture medium, includes: Nicotinamide 4.5-5.5 mM, adrenocorticotropic hormone 15-25 nM, epidermal growth factor 45-55 ng / mL, basic fibroblast growth factor 35-45 ng / mL, and forsocrine 15-25 μM.

[0010] The preferred amounts of growth factors and cell signaling pathway regulators used in this invention can produce adrenocortical organoids with higher consistency with primary tissues.

[0011] Furthermore, the adrenal cortex organoid culture medium also includes one or more of the following: serum substitute, glutamine dipeptide, buffer, antioxidant, or antibiotic; Preferably, the adrenal cortex organoid culture medium further includes: B27 additive, L-alanyl-L-glutamine, HEPES buffer, N-acetyl-L-cysteine, and penicillin-streptomycin bispecific antibody.

[0012] Furthermore, the adrenal cortex organoid culture medium further includes: B27 additive 0.5-5 v / v%, L-alanyl-L-glutamine 0.2-4 v / v%, HEPES buffer 0.2-4 v / v%, N-acetyl-L-cysteine ​​0.2-4 mM, and penicillin-streptomycin antibiotic 0.05-0.5 v / v%. Preferably, the culture medium further includes: B27 additive 1-4 v / v%, L-alanyl-L-glutamine 0.5-3 v / v%, HEPES buffer 0.5-3 v / v%, N-acetyl-L-cysteine ​​0.5-3 mM, and penicillin-streptomycin antibiotic 0.1-0.4 v / v.

[0013] The present invention preferably uses the above-mentioned nutritional maintenance components, which can help maintain the structural and functional integrity of adrenal cortical organoids and ensure a high cell survival rate during culture.

[0014] Furthermore, the basal culture medium is selected from one of DMEM, DMEM / F12, Advanced DMEM / F12, or RPMI 1640 medium; Preferably, the basal culture medium is DMEM / F12.

[0015] Furthermore, the adrenal cortex organoid culture medium, based on DMEM / F12 medium, also includes: B27 additive 1-3 v / v% L-Alanyl-L-Glutamine 0.5-2 v / v% HEPES buffer 0.5-2 v / v% N-acetyl-L-cysteine ​​0.5-2 mM; Penicillin-streptomycin bispecific antibody 0.1-0.3 v / v% Nicotinamide 4.5-5.5 mM; Adrenocorticotropic hormone 15-25 nM; Epidermal growth factor 45-55 ng / mL; Basic fibroblast growth factor 35-45 ng / mL; Forscolin 15-25 μM.

[0016] In a second aspect, the present invention provides a method for constructing adrenal cortex organoids, comprising: Primary adrenal cells were seeded and cured in a mixture of the aforementioned adrenal cortical organoid culture medium and matrix gel, wherein the mass ratio of the adrenal cortical organoid culture medium to matrix gel in the mixture was 1:(0.5-2), and the curing conditions included treatment at 35-40°C for 20-45 min. The adrenal cortex organoids were cultured in the aforementioned adrenal cortex organoid culture medium.

[0017] Furthermore, the primary adrenal cells were prepared by the following method: The adrenal gland tissue was shredded, digested with type II collagenase, filtered, centrifuged, and resuspended to remove the precipitate.

[0018] Furthermore, the concentration of the type II collagenase is 0.5-4 mg / ml, preferably 1-3 mg / ml.

[0019] Furthermore, the digestion is carried out in a water bath shaker at a speed of 100-300 rpm, a water bath temperature of 30-40°C, and a digestion time of 30-50 min. Preferably, the speed is 150-250 rpm, the water bath temperature is 32-38°C, and the digestion time is 35-45 min.

[0020] Furthermore, the filter used in the filtration process has a pore size of 40-80 μm.

[0021] Furthermore, the resuspension of the precipitate is carried out at 0-10°C, and the culture dishes inoculated after resuspension are preheated at 35-40°C.

[0022] Furthermore, the cultivation conditions include: cultivation at 35-40℃ for 6-10 days.

[0023] Furthermore, the primary adrenal cells are derived from humans.

[0024] Furthermore, the primary adrenal cells are derived from adrenal tumor tissue or normal tissue adjacent to an adrenal tumor.

[0025] The culture method provided by this invention, combined with the aforementioned culture medium, can construct adrenal cortex organoids that can highly simulate the physiological microenvironment and tissue specificity of the real adrenal cortex. These organoids can be further applied to the research of adrenal cortex-related diseases and have important application value.

