Adrenal organoid culture medium and application thereof

Abstract

The present invention relates to organoid culture. The present invention provides an adrenal organoid culture medium and its application, wherein the culture medium components include FGF2, Wnt-3a and ACTH, and when the culture medium contains any one of FGF2, Wnt-3a and ACTH, the growth of adrenal organoids is significantly promoted, and the cell activity of adrenal organoids can be improved. In addition, through lentiviral transfection technology, an adrenal cortical adenoma disease model with PRKACA protein L206R mutation is successfully constructed based on the adrenal organoids, which lays a foundation for in vitro observation, interference with adrenal tissue occurrence and hormone secretion, and research on life activities such as adrenal disease generation and damage repair.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to an adrenal organoid culture medium and application thereof. Background Art

[0002] The adrenal gland is an important organ in the human endocrine system. It is located above the two kidneys, one on each side, and is wrapped by renal fascia and adipose tissue. The adrenal gland is composed of the outer adrenal cortex and the inner adrenal medulla, and the two are different in development, structure and function. The adrenal medulla from the ectoderm is wrapped by the cortex from the mesoderm. The chromaffin cells in the medulla provide the body with adrenal hormones, etc. The adrenal cortex is responsible for synthesizing corticosteroid hormones using cholesterol. The two structures interact with each other and coordinately regulate the body's various life activities to maintain human homeostasis. Therefore, the adrenal gland is an important organ in the human body. If dysfunction occurs or lack of timely repair, it will lead to the occurrence of a variety of metabolic disorders. The hyperplasia and tumorization of the gland itself, or the influence of other pathogenic tissues will also cause hormone imbalance, ultimately endangering life safety.

[0003] Organoids are cultures of stem cells or precursor cells in vitro in three dimensions, which are formed through cell self-proliferation, directed differentiation, lineage commitment and self-assembly, and have organ-specific cell composition, key structures and functional characteristics. Organoids can partially or even completely restore the cell composition and structural functions of organs in the body, and maintain genetic and phenotypic stability during long-term expansion, solving the difficulty that traditional biological models cannot simulate tissues and organs, and shifting the research of in vitro tissues and organs from the surface level of two-dimensional cells to the deep level of three-dimensional tissues, providing a good model for basic research such as organ development, and opening up an innovative path for medical transformation and disease treatment.

[0004] There is currently a lack of adrenal organoid culture media on the market, so there are no adrenal organoids and derived disease models. Due to the lack of research models, the world's research on the adrenal glands and related diseases is very limited. People cannot observe and interfere with the life activities of the adrenal glands such as tissue development and hormone secretion in vitro, nor can they study the occurrence and damage repair of adrenal gland diseases, which also makes it difficult to improve the clinical treatment of adrenal diseases. Therefore, it is urgent to develop a culture method suitable for the stable growth of adrenal organoids. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0006] Therefore, the first aspect of the present invention provides an adrenal organoid culture medium, the culture medium comprising a basic culture medium component and an additive, wherein:

[0007] The additives include FGF2, Wnt-3a and ATCH.

[0008] The present invention provides a culture method suitable for the stable growth of adrenal organoids. The culture medium of the present invention is used to culture adrenal organoids, which has the advantages of short culture cycle, rapid proliferation, high cell number and cell survival rate, and long-term stable passage. It lays a foundation for in vitro observation, interference with adrenal tissue development and hormone secretion, and research on life activities such as disease generation and injury repair of the adrenal glands.

[0009] In order to improve the success rate of primary culture and passage expansion of adrenal organoids and meet the nutritional needs of adrenal organoids, the present invention proposes a culture medium for adrenal organoids, the culture medium contains FGF2, Wnt-3a and ATCH factors. Through experimental verification, these three factors are particularly important for the growth of adrenal organoids, among which FGF2 is a member of the fibroblast growth factor family, which can bind to specific fibroblast growth factor receptors (FGFRs), promote cell proliferation, tissue repair, and participate in angiogenesis; Wnt-3a is the transcription product of the wnt gene, a key factor in the Wnt signaling pathway, which can regulate β-catenin through positive feedback by binding to the Wnt receptor, and can maintain cell stemness; ATCH is adrenocortical hormone, a polypeptide hormone secreted by the pituitary gland of vertebrates, which can maintain tissue hyperplasia of the adrenal cortex, promote the self-organizing ability of adrenal organoids, and assist in the improvement of adrenal function. The inventors found that during the culture process of adrenal organoids, FGF2 can support the formation of a tight spherical structure of adrenal organoids, Wnt-3a can support the growth of adrenal organoids in higher quantities, and ATCH can induce adrenal organoids to secrete granules. These three factors and their corresponding functions are crucial to the self-organizing ability of adrenal cells and the restoration of the microenvironment.

[0010] According to a specific embodiment of the present invention, the concentration of FGF2 is 2-100 ng / mL, preferably 5-50 ng / mL, and further preferably 10-30 ng / mL. When the FGF2 in the culture medium is within this concentration range, it can ensure a better growth state of adrenal organoids, promote cell survival and cell proliferation, and enable adrenal organoids to form a tight spherical structure.

[0011] According to a specific embodiment of the present invention, the concentration of Wnt-3a is 50-500 ng / mL, preferably 70-400 ng / mL, and further preferably 100-300 ng / mL. When Wnt-3a in the culture medium is within this concentration range, it can upregulate the Wnt / β-catenin pathway, ensure the good growth state of adrenal organoids, and maintain the stemness of cells.

[0012] According to a specific embodiment of the present invention, the concentration of ATCH is 0.5-10nM, preferably 1-8nM, and further preferably 1-5nM. When ATCH in the culture medium is within this concentration range, it can maintain the proliferation of adrenal cells, promote the self-organizing ability of adrenal organoids, and induce the formation of secretory granules.

[0013] According to a specific embodiment of the present invention, the additive further includes EGF, FGF7, and FGF10.

[0014] According to a specific embodiment of the present invention, the concentration of EGF is 50-500 ng / mL, preferably 70-200 ng / mL.

[0015] According to a specific embodiment of the present invention, the concentration of FGF7 is 1-100 ng / mL, preferably 10-50 ng / mL.

[0016] According to a specific embodiment of the present invention, the concentration of FGF10 is 50-500 ng / mL, preferably 70-200 ng / mL.

[0017] According to a specific embodiment of the present invention, the additive further includes an anti-apoptotic factor ROCK inhibitor (Y27632), an ALK-5 inhibitor (A83-01), N-acetylcysteine, R-spondin, and Nicotinamid.

[0018] According to a specific embodiment of the present invention, the concentration of the anti-apoptotic factor ROCK inhibitor (Y27632) is 2-20 μM, preferably 5-15 μM.

[0019] According to a specific embodiment of the present invention, the concentration of the ALK-5 inhibitor (A83-01) is 100-1000 nM, preferably 300-800 nM.

[0020] According to a specific embodiment of the present invention, the concentration of N-acetylcysteine ​​is 0.1-3 mM, preferably 0.5-2 mM.

[0021] According to a specific embodiment of the present invention, the concentration of R-spondin is 100-1000 ng / mL, preferably 300-800 ng / mL.

[0022] According to a specific embodiment of the present invention, the concentration of Nicotinamid is 50-500 ng / mL, preferably 50-200 mM.

[0023] According to a specific embodiment of the present invention, the components of the additive include at least one of FGF2, Wnt-3a and ATCH, as well as N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, and FGF10. The inventors have found through continuous experiments that when the culture medium contains the above additives, it has a significant promoting effect on the growth of adrenal organoids and improves the cell activity of adrenal organoids.

[0024] According to an embodiment of the present invention, the additives include FGF2, N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, and FGF10. The inventors have found through continuous experiments that when the culture medium contains the additives, it has a significant promoting effect on the growth of adrenal organoids and improves the cell activity of adrenal organoids.

