Organoid culture medium and use thereof

Abstract

An organoid culture medium and a use thereof, relating to the field of organoid culture. The culture medium comprises a basic culture medium and a specific additive factor, wherein the specific additive factor comprises an ALK-5 inhibitor and an FGF10 protein. The culture medium can culture, from various tissue cells, multiple types of organoids, such as kidney organoids, retinal organoids and related organoid disease models thereof, and has high organoid formation rate, high biological consistency, high research value and wide application prospects.

Description

Organoid culture medium and its applications

[0001] Priority information

[0002] This application claims priority to Chinese patent applications with application number 2023118636491, entitled “Kidney Organoid Culture Medium and Its Applications”, and application number 2023118648766, entitled “Retina Organoid Culture Medium and Its Applications”, filed with the Chinese Patent Office on December 29, 2023, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of organoid culture, and in particular to an organoid culture medium and applications thereof. Background Art

[0004] Organoids are miniature tissue cultures formed by stem cells through proliferation, differentiation, and self-organization under in vitro 3D culture conditions, which are similar to the corresponding normal / disease tissues and organs in the body in terms of cell type, structure, and function. Organoids are miniature organs formed by inoculating pluripotent stem cells, adult stem cells, precursor cells, or tumor cells into matrix gel for in vitro culture, through cell self-proliferation, directed differentiation, lineage commitment, and self-assembly. The organoid culture of normal tissues mainly includes two major sources: adult stem cells and pluripotent stem cells. Organoids can partially or even completely restore the cellular composition and structural function of organs in the body, and maintain genetic and phenotypic stability during long-term expansion. They have a high degree of tissue memory for the restoration of tissues and organs in the body and a strong self-assembly ability, and have broad application prospects.

[0005] The retina is the human body's visual organ. Light enters the eye and is projected onto the retina at the base of the eye. The photoreceptor cells on the retina convert the light signals into electrical signals, which are then transmitted to other cells of the retina for preliminary information integration and processing. The integrated signals are ultimately transmitted to the retinal ganglion cells, which transmit the visual information through the optic nerve into the brain. Organoids derived from human retinal tissue can reflect real developmental events in vivo and have unique advantages, making them a good model for studying retinal development in vitro. Currently, retinal organoids are mainly induced to differentiate into pluripotent stem cells, which are difficult to obtain.

[0006] Optoblastoma is a malignant pediatric ocular tumor clinically classified into two subtypes: RB1 gene deletion and MYCN gene amplification. The MYCN amplification subtype accounts for approximately 2%, and studies on opticoblastomas caused by MYCN amplification are limited.

[0007] Furthermore, culturing renal organoids from renal tissue cells has a low organoid formation rate, and it is difficult to obtain renal organoids with multi-lineage characteristics of nephrons and ureteric buds, as these organoids differ significantly from the biological characteristics of the human kidney. Consequently, renal organoid disease models constructed in this way cannot truly reproduce the development and progression of renal disease.

[0008] Therefore, developing kidney and retinal organoid culture media with high organoid formation rates and little difference in biological characteristics from human kidneys remains a technical challenge in this field and needs to be solved urgently. Furthermore, constructing kidney organoid disease and MYCN-amplified retinoblastoma organoid models also requires further research and solutions. Summary of the Invention

[0009] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0010] In a first aspect, the present invention provides an organoid culture medium. According to an embodiment of the present invention, the culture medium comprises: a basal culture medium and specific additive factors, wherein the specific additive factors comprise: an ALK-5 inhibitor and an FGF10 protein.

[0011] According to an embodiment of the present invention, the above-mentioned organoid culture medium may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the ALK-5 inhibitor is A83-01.

[0013] According to an embodiment of the present invention, the concentration of A83-01 is 0.2 to 2 μM, preferably 0.25 to 1 μM.

[0014] According to an embodiment of the present invention, the concentration of the FGF10 protein is 10-100 ng / mL, preferably 50-100 ng / mL.

[0015] According to an embodiment of the present invention, the specific additive factor further comprises at least one of the following: N-acetylcysteine, nicotinamide, p38 MAPK inhibitor and EGF protein.

[0016] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​is 0.5-2.5 mM, preferably 0.75-2.5 mM.

[0017] According to an embodiment of the present invention, the concentration of nicotinamide is 5 to 15 mM, preferably 2.5 to 10 mM.

[0018] According to an embodiment of the present invention, the p38 MAPK inhibitor is SB202190.

[0019] According to an embodiment of the present invention, the concentration of SB202190 is 0.2 to 2 μM, preferably 0.25 to 1 μM.

[0020] According to an embodiment of the present invention, the concentration of the EGF protein is 10-100 ng / mL, preferably 50 ng / mL.

[0021] According to an embodiment of the present invention, the specific additive factor includes at least one of the following: Forskolin, Prostaglandin E2, R-Spondin protein and Noggin protein.

[0022] According to an embodiment of the present invention, the concentration of Forskolin is 0.5-2 μM, preferably 0.8-1.2 μM, and more preferably 1 μM.

[0023] According to an embodiment of the present invention, the concentration of Prostaglandin E2 is 0.5-2 μM, preferably 0.8-1.2 μM, and more preferably 1 μM.

[0024] According to an embodiment of the present invention, the concentration of the R-Spondin protein is 250-1000 ng / mL, preferably 400-600 ng / mL, and more preferably 500 ng / mL.

[0025] According to an embodiment of the present invention, the concentration of the Noggin protein is 50-200 ng / mL, preferably 75-125 ng / mL, and more preferably 100 ng / mL.

[0026] According to an embodiment of the present invention, the specific additive factor further includes a GSK-3β inhibitor.

[0027] According to an embodiment of the present invention, the GSK-3β inhibitor is CHIR-99021.