[0026] Thirdly, the present invention provides an adrenal cortex organoid, which is constructed by the aforementioned method.

[0027] Fourthly, the present invention provides the application of the aforementioned adrenal cortical organoids in any of the following: (1) Construct an adrenal cortex organoid xenotransplantation model; (2) Constructing an adrenal cortex organoid gene editing model; (3) Construct an adrenocortical organoid disease model; (4) Construct an adrenal cortex organoid sample library; (5) Adrenal gland development and regenerative medicine research; (6) Research on adrenocortical tumors; (7) To study the molecular mechanisms of adrenal gland development and regeneration; (8) To study the pathogenesis and development mechanism of Cushing's syndrome / cortisol adenoma; (9) Prepare or screen drugs for the treatment of Cushing's syndrome / cortisol adenoma; (10) Screening for relevant biomarkers of Cushing's syndrome / cortisol adenoma.

[0028] The adrenal cortex organoids prepared by this invention can be widely applied in various fields of basic and translational medicine. They self-assemble in three-dimensional space, reproducing the complex multi-level tissue structure of the adrenal cortex in vivo, providing a realistic research platform for studying intercellular interactions and the microenvironment. Furthermore, these organoids not only express specific cortical markers but also possess a complete steroid hormone synthase system and multiple hormone secretion functions. In addition, these organoids can be stably expanded in vitro for extended periods without losing the genetic characteristics of the original cells. The highly biomimetic characteristics of these organoids in terms of tissue morphology, endocrine function, and genetic background collectively constitute the basis for their feasible application in multiple downstream fields.

[0029] Based on the above principles, the organoids of this invention can be used as highly accurate in vitro models for disease modeling and drug screening. By constructing organoids using patient-derived stem cells carrying pathogenic mutations, or by applying specific pathological stimuli to healthy organoids in vitro, the pathological phenotypes of Cushing's syndrome or corticosteroid adenomas can be reconstructed in vitro. Based on this, researchers can screen for novel disease-related biomarkers through multi-omics sequencing comparisons. More importantly, by adding candidate drugs to the culture system and detecting phenotypic changes in organoid structure and function, targeted drugs for treating Cushing's syndrome or corticosteroid adenomas can be screened and evaluated with high throughput and high precision.

[0030] In terms of in vivo applications and mechanistic studies, the organoids of this invention provide an excellent vehicle for xenotransplantation and developmental regeneration research. When the organoids prepared according to this invention are transplanted into immunodeficient animals (such as the subcapsular region of mouse kidneys), they can rapidly aggregate in host microvessels, achieving vascularization and long-term survival. Using this xenotransplantation model, on the one hand, the colonization and tissue regeneration process of cortical progenitor cells in vivo can be dynamically tracked, allowing for in-depth analysis of the molecular mechanisms of adrenal gland development; on the other hand, this model can realistically simulate the invasive characteristics of cortical tumors in vivo and their systemic effects on the endocrine axis, providing a living in vivo testing environment closest to that of humans for preclinical mechanistic studies and drug efficacy evaluation.

[0031] Furthermore, thanks to the optimized culture system of this invention, the organoids exhibit excellent in vitro manipulability and survival stability. Technicians can utilize gene editing technologies (such as CRISPR / Cas9) to precisely modify specific genes in the organoids, thereby constructing gene-editing models for target validation. Simultaneously, the organoids prepared by this invention demonstrate good cryopreservation and resuscitation tolerance, supporting the standardization of organoids from diverse genetic backgrounds (including normal donors and patients with various adrenal diseases). This allows for the construction of a living adrenal cortical organoid sample library with multidimensional phenotypes and genome annotations, enabling the long-term preservation and shared application of valuable biomedical resources.

[0032] The present invention has the following beneficial effects: This invention addresses the cellular origin and tissue homeostasis maintenance mechanisms of the zona fasciculata in the adrenal cortex, providing a specific culture medium for adrenal cortical organoids. Based on this medium, zona fasciculata organoids can be successfully constructed in Matrigel, forming three-dimensional organoids that highly resemble the real zona fasciculata in morphology and physiological function within a short time. Furthermore, the prepared zona fasciculata organoids also possess a high aldosterone secretion capacity.