[0025] According to an embodiment of the present invention, the additives include Wnt-3a, N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, and FGF10. The inventors have found through continuous experiments that when the culture medium contains the additives, it has a significant promoting effect on the growth of adrenal organoids and improves the cell activity of adrenal organoids.

[0026] According to an embodiment of the present invention, the additives include ATCH, N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, and FGF10. The inventors have found through continuous experiments that when the culture medium contains the additives, it has a significant promoting effect on the growth of adrenal organoids and improves the cell activity of adrenal organoids.

[0027] According to an embodiment of the present invention, the additives include FGF2, Wnt-3a, ATCH, N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, and FGF10. The inventors have found through continuous experiments that when the culture medium contains the additives, it has a significant promoting effect on the growth of adrenal organoids and improves the cell activity of adrenal organoids.

[0028] According to the specific implementation scheme of the present invention, the "FGF2" involved in the present application is a member of the fibroblast growth factor family, which can bind to a specific fibroblast growth factor receptor (FGFR) and has the function of promoting cell proliferation, tissue repair and participating in angiogenesis; the "Wnt-3a" involved in the present application is the transcription product of the wnt gene, a key factor in the Wnt signaling pathway, which regulates β-catenin by positive feedback through binding to the Wnt receptor to maintain cell stemness; ATCH is adrenocortical hormone, a hormone secreted by the pituitary gland of vertebrates. A polypeptide hormone that can promote adrenal cortical tissue proliferation, assist in the improvement of adrenal function, and induce the formation of secretory granules; the "anti-apoptotic factor ROCK inhibitor" involved in this application is Y27632 (ROCK is a serine / threonine protein kinase that plays a key role in the regulation of cell autophagy and apoptosis); the "ALK-5 inhibitor" involved in this application is A83-01 (A83-01 is a powerful TGF-β type I receptor ALK-5 (IC50: 12nm) / ALK4 ((IC50: 45nm) / ALK 7 (IC50: 7.5nm) kinase inhibitor, A83-01 has a strong inhibitory effect on ALK-5); the "EGF" described in this application is epidermal growth factor; the "R-spondin" described in this application is an agonist of the Wnt / β-catenin signaling pathway; the "FGF7" described in this application is fibroblast growth factor 7, a ligand of FGFR2b; the "FGF10" described in this application is fibroblast growth factor 10, a ligand of FGFR2b.

[0029] According to a specific embodiment of the present invention, the additive further includes a hydrogen ion buffer (HEPES), L-glutamine, and a serum substitute (B plus).

[0030] According to a specific embodiment of the present invention, the volume fraction of the hydrogen ion buffer (HEPES) is 0.1-5%, preferably 0.5-2%.

[0031] According to a specific embodiment of the present invention, the volume fraction of L-glutamine is 0.1-5%, preferably 0.5-2%.

[0032] According to a specific embodiment of the present invention, the volume fraction of the hydrogen ion buffer in the culture medium is 1%.

[0033] It should be noted that the component of the "hydrogen ion buffer" stock solution described in the present application is 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and the concentration of the component is 1M.

[0034] According to an embodiment of the present invention, the volume fraction of L-glutamine in the culture medium is 0.5-2%.

[0035] According to a specific embodiment of the present invention, the volume fraction of L-glutamine in the culture medium is 1%.

[0036] It should be noted that the concentration of the "L-glutamine" stock solution described in this application is 200 mM.

[0037] The volume fraction of the serum substitute (B plus) is 0.5-10%, preferably 1-5%

[0038] The "serum replacement (B plus)" described in the present application is an improved serum component replacement based on B27, which can be added to the culture medium to promote the growth and reproduction of cells.

[0039] According to a specific embodiment of the present invention, the culture medium further includes an antibacterial component, which can prevent the adrenal organoid from being contaminated.

[0040] According to a specific embodiment of the present invention, the antibacterial component can be any effective dose of antibiotics or other substances with antibacterial effects known in the art that do not affect cell growth, including penicillin / streptomycin dual antibiotic solution.

[0041] According to a specific embodiment of the present invention, the concentration of penicillin in the culture medium is 50-200 U / mL, preferably 90-110 U / mL.

[0042] According to a specific embodiment of the present invention, the concentration of streptomycin in the culture medium is 0.01-1 mg / ml, preferably 0.05-0.5 mg / ml.

[0043] According to a specific embodiment of the present invention, the basic culture medium includes any one selected from Advanced DMEM / F12, Ham's F-12, SILAC Advanced DMEM / F-12Flex, DMEM / F-12, and William's E culture medium.

[0044] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​in the culture medium is 1-1.5mM, the concentration of nicotinamide in the culture medium is 0.05-1μg / mL, the concentration of anti-apoptotic factor ROCK inhibitor in the culture medium is 5-20μM, the concentration of ALK-5 inhibitor in the culture medium is 0.1-1μM, the concentration of EGF in the culture medium is 0.05-1μg / mL, the concentration of R-spondin in the culture medium is 0.5-1μg / mL, the concentration of FGF7 in the culture medium is 20-30ng / mL, the concentration of FGF10 in the culture medium is 50-150ng / mL, and the volume fraction of serum substitute B plus in the culture medium is 1%-3%. The inventors have found through continuous experiments that when the components in the culture medium are within this concentration range, the growth state of adrenal organoids can be guaranteed to be better and the cell activity is higher, which lays a foundation for subsequent research on the adrenal gland.

[0045] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​in the culture medium is 1-1.5mM, the concentration of nicotinamide in the culture medium is 0.05-0.2μg / mL, the concentration of anti-apoptotic factor ROCK inhibitor in the culture medium is 5-20μM, the concentration of ALK-5 inhibitor in the culture medium is 0.2-0.8μM, the concentration of EGF in the culture medium is 0.05-1μg / mL, the concentration of R-spondin in the culture medium is 0.5-1μg / mL, the concentration of FGF7 in the culture medium is 20-30ng / mL, the concentration of FGF10 in the culture medium is 50-150ng / mL, and the volume fraction of serum substitute B plus in the culture medium is 1-3%. The inventors have found through continuous experiments that when the components in the culture medium are within this concentration range, the growth state of adrenal organoids can be guaranteed to be better and the cell activity is higher, which lays a foundation for subsequent research on the adrenal gland.

[0046] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​in the culture medium is 1-1.5mM, the concentration of nicotinamide in the culture medium is 0.05-1μg / mL, the concentration of Y27632 in the culture medium is 5-20μM, the concentration of A83-01 in the culture medium is 0.1-1μM, the concentration of EGF in the culture medium is 0.05-1μg / mL, the concentration of R-spondin in the culture medium is 0.5-1μg / mL, the concentration of FGF7 in the culture medium is 20-30ng / mL, the concentration of FGF10 in the culture medium is 50-150ng / mL, and the concentration of serum substitute B plus in the culture medium is 1-3%. The inventors have found through continuous experiments that when the components in the culture medium are within this concentration range, the growth state of adrenal organoids can be guaranteed to be better and the cell activity is higher, which lays a foundation for subsequent research on the adrenal gland.

[0047] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​in the culture medium is 1-1.5mM, the concentration of nicotinamide in the culture medium is 0.05-0.2μg / mL, the concentration of Y27632 in the culture medium is 5-20μM, the concentration of A83-01 in the culture medium is 0.2-0.8μM, the concentration of EGF in the culture medium is 0.05-1μg / mL, the concentration of R-spondin in the culture medium is 0.5-1μg / mL, the concentration of FGF7 in the culture medium is 20-30ng / mL, the concentration of FGF10 in the culture medium is 50-150ng / mL, and the concentration of serum substitute B plus in the culture medium is 1%-3%. The inventors have found through continuous experiments that when the components in the culture medium are within this concentration range, the growth state of adrenal organoids can be guaranteed to be better and the cell activity is higher, which lays a foundation for subsequent research on the adrenal gland.