[0028] According to an embodiment of the present invention, the concentration of CHIR-99021 is 1-5 μM, preferably 1 μM.

[0029] According to an embodiment of the present invention, the specific additive factor further includes: Y-27632.

[0030] According to an embodiment of the present invention, the concentration of Y-27632 is 2.5 to 10 μM, preferably 4 to 6 μM, and more preferably 5 μM.

[0031] According to an embodiment of the present invention, the specific additive factor further includes: L-glutamine.

[0032] According to an embodiment of the present invention, the concentration of L-glutamine is 1-4 mM, preferably 1.5-2.5 mM, and more preferably 2 mM.

[0033] According to an embodiment of the present invention, the basal culture medium is DMEM / F12 reduced serum culture medium.

[0034] According to an embodiment of the present invention, the basic culture medium further includes: a hydrogen ion buffer, penicillin and streptomycin.

[0035] According to an embodiment of the present invention, the hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid.

[0036] According to an embodiment of the present invention, the concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 25 mM, preferably 5 to 15 mM, preferably 10 mM.

[0037] According to an embodiment of the present invention, the concentration of penicillin is 10-150 U / mL, preferably 20-70 U / mL, and the concentration of streptomycin is 0.075-0.15 mg / mL.

[0038] According to an embodiment of the present invention, the basal culture medium further includes: B27 additive.

[0039] According to an embodiment of the present invention, the concentration of the B27 additive is 0.5% to 2% by volume, preferably 0.75% to 1.5% by volume, and more preferably 1% by volume.

[0040] In a second aspect, the present invention provides an organoid culture medium. According to an embodiment of the present invention, the culture medium comprises: a basal culture medium and specific additives, wherein the specific additives include: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, FGF10 protein, and R-Spondin protein, wherein the ALK-5 inhibitor is A83-01, and the p38 MAPK inhibitor is SB202190. The basal culture medium comprises: DMEM / F12 reduced serum medium, a hydrogen ion buffer, penicillin and streptomycin, and a B27 additive.

[0041] According to an embodiment of the present invention, the hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid.

[0042] According to an embodiment of the present invention, based on the total volume of the culture medium, the culture medium includes the following concentrations of the basal culture medium and specific additive factors: 98% to 99% by volume of the DMEM / F12 reduced serum medium, 5 to 15 mM of the 4-hydroxyethylpiperazineethanesulfonic acid, 20 to 100 U / mL of the penicillin, 0.1 to 0.2 mg / mL of the streptomycin, 1% to 2% by volume of the B27 additive, 0.5 to 2.5 mM of the N-acetylcysteine, 5 to 15 mM of the nicotinamide, 0.2 to 2 μM of the A83-01, 0.2 to 2 μM of the SB202190, 10 to 100 ng / mL of the FGF10 protein, and 100 to 900 g / mL of the R-Spondin protein.

[0043] According to an embodiment of the present invention, the specific additive factors further include: EGF protein and GSK-3β inhibitor.

[0044] According to an embodiment of the present invention, the GSK-3β inhibitor is CHIR-99021.

[0045] According to an embodiment of the present invention, based on the total volume of the culture medium, the culture medium includes the following concentrations of the specific additive factors: 10-100 ng / mL of the EGF protein and 1-5 μM of the CHIR-99021.

[0046] According to an embodiment of the present invention, the specific additive factors further include: Forskolin, Prostaglandin E2, Noggin protein, L-glutamine and Y-27632.

[0047] According to an embodiment of the present invention, based on the total volume of the culture medium, the culture medium includes the specific additive factor at the following concentrations:

[0048] 1 μM of the Forskolin, 1 μM of the Prostaglandin E2, 100 ng / mL of the Noggin protein, 2 mM of the L-glutamine, and 5 μM of the Y-27632.

[0049] According to an embodiment of the present invention, based on the total volume of the culture medium, the culture medium includes the following concentrations of the basal medium and specific additive factors: 99% by volume of the DMEM / F12 reduced serum medium, 10 mM of the 4-hydroxyethylpiperazineethanesulfonic acid, 50 U / mL of the penicillin, 0.05 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1.25 mM of the N-acetylcysteine, 10 mM of the nicotinamide, 1 μM of the A83-01, 1 μM of the SB202190, 1 μM of the CHIR-99021, 50 ng / mL of the EGF protein, 100 ng / mL of the FGF10 protein, and 500 g / mL of the R-Spondin protein.

[0050] According to an embodiment of the present invention, based on the total volume of the culture medium, the culture medium includes the following concentrations of the basal medium and specific additive factors: 99% by volume of the DMEM / F12 reduced serum medium, 10 mM of the 4-hydroxyethylpiperazineethanesulfonic acid, 100 U / mL of the penicillin, 0.1 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1 μM of the Forskolin, 1 μM of the Prostaglandin E2, 500 ng / mL of the R-Spondin protein, 100 ng / mL of the Noggin protein, 100 ng / mL of the FGF10 protein, 2 mM of the L-glutamine, 1.25 mM of the N-acetylcysteine, 5 mM of the nicotinamide, 5 μM of the Y-27632, and 500 nM of the SB202190.

[0051] In the third aspect of the present invention, the present invention proposes use of the culture medium described in the first aspect or the second aspect in constructing an organoid or an organoid disease model.

[0052] According to an embodiment of the present invention, the organoid comprises at least one of a kidney organoid and a retinal organoid.

[0053] According to an embodiment of the present invention, the organoid disease model includes at least one of a kidney organoid disease model and a retinal organoid disease model.

[0054] In a fourth aspect, the present invention provides a method for constructing an organoid. According to an embodiment of the present invention, the method comprises:

[0055] The tissue cells are cultured in the culture medium of the first aspect or the second aspect to obtain the organoid.