[0033] The three-dimensional adrenal cortex organoids obtained using the culture medium and method provided in this invention can highly simulate the physiological microenvironment of the real adrenal cortex, effectively maintain cell tissue specificity, and maintain a high degree of consistency with the real adrenal cortex in terms of tissue structure, morphology, and gene and protein expression profiles. They have multiple application values: on the one hand, they can serve as heritable disease models for mechanistic studies of adrenal cortex-related diseases (such as Cushing's syndrome); on the other hand, they provide an experimental pathway for organoid transplantation-guided adrenal tissue regeneration and functional reconstruction, further laying a strategic foundation with both translational potential and platform function for the restoration of endocrine homeostasis in patients with adrenal insufficiency. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a photograph of the seeding process on a culture plate, as provided in an embodiment of the present invention.

[0036] Figure 2 These are the microscopic views (magnification 4X) of primary organoids cultured on days 2 and 10, as provided in this embodiment of the invention.

[0037] Figure 3 These are the microscopic views (magnification 4X) of the second-generation primary organoids cultured on days 3 and 6, as provided in this embodiment of the invention.

[0038] Figure 4 These are the microscopic views (magnification 4X) of the third-generation primary organoids cultured on day 0 and day 9 of this invention.

[0039] Figure 5 This is the microscopic appearance of the primary organoids cultured on day 12 (magnification 10X) provided in this embodiment of the invention.

[0040] Figure 6 This is the microscopic appearance of the primary organoids on day 12 of the first generation of culture provided in this embodiment of the invention (magnification 10X).

[0041] Figure 7 These are the microscopic views (magnification 4X) of primary organoids cultured on days 3 and 9, as provided in this embodiment of the invention.

[0042] Figure 8 These are the microscopic views (magnification 4X) of the second-generation primary organoid culture on day 0 and day 15 provided in this embodiment of the invention.

[0043] Figure 9 This is the microscopic appearance (magnification 4X) of the third-generation primary organoid culture on day 14 provided in this embodiment of the invention.

[0044] Figure 10 This is the microscopic appearance of primary organoid culture on day 9 (magnification 10X) provided in this embodiment of the invention.

[0045] Figure 11 The results are immunofluorescence staining (CYP11B1 and CYP11B2) on day 11 of the second-generation primary organoid culture provided in this embodiment of the invention.

[0046] Figure 12 This is the immunofluorescence staining result (KI67) on day 11 of the second-generation primary organoid culture provided in this embodiment of the invention.

[0047] Figure 13 The results of primary organoid proliferation detection based on ATP content are provided in the embodiments of the present invention.

[0048] Figure 14 These are the results of cortisol and aldosterone detection in primary organoids provided in this embodiment of the invention.

[0049] Figure 15 These are the results of cortisol and aldosterone detection in the fourth and third generations of primary organoid cultures provided in this embodiment of the invention.

[0050] Figure 16 The results are from the second-generation qPCR detection of primary organoid culture provided in this embodiment of the invention.

[0051] Figure 17 The image shows the results of culturing adrenal cortical organoids in the culture medium of Comparative Example 2 provided in this embodiment of the invention; where A is the light microscopy result on the first day and B is the light microscopy result on the eleventh day.

[0052] Figure 18 This is a light micrograph of the culture medium used in Comparative Example 3 of the present invention to culture adrenocortical organoids (the result on day 9); where A is the complete culture medium of Example 1, B is A without adrenocorticotropic hormone, C is A without basic fibroblast growth factor, D is A without epidermal growth factor, and E is A without foscorine. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0055] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0056] The matrix adhesive used in the following examples is Corning Matrigel.

[0057] Example 1: Method for preparing adrenal cortical organoids 1. Adrenal gland specimen processing: (1) Quickly transfer the surgical sample into a centrifuge tube containing tissue preservation solution. The main component of the preservation solution is DMEM / F12. Add 0.2% Penicillin-Streptomycin bispecific antibody to the preservation solution. Place the centrifuge tube in an ice pack and send it to the laboratory.

[0058] (2) Perform the procedure in a biosafety cabinet, transferring the adrenal gland tissue to a culture dish and washing it three times with DMEM / F12 solution containing 0.2% penicillin-streptomycin. Transfer the tissue to a new culture dish containing the above tissue washing solution and remove any necrotic tissue. Cut the tissue into pieces approximately 0.5 mm in size. 3 Small and large tissue fragments.