[0048] According to an embodiment of the present invention, the concentration of FGF2 in the culture medium is 0.01-0.1 μg / mL, the concentration of Wnt-3a in the culture medium is 0.1-1 μg / mL, and the concentration of ATCH in the culture medium is 1-5 nM. Preferably, the concentration of FGF2 in the culture medium is 0.01-0.03 μg / mL, the concentration of Wnt-3a in the culture medium is 0.1-0.5 μg / mL, and the concentration of ATCH in the culture medium is 2-3 nM. When the FGF2, Wnt-3a and ATCH in the culture medium are within this concentration range, it can ensure that the growth state of adrenal organoids is better, the cell activity is higher, and the organoid biomimetic ability is stronger.

[0049] The second aspect of the present invention provides use of the culture medium described in the first aspect in culturing adrenal organoids.

[0050] A third aspect of the present invention provides a method for culturing adrenal organoids, the method comprising: culturing a sample containing adrenal adult stem cells in the culture medium described in the first aspect.

[0051] According to a specific embodiment of the present invention, the method further comprises:

[0052] (a) digesting the sample containing adrenal adult stem cells to obtain adrenal cell clusters;

[0053] (b) culturing the adrenal cell cluster using the culture medium described in the first aspect.

[0054] According to an embodiment of the present invention, the method comprises: suspending and culturing adrenal cells in the aforementioned culture medium to obtain adrenal organoids. The adrenal organoids cultured by this method have good morphology, can promote the rapid growth of adrenal organoids, significantly increase the number of organoids, and greatly improve cell survival rate.

[0055] According to an embodiment of the present invention, the adrenal cells are isolated from adrenal tissue.

[0056] In a fourth aspect, the present invention provides a method for constructing an adrenal cortical adenoma organoid, the method comprising: causing adrenal single cells and / or adrenal organoids to express a PRKACA protein with an L206R mutation. The method for constructing an adrenal cortical adenoma organoid according to a specific embodiment of the present invention can successfully obtain an adrenal cortical adenoma organoid model, providing a useful tool for scientific research and treatment of adrenal cortical adenoma diseases.

[0057] PRKACA protein is a regulatory subunit of cAMP. Its 206-position mutation will lead to continuous activation of the cAMP signaling pathway, causing tumorigenesis and excessive production of glucocorticoids. PRKACA-L206R protein mutation is the main cause of adrenal cortical adenoma that produces excessive endogenous cortisol.

[0058] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings. In the electron microscope image of the adrenal organoid structure, particles with a pure black color (pure black indicates that they contain hormones) and a diameter of less than 2 μm are functional secretory granules that can secrete hormones; particles with a grayish color are non-functional secretory granules that do not contain hormones; and round objects with a brownish color, a diameter of more than 3 μm, and rough edges are liposomes, wherein:

[0060] Figure 1 This is a seven-day culture image of the adrenal organoids in Example 1 of the present invention, with a magnification of 4 times ( Figure 1 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 1 In B, scale bar: 5 μm);

[0061] Figure 2 This is a seven-day culture image of the adrenal organoid in Example 2 of the present invention, with a magnification of 4 times ( Figure 2 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 2 In B, scale bar: 5 μm);

[0062] Figure 3 This is a seven-day culture image of the adrenal organoids in Example 3 of the present invention, with a magnification of 4 times ( Figure 3 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 3 In B, scale bar: 5 μm);

[0063] Figure 4 This is a seven-day culture image of the adrenal organoid in Example 4 of the present invention, with a magnification of 4 times ( Figure 4 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 4 In B, scale bar: 5 μm);

[0064] Figure 5 This is a seven-day culture image of the adrenal organoid in Example 5 of the present invention, with a magnification of 4 times ( Figure 5 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 5 In B, scale bar: 5 μm);

[0065] Figure 6 This is a picture of the adrenal organoid cultured for seven days in Comparative Example 6 of the present invention, with a magnification of 4 times ( Figure 6 A in the figure, scale bar: 100 μm), and electron micrograph of adrenal organoid structure ( Figure 6 In B, scale bar: 2 μm);

[0066] Figure 7 It is the statistical result of the average number of functional secretory granules of single cells according to 7 examples;

[0067] Figure 8 is the immunofluorescence staining result of the adrenal organoid in Comparative Example 7 of the present invention;

[0068] Fig. 9 This is a culture picture of the adrenal cortical adenoma organoids with L206R mutation of PRKACA protein in Example 8 of the present invention (right picture, PRKACA L206R group, scale bar: 100 μm) and the normal adrenal organoids as a control (left picture, CTRL group, scale bar: 100 μm) cultured for seven days, with a magnification of 4 times;

[0069] Fig.10 is the statistical result of the average area of ​​the adrenal organoids (CTRL group) and adrenal cortical adenoma organoids (PRKACA L206R group) in Example 8 of the present invention;

[0070] Fig.11 are the statistical results of the expression levels of adrenal cortical adenoma pathological markers PRKACA and STAR in the adrenal organoids (CTRL group) and adrenal cortical adenoma organoids (PRKACA L206R group) in Example 9 of the present invention;

[0071] Fig.12 These are statistical data on the secretion of glucocorticoids (cortisol, cortisone) by the adrenal organoids (CTRL group) and adrenal cortical adenoma organoids (PRKACA L206R group) in Example 9 of the present invention. DETAILED DESCRIPTION

[0072] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0073] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0074] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0075] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0076] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0077] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0078] In this article, the term "organoid" refers to an in vitro 3D culture that can simulate the three-dimensional structure and physiological function of organs (or tissues) in the body under normal (or disease) conditions. In layman's terms, organoids are three-dimensional cell cultures. Stem cells are cultured in matrix gel. Under the action of chemical small molecule inhibitors / activators, cytokines, culture medium additives and other substances, the resulting tissue structure is similar to the corresponding organ. It has the characteristics of cell proliferation and differentiation, self-renewal, self-assembly, long-term culture, and genetic stability.

[0079] In this article, the term "adult stem cell" refers to an undifferentiated cell present in a differentiated tissue that is capable of self-renewal and specialization to form cells that make up that type of tissue. Adult stem cells are present in various tissues and organs of the body. Under certain conditions, adult stem cells either produce new stem cells or differentiate according to a certain procedure to form new functional APSC pluripotent cells, thereby maintaining a dynamic balance of growth and decline in tissues and organs. Adult stem cells in adult individual tissues are mostly dormant under normal circumstances, and can show varying degrees of regeneration and renewal capabilities under pathological conditions or under the induction of external factors.

[0080] In this article, the concentration of all reagents refers to the final concentration of the reagent in the culture medium. For example, in the adrenal organoid culture medium, the concentration of FGF2 is 2-100 ng / mL, which means that the final concentration of FGF2 in the adrenal organoid culture medium is 2-100 ng / mL.

[0081] There is no culture medium suitable for adrenal organoid culture in the existing technology, and therefore no adrenal organoid model, which makes the current research on the adrenal gland and adrenal-related diseases very limited.

[0082] To this end, the present invention provides, on one hand, a culture medium suitable for culturing adrenal organoids, the culture medium comprising a basal culture medium component and an additive component, wherein the additive comprises at least one of FGF2, Wnt-3a, and ATCH.

[0083] The purpose of constructing organoids is to simulate the various morphological structures and functions of tissues and organs in the body. If the purpose of life simulation cannot be achieved, it cannot be said to be successful. Similarly, as an in vitro model, organoids must have the ability to be continuously propagated. Losing this characteristic is also unacceptable. In adrenal organoids, two characteristics must appear: one is a spherical appearance (reflecting the close cell connection in the organoid), and the other is good proliferation ability and clone formation rate (indicating the ability to be continuously propagated). When the above two characteristics appear, it can be said that an adrenal organoid without "function" has been constructed, and when the third characteristic: the presence of a large number of secretory granules, it can be said that the best adrenal organoid has been constructed.