[0056] According to an embodiment of the present invention, the organoid comprises at least one of a kidney organoid and a retinal organoid.

[0057] According to an embodiment of the present invention, the tissue cells include at least one of kidney tissue cells and retinal tissue cells.

[0058] According to an embodiment of the present invention, the retinal tissue cells include: retinal progenitor cells and / or precursor cells.

[0059] In a fifth aspect, the present invention provides a method for constructing an organoid disease model. According to an embodiment of the present invention, the method comprises: obtaining an organoid according to the method described in the fourth aspect; and performing gene editing on the organoid to obtain the organoid disease model.

[0060] According to an embodiment of the present invention, the gene editing process includes: gene silencing or gene knockout process or gene overexpression process;

[0061] According to an embodiment of the present invention, the genes include: disease-related genes.

[0062] According to an embodiment of the present invention, the gene editing treatment includes: digesting the organoid to obtain organoid single cells, introducing a nucleic acid with gene silencing or gene knockout activity or a nucleic acid construct containing the same into the organoid single cells to obtain organoid single cells after gene silencing or gene knockout, wherein the nucleic acid targets the disease-related gene; and culturing the organoid single cells after gene silencing or gene knockout to obtain the organoid disease model.

[0063] According to an embodiment of the present invention, the gene-edited organoid single cell is cultured in the culture medium according to any one of claims 1 to 12 to obtain the organoid disease model.

[0064] According to an embodiment of the present invention, the nucleic acid comprises at least one of shRNA, siRNA and sgRNA.

[0065] According to an embodiment of the present invention, the organoid comprises at least one of a kidney organoid and a retinal organoid.

[0066] According to an embodiment of the present invention, the disease includes at least one of Wilms' tumor and opticoblastoma.

[0067] According to an embodiment of the present invention, the opticoblastoma is a MYCN-amplified opticoblastoma.

[0068] According to an embodiment of the present invention, the organoid disease model comprises at least one of a Wilms tumor organoid disease model and an opticoblastoma organoid disease model.

[0069] According to an embodiment of the present invention, the opticoblastoma organoid disease model is a MYCN-amplified opticoblastoma organoid disease model.

[0070] According to an embodiment of the present invention, the method includes: the gene editing treatment is gene silencing treatment, and the disease-related gene is at least one of WTX, P53, WT1 and Trim28.

[0071] According to an embodiment of the present invention, the disease-related gene is WTX and / or P53.

[0072] According to an embodiment of the present invention, the nucleic acid is shRNA.

[0073] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing WTX gene activity.

[0074] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing the activity of the P53 gene.

[0075] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing WT1 gene activity.

[0076] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing Trim28 gene activity.

[0077] According to an embodiment of the present invention, the method includes: the disease-related gene is the MYCN gene.

[0078] According to an embodiment of the present invention, the gene editing treatment includes: introducing a nucleic acid or nucleic acid construct containing the MYCN gene sequence into the retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells or retinal organoid tissue digestion cells to obtain retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells or retinal organoid tissue digestion cells in which the MYCN gene is overexpressed.

[0079] According to an embodiment of the present invention, the method includes: the nucleic acid construct is a viral vector.

[0080] According to an embodiment of the present invention, the viral vector is a non-pathogenic viral vector.

[0081] According to an embodiment of the present invention, the non-pathogenic viral vector is selected from one of a retroviral vector, a poxvirus vector, a herpes simplex virus I vector, a chronic toxic vector, an adenovirus vector and an adenovirus-associated virus vector.

[0082] According to an embodiment of the present invention, the non-pathogenic viral vector is a lentiviral vector.

[0083] According to an embodiment of the present invention, the lentiviral vector infection titer is 10 7 ~10 9 TU / mL.

[0084] In a sixth aspect, the present invention provides a method for constructing an animal disease model. According to an embodiment of the present invention, the method comprises: obtaining an organoid disease model according to the method of the fifth aspect, and transplanting the organoid disease model into an animal to obtain the animal disease model.

[0085] According to an embodiment of the present invention, the animal includes: mouse, rat, guinea pig, rabbit, cat, dog, monkey, pig or sheep.

[0086] In a seventh aspect, the present invention provides a method for constructing an organoid bank or an organoid disease model bank. According to an embodiment of the present invention, the method comprises: obtaining an organoid according to the method of the fourth aspect or obtaining an organoid disease model according to the method of the fifth aspect, subculturing the organoid or organoid disease model to obtain the subcultured organoid or organoid disease model, and cryopreserving the subcultured organoid or organoid disease model to obtain the organoid bank or organoid disease model bank.

[0087] In an eighth aspect, the present invention provides an organoid disease model. According to an embodiment of the present invention, the organoid disease model is constructed according to the method described in the fifth aspect.

[0088] According to an embodiment of the present invention, the organoid disease model comprises at least one of a Wilms tumor organoid disease model and an opticoblastoma organoid disease model.

[0089] According to an embodiment of the present invention, the opticoblastoma organoid disease model is a MYCN-amplified opticoblastoma organoid disease model.

[0090] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0092] FIG1 is a bright field image of a kidney organoid obtained by inverted microscopy after culturing human kidney tissue cells in M1-M9 medium on day 6 (D6) and day 12 (D12) in Example 1 of the present invention. Scale bar: 200 μm.