[0059] (3) Place the surgical specimen (adrenocortical adenoma tissue or adjacent normal adrenal tissue) into a 1.5 ml EP tube containing 500 μl of 0.2% Penicillin-Streptomycin in DMEM / F12 medium, and mince the tissue with sterile tissue scissors. Transfer to a 50 ml centrifuge tube.

[0060] (4) Add digestive enzyme (2 mg / ml type II collagenase) to a 50 ml centrifuge tube, and digest at 37℃ and 120 rpm for 50 min until no obvious tissue block remains. Then pipette up and down 10 to 20 times to further promote tissue digestion.

[0061] (5) Filter the digested suspension through a 40 μm filter to obtain a cell suspension; centrifuge at 258g for 5 min, discard the supernatant, add 1 ml of red blood cell lysis buffer to resuspend, pipette and shake to lyse for 1 min, add 10 times the volume of DMEM / F12 medium to dilute, terminate the lysis, centrifuge at 258g for 3 min, and discard the supernatant.

[0062] (6) After resuspending the cell pellet in DMEM / F12 containing 0.2% Penicillin-Streptomycin, count the cells with a cell counter. Based on the counting results, centrifuge again and add an appropriate amount of organoid culture medium to resuspend the cell pellet. Place it on ice.

[0063] (7) Wherein, the composition ratio of the culture medium is: 500 mL DMEM / F12, and the following components based on the volume of DMEM / F12: 1% GlutaMax; 0.2% Penicillin-Streptomycin; 1% HEPES buffer; 2% B27 additive; 50 ng / ml human epidermal growth factor (EGF); 40 ng / ml human basic fibroblast growth factor FGF2; 1 mM N-acetyl-L-cysteine; 5 mM nicotinamide; 20 nM ACTH and 20 μM Forskolin.

[0064] In this culture medium, except for DMEM / F12, the amount or concentration of other components is based on DMEM / F12; the B27 additive is 50X B27 additive.

[0065] 2. Adrenal primary cell seeding and 3D culture: (1) Take an appropriate amount of the final cell suspension from step 1 and mix it with matrix gel, wherein each 20 μl of matrix gel-cell suspension contains approximately 1 x 102 cells. 5 10 cells were seeded into 24-well cell culture plates, with 20 μl of Matrigel-cell suspension added to each well, forming hemispherical droplets in the center of the plate. After seeding... Figure 1 As shown. Since the matrix gel is liquid at 0-10℃ and solidifies when the temperature rises, the relevant operations of the matrix gel are performed on ice. At the same time, a pre-cooled pipette tip is used to mix the matrix gel with the cell suspension and to seed the matrix gel-cell suspension to prevent the matrix gel from solidifying prematurely.

[0066] (2) After the gel is seeded, place the 24-well plate in a cell culture incubator (37°C, 5% CO2) for 30 min to allow the matrix gel-cell suspension to solidify.

[0067] 3. Culture medium induces adrenal cortical organoid formation: (1) Take out the 24-well plate from the incubator and slowly add 500 μl of organoid culture medium along the side wall of the well. After adding the medium, put the 24-well plate back into the cell culture incubator (37℃, 5% CO2) for culture.

[0068] (2) The time for changing the medium is determined according to the growth status of the organoids and the color of the culture medium. Generally, the medium is changed every 2-3 days until the organoids mature.

[0069] During this period, the organoids are observed under an inverted microscope. Generally, if the organoids stop growing after 9-10 days, they are passaged. The passage ratio is 1:2 in a 24-well plate (i.e., the primary adrenocortical organoids from one well are passaged into two wells in a new 24-well plate).

[0070] During organoid passage, discard the original culture medium and melt the matrix gel with pre-cooled Y-27632 organoid culture medium containing 20 μM. Gently pipette the mixture of matrix gel and organoids and transfer it to a centrifuge tube. Centrifuge, discard the supernatant, and resuspend the pellet in an appropriate amount of organoid culture medium. Mix an appropriate amount of matrix gel with the cell suspension, and then inoculate and culture the cells, following the same steps as for primary adrenal cortex organoids.