[0084] According to a specific embodiment of the present invention, the present invention provides an adrenal organoid culture medium, which consists of a basal culture medium component and an additive component, wherein the basal culture medium includes any culture medium suitable for organoid culture known in the art, such as Advanced DMEM / F12 and DMEM / F12; the additive includes at least one of FGF2, Wnt-3a and ATCH, wherein the concentration of FGF2 is 2-100 ng / mL, preferably 5-50 ng / mL, and more preferably 10-30 ng / mL, such as 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, and 30 ng / mL; the concentration of Wnt-3a is 50-500 ng / mL, preferably 70-400 ng / mL, and more preferably 100-300 ng / mL, such as 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL. g / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 210ng / mL, 230ng / mL, 250ng / mL, 300ng / mL, 310ng / mL, 340ng / mL, 350ng / mL, 390ng / mL, 400ng / mL; the concentration of ATCH is 0.5-10nM, preferably 1-8nM, and more preferably 1-5nM, such as 1nM, 2nM, 3nM, 4nM, 5nM; further, the additives include N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, FGF10 and serum substitute B plus, the concentration of N-acetylcysteine ​​is 0.1-3mM, preferably 0.5-2mM, for example 0.5mM, 0.75mM, 1mM, 1.25mM, 1.5mM, 1.75mM, 2mM; the concentration of Nicotinamid is 50-500ng / mL, preferably 50-200mM, such as 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM; the anti-apoptotic factor ROCK inhibitor is Y27632, and the concentration of Y27632 is 2-20μM, preferably 5-15μM, such as 5μM, 10μM, 15μM; The ALK-5 inhibitor is A83-01, and the concentration of A83-01 is 100-1000 nM, preferably 300-800 nM, such as 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM; the concentration of EGF is 50-500 ng / mL, preferably 70-200 ng / mL, such as 70 ng / mL, 80 ng / mL, 90 ng / mL, 10 ... 50ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL; the concentration of R-spondin is 100-1000ng / mL, preferably 300-800ng / mL, for example 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL, 700ng / mL, 800ng / mL; the concentration of FGF7 is 1-100ng / mL , preferably 10-50ng / mL, such as 10ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 40ng / mL, 50ng / mL; the concentration of FGF10 is 50-500ng / mL, preferably 70-200ng / mL, such as 70ng / mL, 80ng / mL, 100ng / mL, 150ng / mL, 180ng / mL, 200ng / mL; the serum substitute B The volume fraction of plus is 0.5%-10%, preferably 1%-5%, for example 1%, 2%, 3%, 4%, 5%; further, the additive includes a hydrogen ion buffer, L-glutamine, and a penicillin / streptomycin dual antibody solution, wherein the hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid, and the volume fraction of the 4-hydroxyethylpiperazineethanesulfonic acid is 0.1%-5%, preferably 0.5%-2%, for example 0.5%, 1%, 1.5%, 2%; the L-glutamine The volume fraction of the amide is 0.1%-5%, preferably 0.5-2%, such as 0.5%, 1%, 1.5%, 2%; in the penicillin / streptomycin dual antibody solution, the concentration of penicillin is 50-200U / mL, preferably 90-110U / mL, such as 90U / mL, 100U / mL, 110U / mL, and the concentration of streptomycin is 0.01-1mg / mL, preferably 0.05-0.5mg / mL, such as 0.05mg / mL, 0.08mg / mL, 0.1mg / mL, 0.12mg / mL, 0.2mg / mL, 0.3mg / mL, 0.5mg / mL. When the components in the culture medium are within this concentration range, the growth state of adrenal organoids can be guaranteed to be good and the cell activity is high, laying the foundation for subsequent research on the adrenal gland. .

[0085] The present invention provides a culture method suitable for the stable growth of adrenal organoids. The culture medium of the present invention is used to culture adrenal organoids, which has the advantages of short culture cycle, rapid proliferation, high cell number and cell survival rate, and long-term stable passage. It lays a foundation for in vitro observation, interference with adrenal tissue development and hormone secretion, and research on life activities such as disease generation and injury repair of the adrenal glands.

[0086] In yet another aspect, the present invention provides a method for culturing adrenal organoids, the method comprising: culturing a sample containing adrenal adult stem cells in the culture medium described in the first aspect.

[0087] According to a specific embodiment of the present invention, the method further comprises:

[0088] (a) digesting the sample containing adrenal adult stem cells to obtain adrenal cell clusters;

[0089] (b) culturing the adrenal cell mass using the aforementioned culture medium.

[0090] According to a specific embodiment of the present invention, the reagent used in the digestion treatment is any reagent or reagent combination formula known in the art that can digest a sample containing adrenal adult stem cells and form adrenal cell clusters.

[0091] Another aspect of the present invention provides a method for constructing an adrenal cortical adenoma organoid, the method comprising: causing adrenal single cells and / or adrenal organoids to express a PRKACA protein with an L206R mutation. The method for constructing an adrenal cortical adenoma organoid according to a specific embodiment of the present invention can successfully obtain an adrenal cortical adenoma organoid model, providing a useful tool for scientific research and treatment of adrenal cortical adenoma diseases.

[0092] According to a specific embodiment of the present invention, the method further includes at least one of the following additional technical features:

[0093] According to an embodiment of the present invention, the adrenal organoid is obtained by the method for culturing adrenal organoids as described above, and the adrenal single cell is obtained by digesting a sample containing adrenal adult stem cells or an adrenal organoid obtained by the method for culturing adrenal organoids as described above. It can be understood by those skilled in the art that the digestion treatment is performed by conventional methods in the art, and any reagent that can digest the sample containing adrenal adult stem cells or the adrenal organoid is included in the scope of the present invention.

[0094] According to an embodiment of the present invention, the expression of the PRKACA protein with the L206R mutation in the adrenal single cell and / or the adrenal organoid is achieved by transfecting the adrenal single cell and / or the adrenal organoid with a vector expressing the PRKACA protein with the L206R mutation.

[0095] According to an embodiment of the present invention, the method further comprises using the aforementioned adrenal organoid culture medium to culture the adrenal single cell and / or adrenal organoid expressing the PRKACA protein with the L206R mutation, wherein the culture medium does not contain ATCH. ACTH can promote the activation of the cAMP pathway of adrenal cortical cells and can promote the differentiation and maturation of normal adrenal cells, so a culture medium without ACTH is more conducive to the growth of cortical adenoma organoids.

[0096] According to an embodiment of the present invention, the vector may be a mammalian vector or a lentiviral vector.

[0097] According to an embodiment of the present invention, the vector comprises: at least one of human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), sheep myelin shedding virus (MVV), and pLVX plasmid.

[0098] The fifth aspect of the present invention provides a culture medium for culturing the adrenal cortical adenoma organoids described in the fourth aspect, the culture medium comprising a basal culture medium component and an additive, wherein the additive comprises FGF2 and Wnt-3a. These two factors are particularly important for the growth of adrenal cortical adenoma organoids, wherein FGF2 is a member of the fibroblast growth factor family, can bind to a specific fibroblast growth factor receptor (FGFR), can promote cell proliferation, tissue repair, and participate in angiogenesis; Wnt-3a is the transcription product of the wnt gene, a key factor in the Wnt signaling pathway, which can maintain cell stemness by binding to the Wnt receptor to positively feedback regulate β-catenin. The inventors found that the use of this culture medium can successfully obtain adrenal cortical adenoma organoids with high expression of PRKACA and STAR.