[0093] FIG2 is a graph showing the corresponding organoid formation rate results of kidney organoids cultured in M1 to M9 culture media in Example 1 of the present invention;

[0094] FIG3 is a graph showing the quantification results of mRNA expression in kidney organoids cultured in M8-M9 medium in Example 1 of the present invention;

[0095] FIG4 is an inverted microscope bright field image of the human kidney organoids from the 1st to the 5th passages in Example 2 of the present invention, scale bar: 200 μm;

[0096] FIG5 is a graph showing the quantification of mRNA expression in kidney organoids cultured in M9 medium for five passages in Example 2 of the present invention;

[0097] Figure 6 is a bright field image of four human Wilms tumor models and human kidney organoids obtained by lentiviral transfection of shRNA and knockdown of WT1, WTX, Trim28 and P53 genes, respectively, in Example 3 of the present invention, wherein Control represents human kidney organoids that have not undergone gene editing treatment, WT1-KD represents a human Wilms tumor model in which the WT1 gene has been knocked down, WTX-KD represents a human Wilms tumor model in which the WTX gene has been knocked down, Trim28-KD represents a human Wilms tumor model in which the Trim28 gene has been knocked down, and P53-KD represents a human Wilms tumor model in which the P53 gene has been knocked down;

[0098] Figure 7 is an image J statistical result of the organoid expansion area of ​​four human Wilms' tumor models and human kidney organoids obtained by lentiviral transfection of shRNA and knockdown of WT1, WTX, Trim28 and P53 genes, respectively, in Example 3 of the present invention, wherein Control represents human kidney organoids that have not undergone gene editing treatment, WT1-KD represents a human Wilms' tumor model with knockdown of the WT1 gene, WTX-KD represents a human Wilms' tumor model with knockdown of the WTX gene, Trim28-KD represents a human Wilms' tumor model with knockdown of the Trim28 gene, and P53-KD represents a human Wilms' tumor model with knockdown of the P53 gene;

[0099] FIG8 is a diagram showing the growth of kidney tumors in mice after orthotopic transplantation of the Wilms tumor model mice in Example 4 of the present invention;

[0100] FIG9 is a graph showing the results of specific analysis of tumor origin in mice after orthotopic transplantation of the Wilms tumor model mice in Example 4 of the present invention;

[0101] Figure 10 is an inverted microscope bright field image of the human retinal organoids cultured on the 7th day (Day 7), the 14th day (Day 14), and the 21st day (Day 21) in Example 5 of the present invention. Scale bar: 100 μm;

[0102] FIG11 is a diagram showing the immunofluorescence staining results of human retinal organoids in Example 6 of the present invention, wherein “Tissue” represents a clinical retinal sample and “Organoid” represents a human retinal organoid sample;

[0103] FIG12 is a bright field image of an inverted microscope of an opticoblastoma organoid in Example 7 of the present invention, wherein MYCN OE indicates overexpression of the MYCN gene, CTRL indicates the control group (GFP group), and FIG12 is a bright field image of an opticoblastoma organoid in Example 7 of the present invention, wherein MYCN OE indicates overexpression of the MYCN gene, CTRL indicates the control group (GFP group), Scale bar: 100 μm;

[0104] Figure 13 is an inverted microscope bright field image of the MYCN retinoblastoma organoids from passage 0 to passage 4 in Example 8 of the present invention, wherein P0 is passage 0, P1 is passage 1, P2 is passage 2, P3 is passage 3, P4 is passage 4, and P5 is passage 5. Scale bar: 100 μm;

[0105] FIG14 is a diagram showing the growth of tumor-like masses in the mouse eyeball after orthotopic transplantation of MYCN opticoblastoma organoids in mice;

[0106] FIG15 is a flowchart of the operation of high-throughput drug screening using the MYCN retinoblastoma model in Example 9 of the present invention. DETAILED DESCRIPTION

[0107] 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.

[0108] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0109] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0110] Terms and Definitions

[0111] 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 meaning commonly understood by those skilled in the art to which the present invention belongs.

[0112] 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.

[0113] 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.

[0114] As used herein, the term "Y-27632" is trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, having the chemical structure of Formula I:

[0115] As used herein, the term "A83-01" is a TGF-β type I receptor inhibitor having the chemical structure of Formula II:

[0116] As used herein, the term "SB202190" is a p38 MAPK inhibitor having the chemical structure of Formula III:

[0117] As used herein, "DMEM / F12 Reduced Serum Medium" refers to classic DMEM / F-12 medium supplemented with ethanolamine, glutathione, ascorbic acid, insulin, transferrin, bovine serum albumin, and trace elements. It is commercially available and can be used to maintain cell growth in a low-serum environment. Advanced DMEM / F12 is a variant of DMEM / F12 Reduced Serum Medium.

[0118] In this article, the reagent "Penicillin-Streptomycin Dual Antibody" is a 100-fold diluted stock solution of penicillin and streptomycin. The recommended working concentrations for cell culture are 100 U / mL for penicillin and 0.1 mg / mL for streptomycin. This mixture can be diluted 100-fold from the 100-fold stock solution.

[0119] In this article, the reagent "complete medium" is a medium obtained by adding serum, antibiotics and other substances to the basic medium.

[0120] As used herein, the term "MYCN-amplified opticoblastoma" is equivalent to "MYCN opticoblastoma."

[0121] As used herein, the term "nucleic acid" is equivalent to "polynucleotide" and "isolated nucleic acid", which can be obtained by synthetic methods.

[0122] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0123] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0124] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Many virus-based systems have been developed for gene transfer into mammalian cells. Techniques known in the art can be used to insert the selected gene into a vector and package it into retroviral particles. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. Lentivirus is a genus under the family Retroviridae. Lentiviral vectors are a relatively complex retroviral vector. Reagents for lentiviral packaging are well known in the art, such as conventional lentiviral vector systems including pRsv-REV, pMD1g-pRRE, pMD2G, and target interference plasmids.

[0125] For ease of understanding, the technical solution of the present invention is described in detail below.

[0126] culture medium

[0127] In one aspect, the present invention provides a culture medium comprising a basal medium and specific additives, wherein the specific additives include N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, EGF protein, and FGF10 protein. The culture medium according to the embodiments of the present invention is used to culture kidney organoids, resulting in a high organoid formation rate, high research value, and broad application prospects.