[0071] Example 2 In this invention, the same method as in Example 1 was used to prepare adrenal cortical organoids using different specimens. A total of 8 samples were prepared (Example 1 to Example 8). All specimens in the examples were selected from the Department of Urology, Peking University First Hospital. The tumor specimens were selected from surgically resected specimens of patients with adrenocortical adenomas, and the adrenal tissue adjacent to the tumor was selected from surgically resected specimens of patients with adrenal tumors (e.g., normal tissue adjacent to aldosterone-producing tumors, cortisol tumors, and adrenocortical carcinomas).

[0072] Experimental Example 1: Detection of Adrenal Cortical Organoids This invention examines the adrenal cortical organoids prepared in Examples 1 and 2, specifically by observing and photographing the morphology of adrenal cortical organoids at different growth generations under an inverted microscope.

[0073] Figure 2 The images show the microscopic appearance of the primary organoid cultured in Example 2 on days 2 and 10 (magnification 4X).

[0074] Figure 3 The images show the microscopic appearance of the primary organoids in Example 2 on days 3 and 6 of second-generation culture (magnification 4X).

[0075] Figure 4 The images show the microscopic appearance of the primary organoids in Example 2 on days 0 and 9 of third-generation culture (magnification 4X).

[0076] Figure 5 The image shows the primary organoid culture of Example 2 on day 12 of fourth-generation culture (magnification 10X).

[0077] Figure 6 The image shows the primary organoid culture of Example 3 on day 12 of the first generation culture (magnification 10X).

[0078] Figure 7 The images show the microscopic appearance of the primary organoid cultured in Example 2, on days 3 and 9 (magnification 4X).

[0079] Figure 8 The images show the microscopic appearance of the primary organoids in Example 2, on day 0 and day 15 of second-generation culture (magnification 4X).

[0080] Figure 9 This is the microscopic appearance of the fourth primary organoid in Example 2 on day 14 of third-generation culture (magnification 4X).

[0081] Figure 10 The image shows the primary organoid cultured in Example 2, case 5, on day 9 (magnification 10X).

[0082] Figure 11 The results of immunofluorescence staining on day 11 of the second-generation culture of the primary organoid in Example 2 (Case 6).

[0083] Primary (P1) adrenal cortex organoid morphology as follows Figure 2 and Figure 7 As shown. After passage, second-generation (P2) adrenocortical organoids were obtained. The microscopic appearance of P2 is as follows. Figure 3 and Figure 8 As shown. After passage, third-generation (P3) adrenocortical organoids were obtained. The microscopic appearance of P3 generation is as follows. Figure 4 and Figure 9 As shown. Fourth-generation (P4) adrenocortical organoids were obtained through further subculturing and cultured for 12 days. Microscopic appearance is shown below. Figure 5 .

[0084] Figures 2-5 The results showed that after passage culture, the tissue cell morphology of the P2, P3, and P4 generation adrenocortical organoids was similar to or remained unchanged from that of the primary adrenocortical organoids, indicating high stability.

[0085] Under high magnification, the organoids exhibit a typical grape-bead-like three-dimensional structure composed of multiple cells. Figure 6 and Figure 10 Immunofluorescence staining results further confirmed this structural feature. Figure 11 ).

[0086] 2. This invention further employs the CellTiter-Glo® 3D Cell Viability Assay method to detect proliferation activity, homogeneous time-resolved fluorescence method to detect aldosterone levels, electrochemiluminescence method to detect cortisol levels, qRT-PCR method to detect the expression levels of Mc2r, star, CYP11A1, CYP11B1, CYP11B2, and SULT2A1, and uses immunofluorescence to identify the zona spheroid marker CYP11B2, the zona fasciculata marker CYP11B1, and the proliferation marker KI67. Figure 11 and Figure 12 For each test method, refer to the laboratory manual in this field, or follow the conditions recommended in the manufacturer's instructions.

[0087] Figure 12The results of ATP-based proliferation detection of primary organoids in Example 2, Case 7 (A) and Example 2, Case 8 (B) are shown.

[0088] Figure 13 The results of cortisol and aldosterone testing in primary organoids of Examples 6 and 7 of Example 2 are shown.

[0089] Figure 14 The results of cortisol and aldosterone detection are for the fourth generation of primary organoid culture in Example 2 (Case 2) and the third generation of primary organoid culture in Example 2 (Case 3).

[0090] Figure 15 This is the result of second-generation qPCR detection in the third case of primary organoid culture in Example 2.