[0099] According to a specific embodiment of the present invention, the basal culture medium includes any culture medium suitable for organoid culture known in the art, such as Advanced DMEM / F12 and DMEM / F12; the additive includes at least one of FGF2 and Wnt-3a, wherein the concentration of FGF2 is 2-100 ng / mL, preferably 5-50 ng / mL, and more preferably 10-30 ng / mL, such as 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, and 30 ng / mL; the concentration of Wnt-3a is 50-500 ng / mL, preferably 70-400 ng / mL, and more preferably 100-300 ng / mL, such as 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 210ng / mL, 230ng / mL, 250ng / mL, 300ng / mL, 310ng / mL, 340ng / mL, 350ng / mL, 390ng / mL, 400ng / mL; further, the additive includes N-acetylcysteine, nicotinamide, anti-apoptotic factor ROCK inhibitor, ALK-5 inhibitor, EGF, R-spondin, FGF7, FGF10 and serum substitute B plus, and the concentration of N-acetylcysteine ​​is 0.1-3mM, preferably 0.5-2mM, for example 0.5mM, 0.75mM, 1mM, 1.25mM, 1.5mM, 1.75mM, 2mM; the concentration of the Nicotinamid is 50-500ng / mL, preferably 50-200mM, such as 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM; the anti-apoptotic factor ROCK inhibitor is Y27632, and the concentration of Y27632 is 2-20μM, preferably 5-15μM, such as 5μM, 10μM, 15μM; the ALK-5 inhibitor is A83-01, and the concentration of A83-01 is 100-1000nM, preferably 300-800nM, such as 300nM, 400nM, 500nM, 600nM, 700nM The concentration of EGF is 50-500ng / mL, preferably 70-200ng / mL, such as 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 150ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL; the concentration of R-spondin is 100-1000ng / mL, preferably 300-800ng / mL, such as 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL, 700ng / mL, 800ng / mL; the concentration of FGF7 is 1-100ng / mL, preferably 1 10-50ng / mL, for example, 10ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 40ng / mL, 50ng / mL; the concentration of FGF10 is 50-500ng / mL, preferably 70-200ng / mL, for example, 70ng / mL, 80ng / mL, 100ng / mL, 150ng / mL, 180ng / mL, 200ng / mL; the volume fraction of the serum substitute Bplus is 0.5%-10%, preferably 1%-5%, for example, 1%, 2%, 3%, 4%, 5%; further, the additives include hydrogen ion buffer, L-glutamine, penicillin / streptomycin dual antibody solution, wherein the The hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid, and the volume fraction of the 4-hydroxyethylpiperazineethanesulfonic acid is 0.1%-5%, preferably 0.5%-2%, such as 0.5%, 1%, 1.5%, 2%; the volume fraction of L-glutamine is 0.1%-5%, preferably 0.5%-2%, such as 0.5%, 1%, 1.5%, 2%; in the penicillin / streptomycin dual antibody solution (Penicillin / Streptomycin), the concentration of penicillin is 50-200U / mL, preferably 90-110U / mL, such as 90U / mL, 100U / mL, 110U / mL, and the concentration of streptomycin is 0.01-1mg / mL, preferably 0.05-0.5mg / mL, such as 0.05mg / mL, 0.08mg / mL, 0.1mg / mL, 0.12mg / mL, 0.2mg / mL, 0.3mg / mL, 0.5mg / mL. When the components in the culture medium are within this concentration range, the growth state of adrenal cortical adenoma organoids can be guaranteed to be good and the cell activity is high, which lays the foundation for subsequent research and treatment of adrenal cortical adenoma diseases by scientific researchers. .

[0100] The sixth aspect of the present invention provides use of the culture medium described in the fifth aspect in culturing adrenal cortical adenoma organoids.

[0101] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0102] Source of reagents:

[0103] DMEM / F12: Gibco TM Advanced DMEM / F-12, 12634010;

[0104] HEPES: Gibco TM HEPES (1M), 15630130;

[0105] L-Glutamine (Glutamax): Gibco TM GlutaMAX TM Additive (200 mM), 35050079;

[0106] Penicillin / Streptomycin: Gibco TM Penicillin-streptomycin (10,000 U / mL), 15140163;

[0107] Nicotinamid: Sigma-Aldrich TM Niacinamide, 72340;

[0108] N-Acetylcysteine: Sigma-Aldrich TM N-acetylcysteine ​​amide, A0737;

[0109] Y27632:R&D SYSTEMSTM Y-27632 dihydrochloride, TB1254-GMP;

[0110] A83-01: R&D SYSTEMS TM A 83-01, 2939;

[0111] Serum replacement B27 supplement: Gibco TM B-27 TM Plus supplement, A3582801;

[0112] EGF: R&D SYSTEMS TM Recombinant Human EGF Protein, CF, 236-EG;

[0113] FGF10: R&D SYSTEMS TM Recombinant Human FGF-10 Protein, 345-FG;

[0114] FGF7: R&D SYSTEMS TM Recombinant Human KGF / FGF-7 Protein, 251-KG;

[0115] R-Spondin 1: R&D SYSTEMS TM Recombinant Human R-Spondin 1 Protein, 4645-RS;

[0116] FGF2: R&D SYSTEMS TM Recombinant Human FGF basic / FGF2 / bFGF(145aa) Protein, CF, 3718-FB;

[0117] Wnt3a: R&D SYSTEMS TM Recombinant Human Wnt-3a Protein, 5036-WN;

[0118] ATCH: Beyotime TM ACTH(1-39) human, P9023;

[0119] SF1(Rabbit): SANTA CRUZ BIOTECHNOLOGY TM , sc-393592;

[0120] CYP11B1 Rabbit pAb:ABclonal TM , A15046;

[0121] Anti-Ki67 antibody(Rabbit):abcam TM , ab16667;

[0122] Cy3, Goat Anti-Rabbit IgG: abbkine TM ,A22220.

[0123] Example 1

[0124] Prepare adrenal organoid culture medium according to the recipe in Table 1:

[0125] Table 1

[0126]

[0127] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0128] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove the adipose tissue and connective tissue, retain only the adrenal tissue sample, and repeatedly wash the adrenal tissue sample with DPBS-PS until the washing solution is clear, generally 3-5 times;

[0129] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3 The tissue fragments are transferred to a 15mL centrifuge tube containing 5-10mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37°C and a speed of 100rpm. The digestion time is generally 20-40min, and then 4% FBS is added to terminate the digestion. The cell solution is passed through a 70μM cell sieve, centrifuged at 300g for 3min, and the supernatant is discarded. 1mL of red blood cell lysis solution is added to the container, and then pipetted up and down, and incubated at room temperature for 3min. After the incubation, centrifuged at 300g for 3min, and the cell precipitate obtained after discarding the supernatant is the adrenal cell;

[0130] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, the cell precipitate was obtained, and then the cell precipitate was mixed with the adrenal medium in Table 1 and inoculated into a low-adhesion 24-well cell culture plate. After the inoculation was completed, the cell culture plate was placed in a 37° C. cell culture incubator for culture;

[0131] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0132] like Figure 1 As shown, Figure 1 A in the figure is a bright field image of organoid growth. The black spheres are adrenal organoids. Figure 1 B is an electron microscope image of a single cell in the organoid. In the single cell, the black spheres are functional secretory granules of the adrenal organoid, and the gray spheres are non-functional secretory granules of the adrenal organoid. Using the culture medium and culture method of this example, a large number of organoid clones can be obtained on the 7th day of culture. The average diameter of the organoid is 100 μm, with a good dense spherical shape, and functional secretory granules in the adrenal gland are distributed in the organoid, indicating that the adrenal organoid constructed by the present invention has good biomimetic ability to the human adrenal gland in both structure and function.