[0128] According to an embodiment of the present invention, the specific additive factor further comprises R-Spondin protein, thereby promoting the growth of kidney organoids.

[0129] According to an embodiment of the present invention, the concentration of the R-Spondin protein is 100-900 ng / mL, preferably 500 ng / mL. The inventors obtained the above optimal protein concentration through extensive experiments, thereby further improving the formation rate of kidney organoids.

[0130] According to an embodiment of the present invention, the specific additive factor further comprises: a GSK-3β inhibitor. The inventors have found that a GSK-3β inhibitor plays an important role in obtaining kidney organoids with multi-lineage characteristics of nephrons and ureteric buds.

[0131] According to an embodiment of the present invention, the GSK-3β inhibitor is CHIR-99021. The inventors further discovered that the GSK-3β inhibitor has a good effect on the formation of multi-lineage characteristics of nephrons and ureteric buds in kidney organoids.

[0132] According to an embodiment of the present invention, the concentration of CHIR-99021 is 1-5 μM, preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0133] According to an embodiment of the present invention, the ALK-5 inhibitor is A83-01. The inventors have found through continuous experiments that when the culture medium contains this additive, it can increase the formation rate of kidney organoids and promote the formation of multi-lineage characteristics of nephrons and ureteric buds.

[0134] According to an embodiment of the present invention, the concentration of A83-01 is 0.2 to 2 μM, preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0135] According to an embodiment of the present invention, the p38 MAPK inhibitor is SB202190. The inventors have found through continuous experiments that when the culture medium contains this additive, it can increase the formation rate of kidney organoids and promote the formation of multi-lineage characteristics of nephrons and ureteric buds.

[0136] According to an embodiment of the present invention, the concentration of SB202190 is 0.2-2 μM, preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0137] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​is 0.5 to 2.5 mM, preferably 1.25 mM. The inventors determined this optimal concentration through extensive optimization experiments. When N-acetylcysteine ​​is present in the culture medium within this concentration range, organoid cell expansion and long-term subculture are possible.

[0138] According to an embodiment of the present invention, the concentration of nicotinamide is 5 to 15 mM, preferably 10 mM. The inventors determined this optimal concentration through extensive optimization experiments. When nicotinamide is present in the culture medium within this concentration range, organoid cell expansion and long-term subculture are possible.

[0139] According to an embodiment of the present invention, the concentration of EGF protein is 10-100 ng / mL, preferably 50 ng / mL. The inventors determined this optimal concentration through extensive optimization experiments. When the EGF protein in the culture medium is within this concentration range, organoid cell expansion and long-term subculture are possible.

[0140] According to an embodiment of the present invention, the concentration of FGF10 protein is 10 to 100 ng / mL, preferably 100 ng / mL. The inventors determined this optimal concentration through extensive optimization experiments. When the FGF10 protein in the culture medium is within this concentration range, organoid cell expansion and long-term subculture are possible.

[0141] According to an embodiment of the present invention, the basal culture medium is DMEM / F12 reduced serum medium. When the basal culture medium is DMEM / F12 reduced serum medium, cell expansion and long-term subculture of organoids can be achieved.

[0142] According to an embodiment of the present invention, the basic culture medium further includes: a hydrogen ion buffer, penicillin and streptomycin.

[0143] According to an embodiment of the present invention, the hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid.

[0144] According to an embodiment of the present invention, the concentration of 4-HEPES is 5 to 15 mM, preferably 10 mM. The inventors determined this optimal concentration through extensive optimization experiments. When 4-HEPES is present in the culture medium within this concentration range, organoid cell expansion and long-term subculture are possible.

[0145] According to an embodiment of the present invention, the concentration of penicillin is 20-70 U / mL, and the concentration of streptomycin is 0.02-0.07 mg / mL. The inventors have determined these optimal concentrations through a large number of optimization experiments.

[0146] According to an embodiment of the present invention, the basal culture medium further includes: B27 additive.

[0147] According to an embodiment of the present invention, the concentration of the B27 additive is 1-2% by volume. The inventors have determined this optimal concentration through a large number of optimization experiments.

[0148] In another aspect, the present invention provides a kidney organoid culture medium, comprising: a basal culture medium and specific additives, wherein the specific additives include: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, EGF protein, FGF10 protein, R-Spondin protein, and a GSK-3β inhibitor; wherein the ALK-5 inhibitor is A83-01, the p38 MAPK inhibitor is SB202190, and the GSK-3β inhibitor is CHIR-99021; the basal culture medium includes: DMEM / F12 reduced serum medium, a hydrogen ion buffer, penicillin and streptomycin, and a B27 additive. The culture medium according to the embodiment of the present invention is used to culture kidney organoids, and the resulting organoids have a high organoid formation rate, and have multi-lineage characteristics of nephrons and ureteric buds, with biological characteristics similar to those of the human kidney. The culture medium is used to construct a kidney organoid bank and has a high number of passages.

[0149] According to an embodiment of the present invention, the hydrogen ion buffer is 4-hydroxyethylpiperazineethanesulfonic acid.

[0150] According to an embodiment of the present invention, based on the total volume of the kidney organoid culture medium, the kidney organoid culture medium includes the following concentrations of the basal culture medium and specific additive factors: 98% to 99% by volume of the DMEM / F12 reduced serum medium, 5 to 15 mM of the 4-hydroxyethylpiperazineethanesulfonic acid, 20 to 70 U / mL of the penicillin, 0.02 to 0.07 mg / mL of the streptomycin, 1% to 2% by volume of the B27 additive, 0.5 to 2.5 mM of the N-acetylcysteine, 5 to 15 mM of the nicotinamide, 0.2 to 2 μM of the A83-01, 0.2 to 2 μM of the SB202190, 1 to 5 μM of the CHIR-99021, 10 to 100 ng / mL of the EGF protein, 10 to 100 ng / mL of the FGF10 protein, and 100 to 900 g / mL of the R-Spondin protein. When the components in the culture medium are within the above concentration range, cell expansion, organoid formation and long-term subculture of kidney organoids can be achieved.