[0091] In the primary adrenal cortex organoid culture process, proliferative activity was measured after harvesting organoids on days 3, 6, and 9. Figure 13 The results showed that organoid proliferation activity was significantly enhanced with prolonged culture time, increasing by 52.34% from day 3 to day 6 and by 103.75% from day 6 to day 9.

[0092] During the culture process, the culture medium was replaced with fresh medium every 3 days, and the levels of accumulated aldosterone and cortisol secreted in the medium were measured. The results showed that the hormone secretion of the organoids increased significantly with prolonged culture time. Figure 14 Aldosterone levels reached 18284.96 pmol / L at day 3, 25957.60 pmol / L at day 6, and 38397.76 pmol / L at day 9; cortisol levels reached 29.2 ng / dL at day 3, 129.0 ng / dL at day 6, and 348.0 ng / dL at day 9. This indicates a continuous enhancement of functional activity, indirectly reflecting the continuous proliferation of organoids. Furthermore, after stable cryopreservation and passage to P3 and P4 generations, the organoids maintained a relatively stable hormone secretion capacity. Figure 15 ).

[0093] qPCR results showed that adrenal cortical organoids highly expressed zona fasciculata-specific markers, represented by CYP11B1. Figure 16 This indicates that it possesses the main molecular characteristics and functional potential of fasciculations.

[0094] As can be seen from the above results, the adrenal cortex zona fasciculata organoids constructed in this invention are highly consistent with the primary tissues in terms of morphology, structure, hormone secretion, and protein expression. They also exhibit high proliferative activity and stability, demonstrating significant application value. Furthermore, while exhibiting obvious characteristics of adrenal cortex zona fasciculata organoids, they also possess a high aldosterone secretion capacity, exceeding general expectations in the field.

[0095] Comparative Example 1 This comparative study used different culture media to construct adrenocortical organoids based on the existing literature: "Human adrenocortical organoids for tissue regeneration and disease modeling". The culture media used were as follows: advanced DMEM / F-12 basal medium supplemented with B27 additive, penicillin-streptomycin 1%, 100 μg / mL Primocin, GlutaMax additive, 10 mM HEPES, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 500 ng / mL R-spondin1, 200 ng / mL WNT3A, 50 ng / mL EGF, 100 ng / mL FGF10, 25 ng / mL FGF7, 50 ng / mL FGF2, 500 nM A8301 and 5 μM Y-27632.

[0096] This paper compares the methods of constructing adrenal cortex organoids using low-adsorption suspension culture and matrix gel embedding. However, their experiments suggest that the proliferative capacity of adult adrenal stem cells is limited. This limitation makes it extremely difficult to grow organoids from single cells in Matrigel, and self-assembly cannot be completed within this space. Although many mature organoids currently use Matrigel, the self-assembly of adrenal organoids in Matrigel remains a technical bottleneck.

[0097] This invention solves this technical problem by successfully constructing adrenal cortex organoids (possessing complete function and normal physiological activity) using adult adrenal stem cells in Matrigel, based on the culture medium described in this application. Compared to the organoids in the literature (which formed functionally complete organoids in about 20 days), the organoids constructed in this invention exhibit stronger proliferative activity (doubling in about 3 days, and obtaining well-formed, highly proliferative, and relatively complete physiological organoids in 6-9 days) and physiological function, and can simultaneously secrete aldosterone and cortisol (aldosterone levels were low in the literature).

[0098] Comparative Example 2 This comparative example uses the same method as Example 1 to prepare adrenal cortical organoids, the only difference being the composition ratio of the culture medium: 500 ml DMEM / F12, and the following components based on the volume of DMEM / F12: 1% GlutaMax; 0.2% Penicillin-Streptomycin; 1% HEPES buffer; 2% B27 additive; 1mM N-acetyl-L-cysteine; 20 ng / ml Wnt3a; 20 ng / ml RSPO1; 50 ng / ml Noggin.

[0099] In this culture medium, except for DMEM / F12, the amount or concentration of other components is based on DMEM / F12; the B27 additive is 50X B27 additive.

[0100] The results are as follows Figure 17 As shown, the culture success rate is extremely low under this culture medium composition, and no obvious three-dimensional structure is observed. Furthermore, no matter how the dosage is optimized, it is difficult to successfully culture organoid structures.