[0133] Example 2

[0134] Prepare adrenal organoid culture medium according to the recipe in Table 2:

[0135] Table 2

[0136]

[0137] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0138] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove adipose tissue and connective tissue as much as possible, retain only the adrenal tissue sample, and wash the adrenal tissue sample repeatedly with DPBS-PS until the washing solution is clear, generally washing 3-5 times;

[0139] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3The tissue fragments are transferred to a 15mL centrifuge tube containing 5-10mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37°C and a speed of 100rpm. The digestion time is generally 20-40min, and then 4% FBS is added to terminate the digestion. The cell solution is passed through a 100μM cell sieve, centrifuged at 300g for 3min, and the supernatant is discarded. 1mL of red blood cell lysis solution is added to the container, and then pipetted up and down, and incubated at room temperature for 3min. After the incubation, centrifuged at 300g for 3min, and the cell precipitate obtained after discarding the supernatant is the adrenal cell;

[0140] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, the cell precipitate was obtained, and then the cell precipitate was mixed with the adrenal medium in Table 2 and inoculated into a low-adhesion 24-well cell culture plate. After the inoculation was completed, the cell culture plate was placed in a 37°C cell culture incubator for culture;

[0141] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0142] like Figure 2 As shown, Figure 2 A in the figure is a bright field image of organoid growth. The spherical object is an adrenal organoid. Figure 2 B is an electron microscope image of a single cell of an organoid. In a single cell, the gray spheres are non-functional secretory granules of the adrenal organoid, the brown particles with rough edges and a diameter of more than 3 μm are liposomes, and the black spheres are functional secretory granules of the adrenal organoid. Using the culture medium and culture method of this example, organoid clones were obtained on the 7th day of culture. The average diameter of the organoids was 100 μm, with good dense spheres and fewer functional secretory granules. The number of organoids and functional secretory granules in Example 2 decreased compared with Example 1, but had good dense spheres, indicating that the use of FGF2 alone can support the formation of a compact spherical structure in adrenal organoids.

[0143] Example 3

[0144] Prepare adrenal organoid culture medium according to the recipe in Table 3:

[0145] Table 3

[0146]

[0147] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0148] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove adipose tissue and connective tissue as much as possible, retain only the adrenal tissue sample, and use DPBS-PS to wash the adrenal tissue sample repeatedly until the washing solution is clear, generally wash 3-5 times.

[0149] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3 The tissue fragments are transferred to a 15 mL centrifuge tube containing 5-10 mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37° C. and a rotation speed of 100 rpm. The digestion time is generally 20-40 minutes. After that, 4% FBS is added to terminate the digestion. The cell solution is passed through a 100 μM cell sieve, centrifuged at 300 g for 3 minutes, and the supernatant is discarded. 1 mL of red blood cell lysis solution is added to the container, and then pipetted up and down with a pipette, and incubated at room temperature for 3 minutes. After the incubation is completed, the cell precipitate obtained by centrifuging at 300 g for 3 minutes and discarding the supernatant is the adrenal cell.

[0150] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal culture medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, a cell precipitate was obtained. The cell precipitate was then mixed with the adrenal culture medium in Table 3 and inoculated into a low-adhesion 24-well cell culture plate. After inoculation, the cell culture plate was placed in a 37° C. cell culture incubator for culture.

[0151] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0152] like Figure 3 As shown, Figure 3 A in the figure is a bright field image of organoid growth. The spherical object is an adrenal organoid. Figure 3B is an electron microscope image of the organoid. The gray spheres are non-functional secretory granules of the adrenal organoids, the brown particles with rough edges and a diameter of more than 3 μm are liposomes, and the black spheres are functional secretory granules of the adrenal organoids. Using the culture medium and culture method of this example, a large number of organoid clones were obtained on the 7th day of culture. The average diameter of the organoids was 100 μm, the organoids could not form a tight spherical structure, and almost no functional secretory granules were contained. Compared with Example 1, the adrenal organoids in Example 3 could not form a tight spherical structure and had fewer functional secretory granules, indicating that Wnt3a alone can increase the number of adrenal organoids.

[0153] Example 4

[0154] Prepare adrenal organoid culture medium according to the recipe in Table 4:

[0155] Table 4

[0156]

[0157] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0158] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove adipose tissue and connective tissue as much as possible, retain only the adrenal tissue sample, and use DPBS-PS to wash the adrenal tissue sample repeatedly until the washing solution is clear, generally wash 3-5 times.

[0159] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3 The tissue fragments are transferred to a 15 mL centrifuge tube containing 5-10 mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37° C. and a rotation speed of 100 rpm. The digestion time is generally 20-40 minutes. After that, 4% FBS is added to terminate the digestion. The cell solution is passed through a 100 μM cell sieve, centrifuged at 300 g for 3 minutes, and the supernatant is discarded. 1 mL of red blood cell lysis solution is added to the container, and then pipetted up and down with a pipette, and incubated at room temperature for 3 minutes. After the incubation is completed, the cell precipitate obtained by centrifuging at 300 g for 3 minutes and discarding the supernatant is the adrenal cell.

[0160] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal culture medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, a cell precipitate was obtained. The cell precipitate was then mixed with the adrenal culture medium in Table 4 and inoculated into a low-adhesion 24-well cell culture plate. After inoculation, the cell culture plate was placed in a 37° C. cell culture incubator for culture.

[0161] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0162] like Figure 4 As shown, Figure 4 A in the figure is a bright field image of organoid growth. The black spheres are adrenal organoids. Figure 4 B is an electron microscope image of the organoid, and the black spheres are functional secretory granules of the adrenal organoid. Using the culture medium and culture method of this example, organoid clones were obtained on the 7th day of culture. The average diameter of the organoids was 100 μm, the number of organoids was small, and the shape was irregular. Compared with Examples 2 and 3, there were more secretory granules distributed in the organoids, indicating that ATCH alone can promote the secretory function of adrenal organoids.

[0163] Example 5

[0164] Prepare adrenal organoid culture medium according to the recipe in Table 5:

[0165] Table 5

[0166]

[0167] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0168] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove adipose tissue and connective tissue as much as possible, retain only the adrenal tissue sample, and use DPBS-PS to wash the adrenal tissue sample repeatedly until the washing solution is clear, generally wash 3-5 times.

[0169] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3The tissue fragments are transferred to a 15 mL centrifuge tube containing 5-10 mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37° C. and a rotation speed of 100 rpm. The digestion time is generally 20-40 minutes. After that, 4% FBS is added to terminate the digestion. The cell solution is passed through a 100 μM cell sieve, centrifuged at 300 g for 3 minutes, and the supernatant is discarded. 1 mL of red blood cell lysis solution is added to the container, and then pipetted up and down with a pipette, and incubated at room temperature for 3 minutes. After the incubation is completed, the cell precipitate obtained by centrifuging at 300 g for 3 minutes and discarding the supernatant is the adrenal cell.

[0170] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal culture medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, a cell precipitate was obtained. The cell precipitate was then mixed with the adrenal culture medium in Table 5 and inoculated into a low-adhesion 24-well cell culture plate. After inoculation, the cell culture plate was placed in a 37° C. cell culture incubator for culture.

[0171] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0172] The culture medium provided in this comparative example is compared with the culture medium of Example 1, except that FGF2, Wnt3a and ATCH are removed, and the culture process is the same as that of Example 1. Figure 5 As shown, Figure 5 A in the figure is a bright field image of organoid growth. Adrenal organoids cultured in this medium grow slowly, are fewer in number, have more thorns on the surface of the organoids, and have poor morphology. Figure 5 B is an electron micrograph of the organoid. No secretory granules appear in the organoid, indicating that cells of the organoid cultured using the culture medium in Example 5 cannot form tight connections with each other and the activity of the organoid is poor.

[0173] Example 6

[0174] Prepare adrenal organoid culture medium according to the recipe in Table 6:

[0175] Table 6

[0176]

[0177] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0178] 1. Transfer the adrenal tissue sample to be cultured into a 3.5 cm or 10 cm culture dish filled with DPBS (DPBS-PS) or normal tissue basal culture medium supplemented with 1% penicillin-streptomycin double antibody solution, remove adipose tissue and connective tissue as much as possible, retain only the adrenal tissue sample, and use DPBS-PS to wash the adrenal tissue sample repeatedly until the washing solution is clear, generally wash 3-5 times.