[0151] According to an embodiment of the present invention, the renal organoid culture medium comprises the following concentrations of the basal medium and specific additives, based on the total volume of the renal organoid culture medium: 99% by volume of the DMEM / F12 reduced serum medium, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid, 50 U / mL of penicillin, 0.05 mg / mL of streptomycin, 1% by volume of the B27 additive, 1.25 mM of N-acetylcysteine, 10 mM of nicotinamide, 1 μM of A83-01, 1 μM of SB202190, 1 μM of CHIR-99021, 50 ng / mL of EGF protein, 100 ng / mL of FGF10 protein, and 500 μg / mL of R-Spondin protein. When the components in the culture medium are within the above preferred concentration ranges, cell expansion, organoid formation, and long-term subculture of renal organoids can be achieved.

[0152] The above-mentioned culture medium of the present invention is suitable for kidney organoid culture, and has a high organoid formation rate; it is conducive to the formation of kidney organoids with multi-lineage characteristics of nephrons and ureteric buds; it is particularly suitable for constructing kidney organoids from human kidney tissue cells. Furthermore, a kidney organoid disease model can be constructed by gene editing means. According to an embodiment of the present invention, the present invention successfully constructed a human nephroblastoma organoid development model through gene silencing technology. The nephroblastoma organoid has genetic epigenetics and disease occurrence characteristics consistent with real nephroblastoma tissue, and can be used as an effective model for the research, drug development and treatment of nephroblastoma (especially infantile nephroblastoma) diseases, with high application value.

[0153] Furthermore, after extensive in vitro research, the inventors successfully cultured retinal organoids using human retinal tissue. Furthermore, through gene editing, they overexpressed the MYCN gene in human retinal tissue cells, thereby generating a novel retinal organoid for opticoblastoma. These retinal and opticoblastoma organoids could serve as new tools and platforms for high-throughput drug screening, with broad potential applications.

[0154] In one aspect, the present invention provides a culture medium comprising a basal medium and specific additives, including A83-01, forskolin, prostaglandin E2, R-spondin protein, Noggin protein, and FGF10 protein. The culture medium according to the embodiments of the present invention can be used to culture retinal organoids from retinal tissue, achieving high organoid formation rates, high biological consistency, high research value, and broad application prospects.

[0155] According to an embodiment of the present invention, the concentration of A83-01 is 250-1000 nM, preferably 400-600 nM, and more preferably 500 nM. The inventors obtained the above-mentioned optimal working concentration of A83-01 through extensive experiments, thereby further promoting the formation of retinal organoids.

[0156] According to an embodiment of the present invention, the concentration of Forskolin is 0.5-2 μM, preferably 0.8-1.2 μM, and more preferably 1 μM. The inventors obtained the above-mentioned optimal working concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0157] According to an embodiment of the present invention, the concentration of prostaglandin E2 is 0.5 to 2 μM, preferably 0.8 to 1.2 μM, and more preferably 1 μM. The inventors obtained the above-mentioned optimal working concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0158] According to an embodiment of the present invention, the concentration of the R-Spondin protein is 250-1000 ng / mL, preferably 400-600 ng / mL, and more preferably 500 ng / mL. The inventors obtained this optimal protein concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0159] According to an embodiment of the present invention, the concentration of Noggin protein is 50-200 ng / mL, preferably 75-125 ng / mL, and more preferably 100 ng / mL. The inventors obtained the above optimal protein concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0160] According to an embodiment of the present invention, the concentration of the FGF10 protein is 50-200 ng / mL, preferably 75-125 ng / mL, and more preferably 100 ng / mL. The inventors obtained this optimal protein concentration through extensive experiments, thereby further promoting the formation and long-term subculture of retinal organoids.

[0161] According to an embodiment of the present invention, the specific additive factors further include: Y-27632 and SB202190, thereby further promoting the growth of retinal organoids.

[0162] According to an embodiment of the present invention, the concentration of Y-27632 is 2.5 to 10 μM, preferably 4 to 6 μM, and more preferably 5 μM. The inventors obtained the above optimal working concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0163] According to an embodiment of the present invention, the concentration of SB202190 is 250-1000 nM, preferably 400-600 nM, and more preferably 500 nM. The inventors obtained the above-mentioned optimal working concentration through extensive experiments, thereby further promoting the formation of retinal organoids.

[0164] According to an embodiment of the present invention, the specific additive factors further include L-glutamine, N-acetylcysteine, and nicotinamide, thereby promoting cell expansion and long-term subculture of organoids.

[0165] According to an embodiment of the present invention, the concentration of L-glutamine is 1-4 mM, preferably 1.5-2.5 mM, and more preferably 2 mM. When the L-glutamine in the culture medium is within this concentration range, cell expansion and long-term subculture of organoids can be achieved.

[0166] According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​is 0.75 to 2.5 mM; preferably 1 to 1.5 mM; and more preferably 1.25 mM. The inventors determined this optimal concentration through extensive optimization experiments. When N-acetylcysteine ​​is present in the culture medium within this concentration range, organoid cell expansion and long-term subculture are possible.

[0167] According to an embodiment of the present invention, the concentration of nicotinamide is 2.5-10 mM, preferably 4-6 mM, and more preferably 5 mM. When the nicotinamide concentration in the culture medium is within this range, cell expansion and long-term subculture of organoids can be achieved.