[0101] Comparative Example 3 This comparative example uses the same method as Example 1 to prepare adrenocortical organoids, the only difference being the removal of adrenocorticotropic hormone, basic fibroblast growth factor, epidermal growth factor and forsocrine from the culture medium.

[0102] The results are as follows Figure 18 As shown, forsocolin is an essential component for organoid formation and growth; organoids cannot form and proliferate in conditioned culture systems without forsocolin. In contrast, while culture media lacking other components can still induce the formation of three-dimensional organoids, their diameter is reduced, the culture period is prolonged, and their proliferative activity is lower.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for adrenal cortex organoids, characterized in that, The adrenal cortex organoid culture medium, based on the basal culture medium, includes: Nicotinamide 4-6 mM, adrenocorticotropic hormone 10-30 nM, epidermal growth factor 40-60 ng / mL, basic fibroblast growth factor 30-50 ng / mL, and forsocrine 10-30 μM.

2. The adrenal cortex organoid culture medium according to claim 1, characterized in that, The adrenal cortex organoid culture medium, based on the basal culture medium, includes: Nicotinamide 4.5-5.5 mM, adrenocorticotropic hormone 15-25 nM, epidermal growth factor 45-55 ng / mL, basic fibroblast growth factor 35-45 ng / mL, and forsocrine 15-25 μM.

3. The adrenal cortex organoid culture medium according to claim 1 or 2, characterized in that, The adrenal cortex organoid culture medium also includes one or more of the following: serum substitutes, glutamine dipeptides, buffers, antioxidants, or antibiotics.

4. The adrenal cortex organoid culture medium according to claim 3, characterized in that, The adrenal cortex organoid culture medium also includes: B27 additive 0.5-5 v / v%, L-alanyl-L-glutamine 0.2-4 v / v%, HEPES buffer 0.2-4 v / v%, N-acetyl-L-cysteine ​​0.2-4 mM, and penicillin-streptomycin antibiotic 0.05-0.5 v / v.

5. The adrenal cortex organoid culture medium according to claim 1 or 2, characterized in that, The basal culture medium is selected from one of DMEM, DMEM / F12, Advanced DMEM / F12 or RPMI 1640 media.

6. The adrenal cortex organoid culture medium according to claim 1, characterized in that, The adrenal cortex organoid culture medium, in addition to the DMEM / F12 medium, also includes: B27 additive 1-3 v / v% L-Alanyl-L-Glutamine 0.5-2 v / v% HEPES buffer 0.5-2 v / v% N-acetyl-L-cysteine ​​0.5-2 mM; Penicillin-streptomycin bispecific antibody 0.1-0.3 v / v% Nicotinamide 4.5-5.5 mM; Adrenocorticotropic hormone 15-25 nM; Epidermal growth factor 45-55 ng / mL; Basic fibroblast growth factor 35-45 ng / mL; Forscolin 15-25 μM.

7. A method for constructing adrenal cortex organoids, characterized in that, include: Primary adrenal cells were seeded and cured in a mixture of adrenal cortical organoid culture medium and matrix gel according to any one of claims 1-6, wherein the mass ratio of adrenal cortical organoid culture medium to matrix gel in the mixture was 1:(0.5-2), and the curing conditions included treatment at 35-40°C for 20-45 min. Adrenal cortical organoids are cultured in the adrenal cortical organoid culture medium as described in any one of claims 1-6.

8. The method according to claim 7, characterized in that, The cultivation conditions include: culturing at 35-40℃ for 6-10 days.

9. An adrenal cortex organoid, characterized in that, The adrenal cortical organoid is constructed by the method described in claim 7 or 8.

10. The use of the adrenal cortex organoid of claim 9 in any of the following: (1) Construct an adrenal cortex organoid xenotransplantation model; (2) Constructing an adrenal cortex organoid gene editing model; (3) Construct an adrenocortical organoid disease model; (4) Construct an adrenal cortex organoid sample library; (5) Adrenal gland development and regenerative medicine research; (6) Research on adrenocortical tumors; (7) To study the molecular mechanisms of adrenal gland development and regeneration; (8) To study the pathogenesis and development mechanism of Cushing's syndrome / cortisol adenoma; (9) Prepare or screen drugs for the treatment of Cushing's syndrome / cortisol adenoma; (10) Screening for relevant biomarkers of Cushing's syndrome / cortisol adenoma.

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