[0179] 2. After removing the DPBS-PS remaining in the centrifuge tube, cut the adrenal tissue sample into 3 mm 3 The tissue fragments are transferred to a 15 mL centrifuge tube containing 5-10 mL normal tissue primary digestion solution (the main components of the digestion solution are collagenase I and nuclease), and digested in a horizontal shaker at a temperature of 37° C. and a rotation speed of 100 rpm. The digestion time is generally 20-40 minutes. After that, 4% FBS is added to terminate the digestion. The cell solution is passed through a 100 μM cell sieve, centrifuged at 300 g for 3 minutes, and the supernatant is discarded. 1 mL of red blood cell lysis solution is added to the container, and then pipetted up and down with a pipette, and incubated at room temperature for 3 minutes. After the incubation is completed, the cell precipitate obtained by centrifuging at 300 g for 3 minutes and discarding the supernatant is the adrenal cell.

[0180] 3. The cell precipitate obtained in step 2 was fully mixed with the normal tissue basal culture medium, resuspended, centrifuged, and the supernatant was discarded. After repeated multiple times, a cell precipitate was obtained. The cell precipitate was then mixed with the adrenal culture medium in Table 6 and inoculated into a low-adhesion 24-well cell culture plate. After inoculation, the cell culture plate was placed in a 37° C. cell culture incubator for culture.

[0181] 4. In addition, DPBS supplemented with 1% penicillin-streptomycin double antibody solution was used for edge sealing treatment; the adrenal organoid culture medium was replaced once every 2 days in the wells where the adrenal cells were suspended and cultured, and the adrenal organoids were obtained after 7 days of culture.

[0182] The culture medium provided in this comparative example has lower concentrations of FGF2, Wnt3a and ATCH than the culture medium in Example 1, and the culture process is the same as in Example 1. Figure 6 As shown, Figure 6 A in the figure is a bright field image of organoid growth. The black spheres are adrenal organoids. Figure 6B is an electron microscope image of the organoid, and the black spheres are secretory granules of the adrenal organoid. Compared with Example 5, the adrenal organoids cultured in this medium have greatly improved in both quantity and morphology, and there are a small number of functional secretory granules in the internal organs; compared with Example 1, the organoids grow more slowly, the average diameter is reduced, the number is small, and the functional secretory granules in the cells are small, indicating that the adrenal organoids cultured in this medium can perform certain adrenal organoid functions, but the activity is lower than that in Example 1.

[0183] Example 7

[0184] The average number of functional secretory granules of single cells in the organoids cultured with different culture medium components in Examples 1-6 was counted. The presence of functional secretory granules represents that the cultured organoids are functional organoids, and also reflects whether the organoids can have good biomimetic capabilities for the human adrenal gland. In the electron micrograph of the adrenal organoid structure, particles with pure black color (pure black indicates that they contain hormones) and a diameter of less than 2 μm are functional secretory granules that can secrete hormones; particles with a grayish color are non-functional secretory granules that do not contain hormones; and round objects with a brownish color, a diameter of more than 3 μm, and rough edges are liposomes. The statistical results of the average number of functional secretory granules of single cells in the organoids are as follows: Figure 7 As shown, the organoids cultured using the culture medium of Example 1 have good biomimetic capabilities.

[0185] The inventors performed immunofluorescence staining on the adrenal organoids cultured in Example 1 and normal adrenal tissue sections, using F-1 to label steroidogenic factors, Cyp11b1 to label proteins in zona fasciculata cells (which function as a key cholesterol side chain cleavage enzyme for cortisol secretion), and Ki67 to label cells with proliferation ability, such as Figure 8 As shown, Figure 8 The first row of pictures are immunofluorescence staining of adrenal tissue sections, the second row of pictures are immunofluorescence staining of primary adrenal organoids, and the third row of pictures are immunofluorescence staining of passaged adrenal organoids. The results show that the organoids cultured using the culture medium of the present invention are adrenal organoids with adrenal function.

[0186] Example 8

[0187] Prepare culture medium for cortical adenoma organoids according to the recipe in Table 7:

[0188] Table 7

[0189]

[0190] Adrenal organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows:

[0191] 1. Prepare HEK 293T cells (passage number <10) in good condition and without mycoplasma contamination for plating. The number of cells to be plated should reach 70%-90% density before transfection. Replace the preheated antibiotic-free culture medium 1-2 hours before transfection.

[0192] 2. Prepare the transfection working solution according to the instructions of Vigofect transfection reagent, gently mix it with the plasmid, and gently and evenly drop the transfection working solution into the cells, shake it gently to mix, and place the cells in the incubator for incubation;

[0193] 3. After incubation for 16-20 hours, remove the transfection working solution and replace with preheated fresh cell culture medium. After incubation for 36 hours, collect the virus supernatant, centrifuge at 4°C, 1000 rpm for 10 minutes, filter with a 0.45 μm filter, and collect the crude extract containing the virus;

[0194] 4. Use Honorgene according to the instructions TM The lentivirus concentrate was mixed with the crude virus extract described in step 3, mixed thoroughly and incubated at 4° C. overnight, and then centrifuged at 4° C., 1500×g for 45 min to obtain a lentivirus having the PRKACA L206R mutant target sequence (SEQ ID NO: 1);

[0195] 5. Drop 15 μL of matrix gel in the center of the 24-well plate, spread it evenly with a pipette tip, cover the bottom of the well plate, and place the 24-well plate in a 37°C incubator for 15 minutes to solidify the gel drop;

[0196] 6. Use the adrenal organoid culture medium and method in Example 1 to culture the adrenal organoids to a diameter of 100-500 μm, and then transfect the organoids. Aspirate the culture medium in the well plate and add an equal volume of basal culture medium. Use the tip of the pipette to gently scrape off the matrix gel and organoid mixture, transfer to a 1.5mL or 15mL centrifuge tube, gently pipette, centrifuge at 100g for 1 minute, and then add Organoid Recovery Solution (please add the merchant and batch number of the reagent purchase) to remove the matrix gel to recover the pure organoid precipitate;

[0197] 7. Resuspend the obtained organoid precipitate with 300 μL of the adrenal organoid culture medium shown in Table 1, add an equal volume of lentivirus solution, mix well and evenly drop it in the well plate with solidified gel droplets, and incubate in a 37°C incubator overnight;

[0198] 8. After the incubation, the organoids were gently suspended from the well plate with a 1 mL pipette, transferred to a centrifuge tube, centrifuged at 100 × g for 1 minute, the supernatant was removed, and the basal medium was used to blow once again. The infected organoid precipitate was resuspended with matrix gel and inoculated into a cell culture well plate. After solidification in a 37° C. incubator for 15 minutes, the adrenal cortical adenoma organoid culture medium was added, and then the cell culture plate was placed in an incubator for culture. After 7 days of culture, the adrenal cortical adenoma organoid with the PRKACA protein L206R mutation (SEQ ID NO: 2) was obtained ( Fig. 9 ), calculate its volume, the result is as follows Fig.10 As shown, the volume of adrenal cortical adenoma organoids is 3 to 4 times that of normal adrenal organoids.

[0199] Nucleotide sequence encoding the PRKACA mutant protein:

[0200]

[0201] The amino acid sequence of the PRKACA mutant protein is:

[0202] MGNAAAAKKGSEQESVKEFLAKAKEDFLKKWESPAQNTAHLDQFERIKTLGTGSFGRVMLVKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLIDQ QGYIQVTDFGFAKRVKGRTWTLCGTPEYRAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF(SEQ ID NO: 2).

[0203] Example 9

[0204] The expression of adrenal cortical adenoma pathological markers PRKACA and STAR and the secretion of glucocorticoids in the obtained adrenal cortical adenoma organoids were detected. The detection experiment was performed by Matt Spectrum, and the detection method was a conventional operation method in the field. PRKACA and STAR were detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) (ACQUITY UPLC-XevoTQS, Waters Corp., Milford, MA, USA), and the raw data files generated by UPLC-MS / MS were processed using MassLynx software (V4.1, Waters, Milford, MA, USA).