[0168] According to an embodiment of the present invention, the basal culture medium is DMEM / F12 reduced serum medium. When the basal culture medium is DMEM / F12 reduced serum medium, cell expansion and long-term subculture of organoids can be achieved.

[0169] According to an embodiment of the present invention, the basic culture medium further comprises: hydrogen ion buffer HEPES, penicillin and streptomycin.

[0170] According to an embodiment of the present invention, the hydrogen ion buffer HEPES is 4-hydroxyethylpiperazineethanesulfonic acid.

[0171] According to an embodiment of the present invention, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 25 mM, preferably 5 to 15 mM, and more preferably 10 mM. The inventors determined this optimal concentration through extensive optimization experiments. When 4-hydroxyethylpiperazineethanesulfonic acid is present in the culture medium within this concentration range, organoid cell expansion and long-term subculture are possible.

[0172] According to an embodiment of the present invention, the concentration of penicillin is 10-150 U / mL, and the concentration of streptomycin is 0.075-0.15 mg / mL; the concentration of penicillin is preferably 75-120 U / mL, more preferably 100 U / mL; the concentration of streptomycin is preferably 0.09-0.125 mg / mL, more preferably 0.1 mg / mL. The inventors determined these optimal concentrations after extensive optimization experiments.

[0173] According to an embodiment of the present invention, the basal culture medium further includes: B27 additive.

[0174] According to an embodiment of the present invention, the concentration of the B27 additive is 0.5-2% by volume, preferably 0.75-1.5% by volume, and more preferably 1% by volume. The inventors have determined this optimal concentration through a large number of optimization experiments.

[0175] In another aspect of the present invention, the present invention provides a retinal organoid culture medium, the culture medium comprising: a basal culture medium and specific additive factors, the specific additive factors comprising: A83-01, Forskolin, Prostaglandin E2, R-Spondin protein, Noggin protein, FGF10 protein, L-glutamine, N-acetylcysteine, nicotinamide, Y-27632 and SB202190; the basal culture medium comprises: DMEM / F12 reduced serum medium, hydrogen ion buffer HEPES, penicillin, streptomycin and B27 additive; the hydrogen ion buffer HEPES is 4-hydroxyethylpiperazineethanesulfonic acid; wherein, based on the total volume of the retinal organoid culture medium, the retinal organoid culture medium comprises the following concentrations of the basal culture medium and specific additive factors: 99% by volume of the DMEM / F12 reduced serum medium, 10 mM of the 4-hydroxyethylpiperazineethanesulfonic acid, 100 U / mL of the penicillin, 0.1 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1 μM of the forskolin, and 1 μM of the prostaglandin E2, 500 ng / mL of the R-Spondin protein, 100 ng / mL of the Noggin protein, 100 ng / mL of the FGF10 protein, 2 mM of the L-glutamine, 1.25 mM of the N-acetylcysteine, 5 mM of the nicotinamide, 5 μM of the Y-27632, and 500 nM of the SB202190.

[0176] The culture medium according to the embodiment of the present invention can obtain retinal organoid culture from retinal tissue culture, has a high organoid formation rate and high biological consistency, is used to construct a retinal organoid library, has a high number of passages, high research value, and broad application prospects.

[0177] use

[0178] In one aspect of the present invention, the present invention provides use of the aforementioned culture medium in constructing kidney organoids or kidney organoid disease models.

[0179] Those skilled in the art will understand that the features and advantages described above for the culture medium are also applicable to this use and will not be described in detail here.

[0180] In another aspect of the present invention, the present invention provides use of the aforementioned culture medium in constructing retinal organoids or retinal organoid disease models.

[0181] Those skilled in the art will understand that the features and advantages described above for the culture medium are also applicable to this use and will not be described in detail here.

[0182] According to an embodiment of the present invention, the retinal organoid disease model is an opticoblastoma organoid disease model.

[0183] According to an embodiment of the present invention, the opticoblastoma organoid disease model is a MYCN-amplified opticoblastoma organoid disease model.

[0184] method

[0185] In one aspect of the present invention, a method for constructing a kidney organoid is provided, comprising: culturing kidney tissue cells in the aforementioned culture medium to obtain the kidney organoid.

[0186] Those skilled in the art will appreciate that the features and advantages described above for the culture medium are also applicable to this method and will not be described in detail here.

[0187] In another aspect of the present invention, a method for constructing a kidney organoid disease model is proposed, the method comprising: obtaining a kidney organoid according to the aforementioned method; and performing gene editing on the kidney organoid to obtain the kidney organoid disease model.

[0188] Those skilled in the art will appreciate that the features and advantages described above for the culture medium also apply to this application and will not be elaborated upon here. The resulting kidney organoid disease model exhibits highly consistent histopathological structure and cellular molecular properties with those of the animals from which the cells were cultured, providing a more accurate research model for the development, progression, and treatment of related human diseases, as well as for drug development.

[0189] According to an embodiment of the present invention, the method for constructing a kidney organoid disease model may further include at least one of the following additional technical features:

[0190] According to an embodiment of the present invention, the gene editing treatment includes: gene silencing or gene knockout treatment or gene overexpression treatment.

[0191] According to an embodiment of the present invention, the genes include: disease-related genes. In some specific embodiments, the dosage form-related genes are pathogenic genes or disease marker genes.

[0192] According to an embodiment of the present invention, the gene silencing or gene knockout treatment includes: digesting the kidney organoid to obtain a kidney organoid single cell, introducing a nucleic acid having gene silencing or gene knockout activity or a nucleic acid construct containing the same into the kidney organoid single cell to obtain a kidney organoid single cell after gene silencing or gene knockout, wherein the nucleic acid targets the disease-related gene; and culturing the kidney organoid single cell after gene silencing or gene knockout to obtain the kidney organoid disease model.