[0205] The results are as follows Fig.11 and 12 As shown, Fig.11 The expression level of PRKACA, a pathological marker of adrenal cortical adenoma, in adrenal cortical adenoma organoids was 6 times that of normal adrenal organoids, and the expression level of STAR was 17 times that of normal adrenal organoids, both of which were significantly higher than those of normal adrenal organoids. Fig.12 The results showed that the secretion of cortisol and corticosterone in adrenal cortical adenoma organoids was significantly higher than that in normal adrenal organoids, indicating that the adrenal cortical adenoma organoid model was successfully constructed.

[0206] In summary, the adrenal organoids cultured in Example 1 have a spherical appearance, good reproduction ability and clone formation rate, and a large number of secretory granules in the cells, indicating that the culture medium in Example 1 can culture functional adrenal organoids; Wnt3a and ATCH were not added to the culture medium of Example 2. When other components and contents were the same, the number of organoids decreased compared with Example 1, and the number of secretory granules was less, but the organoids had a spherical appearance, indicating that the use of FGF2 alone can support the formation of a compact spherical structure of adrenal organoids; Example In the culture medium of Example 3, FGF2 and ATCH were not added. When other components and contents were the same, compared with Example 1, the organoids could not form a compact spheroid structure, and the number of functional secretory granules was small, indicating that the use of Wnt3a alone can support the growth of adrenal organoids in a higher number; in the culture medium of Example 4, FGF2 and Wnt3a were not added. When other components and contents were the same, compared with Examples 2 and 3, the number of secretory granules was large, but the organoids could not form a compact spheroid structure, and the number of organoids was small. FGF2 and Wnt3a were not added to the culture medium of Example 4. When other components and contents were the same, the number of secretory granules was large, but the organoids could not form a compact spheroid structure, and the number of organoids was small. The lack of FGF2, Wnt3a and ATCH in the culture medium of Example 5 resulted in poor organoid activity and incomplete structure, which was reflected in the fact that although the number of secretory granules was large, the lack of a complete microenvironmental support resulted in the secretory granules being inferior to those in Example 1 in terms of morphology and even functionality. In Example 5, FGF2, Wnt3a and ATCH were not added to the culture medium. When other components and contents were the same, compared with Example 1, the number of organoids decreased significantly, the cell survival rate was also greatly reduced, the surviving organoids could not maintain the spheroid structure, and no secretory granules appeared in the organoids, indicating that the lack of FGF2, Wnt3a and ATCH could not successfully culture the organoids. Qualified adrenal organoids were cultured; a small amount of FGF2, Wnt3a and ATCH was added to the culture medium of Example 6. Compared with Example 1, the average diameter of the organoids decreased and the secretory granules were less, but compared with the organoids cultured in Example 5 without adding FGF2, Wnt3a and ATCH, there was a great improvement in terms of organoid morphology, quantity, and secretory granules in the cells of the cultured organoids, indicating that lower concentrations of FGF2, Wnt3a, and ATCH also have a good promoting effect on the culture of adrenal organoids.

[0207] When FGF2 and Wnt3a are added to the adrenal organoid culture medium, adrenal organoids with a spherical appearance (reflecting the close cell connections in the organoids) and good proliferation capacity and clonogenic rate (indicating the ability to be continuously passaged) can be constructed. When the culture medium further contains ATCH, it can promote the differentiation and maturation of the organoids and enable them to secrete cortisol.

[0208] In addition, the adrenal cortical adenoma organoids with the PRKACA protein L206R mutation in Example 8 had a larger average volume than the normal adrenal organoids in the control group, and the expression levels of the case markers PRKACA L206R and STAR were highly upregulated, and they were able to secrete high levels of glucocorticoids, all of which were consistent with the pathological characteristics of the tumor, indicating that the PRKACA L206R mutant adrenal cortical adenoma organoids obtained by lentiviral transfection have a high ability to simulate human diseases.

[0209] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0210] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An adrenal organoid culture medium, characterized in that The culture medium comprises basic culture medium components and additives, wherein: The additives include FGF2, Wnt-3a and ATCH.

2. The culture medium according to claim 1, characterized in that The concentration of FGF2 is 2-100 ng / mL, preferably 5-50 ng / mL, and more preferably 10-30 ng / mL; Optionally, the concentration of Wnt-3a is 50-500 ng / mL, preferably 70-400 ng / mL, and more preferably 100-300 ng / mL; Optionally, the concentration of ATCH is 0.5-10 nM, preferably 1-8 nM, and more preferably 1-5 nM.

3. The culture medium according to claim 1, characterized in that The additive further includes EGF, FGF7, and FGF10; Optionally, the concentration of EGF is 50-500 ng / mL, preferably 70-200 ng / mL; Optionally, the concentration of FGF7 is 1-100 ng / mL, preferably 10-50 ng / mL; Optionally, the concentration of FGF10 is 50-500 ng / mL, preferably 70-200 ng / mL.

4. The culture medium according to claim 1, characterized in that The additives further include Y27632, A83-01, N-acetylcysteine, R-spondin, and Nicotinamid; Optionally, the concentration of Y27632 is 2-20 μM, preferably 5-15 μM; Optionally, the concentration of A83-01 is 100-1000 nM, preferably 300-800 nM; Optionally, the concentration of N-acetylcysteine ​​is 0.1-3 mM, preferably 0.5-2 mM; Optionally, the concentration of R-spondin is 100-1000 ng / mL, preferably 300-800 ng / mL; Optionally, the concentration of Nicotinamid is 50-500 ng / mL, preferably 50-200 mM.

5. The culture medium according to claim 1, characterized in that The additives further include HEPES, L-glutamine, and B plus; Optionally, the volume fraction of HEPES is 0.1-5%, preferably 0.5-2%; Optionally, the volume fraction of L-glutamine is 0.1-5%, preferably 0.5-2%; Optionally, the volume fraction of B plus is 0.5-10%, preferably 1-5%.

6. The culture medium according to claim 1, characterized in that The additive further includes an antimicrobial component; Optionally, the antibacterial component is a penicillin / streptomycin dual antibiotic solution; Optionally, the concentration of penicillin is 50-200 U / mL, preferably 90-110 U / mL; Optionally, the concentration of streptomycin is 0.01-1 mg / mL, preferably 0.05-0.5 mg / mL.

7. The culture medium according to claim 1, characterized in that The basic culture medium includes any one selected from Advanced DMEM / F12, Ham's F-12, SILAC Advanced DMEM / F-12Flex, DMEM / F-12, and William's E culture medium.

8. Use of the culture medium according to any one of claims 1 to 7 in culturing adrenal organoids.

9. A method for culturing adrenal organoids, characterized in that: The method comprises: culturing a sample containing adrenal adult stem cells in the culture medium according to any one of claims 1-7.

10. The method according to claim 9, characterized in that Further including, (a) digesting the sample containing adrenal adult stem cells to obtain adrenal cell clusters or single cells; (b) culturing the adrenal cell cluster or single cell using the culture medium according to any one of claims 1 to 7.

11. A method for constructing adrenal cortical adenoma organoids, characterized in that: The method comprises: causing adrenal single cells and / or adrenal organoids to express a PRKACA protein having an L206R mutation.

12. The method according to claim 11, characterized in that The adrenal organoid is obtained by the method according to claim 9 or 10, and the adrenal single cell is obtained by digesting a sample containing adrenal adult stem cells or an adrenal organoid obtained by the method according to claim 9 or 10; Optionally, the expression of the PRKACA protein with the L206R mutation in the adrenal single cell and / or the adrenal organoid is achieved by transfecting the adrenal single cell and / or the adrenal organoid with a vector expressing the PRKACA protein with the L206R mutation, respectively; Optionally, the method further comprises culturing the adrenal single cell and / or adrenal organoid expressing the PRKACA protein having the L206R mutation using the adrenal organoid culture medium of any one of claims 1 to 7, wherein the culture medium does not contain ATCH; Optionally, the vector comprises a lentiviral vector.

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