[0193] According to an embodiment of the present invention, the gene-silenced or gene-knocked kidney organoid single cell is cultured in the aforementioned culture medium to obtain the kidney organoid disease model.

[0194] According to an embodiment of the present invention, the nucleic acid comprises at least one of shRNA, siRNA and sgRNA.

[0195] According to an embodiment of the present invention, the disease is Wilms' tumor.

[0196] According to an embodiment of the present invention, the gene editing treatment is gene silencing treatment, and the disease-related gene is at least one of WTX, P53, WT1 and Trim28.

[0197] According to an embodiment of the present invention, the disease-related gene is WTX and / or P53.

[0198] According to an embodiment of the present invention, the nucleic acid is shRNA.

[0199] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing WTX gene activity.

[0200] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing the activity of the P53 gene.

[0201] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing WT1 gene activity.

[0202] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence homology thereto and having the function of silencing Trim28 gene activity.

[0203] According to an embodiment of the present invention, the nucleic acid construct is a viral vector.

[0204] According to an embodiment of the present invention, the viral vector is a non-pathogenic viral vector.

[0205] According to an embodiment of the present invention, the non-pathogenic viral vector is selected from one of a retroviral vector, a poxvirus vector, a herpes simplex virus I vector, a chronic toxic vector, an adenovirus vector and an adenovirus-associated virus vector.

[0206] According to an embodiment of the present invention, the non-pathogenic viral vector is a lentiviral vector.

[0207] According to an embodiment of the present invention, the lentiviral vector infection titer is 10 7 ~10 9 TU / mL.

[0208] In one aspect of the present invention, a method for constructing an animal disease model is proposed, comprising: obtaining a kidney organoid disease model according to the aforementioned method, and transplanting the kidney organoid disease model into the animal to obtain the animal disease model.

[0209] Those skilled in the art will appreciate that the features and advantages described above for the culture medium also apply to this method and will not be elaborated upon here. The resulting animal disease model can more accurately simulate the onset and progression of disease in the human body, facilitating disease treatment and drug development.

[0210] According to an embodiment of the present invention, the animal includes: mouse, rat, guinea pig, rabbit, cat, dog, monkey, pig or sheep.

[0211] In another aspect, the present invention provides a method for constructing a kidney organoid library or a kidney organoid disease model library. The method comprises: obtaining kidney organoids or kidney organoid disease models according to the aforementioned method, subculturing the kidney organoids or kidney organoid disease models to obtain subcultured kidney organoids or kidney organoid disease models, and freezing the subcultured kidney organoids or kidney organoid disease models to obtain the kidney organoid library or kidney organoid disease model library.

[0212] Those skilled in the art will appreciate that the features and advantages described above for the culture medium and kidney organoid culture medium are also applicable to this method and will not be elaborated here.

[0213] In one aspect, the present invention provides a method for constructing retinal organoids, comprising culturing retinal tissue cells in the aforementioned culture medium to produce the retinal organoids. The method according to an embodiment of the present invention can produce retinal organoids with high biological consistency.

[0214] According to an embodiment of the present invention, the retinal tissue cells include: retinal progenitor cells and / or precursor cells.

[0215] In another aspect of the present invention, the present invention proposes a method for constructing a retinal organoid disease model, the method comprising: subjecting retinal tissue cells or retinal organoid tissue digestion cells to gene editing to obtain gene-edited cells, and culturing the gene-edited cells in the aforementioned culture medium to obtain the retinal organoid disease model. According to the method of an embodiment of the present invention, a novel retinal organoid disease model with high biological consistency can be constructed. This organoid disease model can accurately simulate the occurrence and development process of the disease, and the research results are reliable. Thus, a reliable research model can be provided for the occurrence, development and treatment of human-related diseases, as well as drug development.

[0216] According to an embodiment of the present invention, the method for constructing a retinal organoid disease model may further include at least one of the following additional technical features:

[0217] According to an embodiment of the present invention, the retinal tissue cells include: retinal progenitor cells and / or precursor cells.

[0218] According to an embodiment of the present invention, the gene editing process includes gene silencing, gene knockout, or gene overexpression. Thus, the organoid disease model constructed is consistent with the organism from which the retinal tissue cells are derived at the genetic level.

[0219] According to an embodiment of the present invention, the genes include: disease-related genes. In some specific embodiments, the dosage form-related genes are pathogenic genes or disease marker genes.

[0220] According to an embodiment of the present invention, the retinal organoid disease model is an opticoblastoma organoid disease model.

[0221] According to an embodiment of the present invention, the opticoblastoma organoid disease model is a MYCN-amplified opticoblastoma organoid disease model.

[0222] According to an embodiment of the present invention, the disease-related gene is the MYCN gene.

[0223] According to an embodiment of the present invention, the gene editing process includes: introducing a nucleic acid or nucleic acid construct containing the MYCN gene sequence into the retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells, or retinal organoid tissue digested cells to obtain retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells, or retinal organoid tissue digested cells that overexpress the MYCN gene. In this way, a MYCN-amplified opticoblastoma organoid model can be constructed.

[0224] According to an embodiment of the present invention, the nucleic acid construct is a viral vector.

[0225] According to an embodiment of the present invention, the viral vector is a non-pathogenic viral vector.

[0226] According to an embodiment of the present invention, the non-pathogenic viral vector is selected from one of a retroviral vector, a poxvirus vector, a herpes simplex virus I vector, a chronic toxic vector, an adenovirus vector and an adenovirus-associated virus vector.

[0227] According to an embodiment of the present invention, the non-pathogenic viral vector is a lentiviral vector.

[0228] According to an embodiment of the present invention, the lentiviral vector infection titer is 10 7 ~10 9 TU / mL.

[0229] In another aspect of the present invention, a method for constructing an animal disease model is proposed, comprising: obtaining a retinal organoid disease model according to the aforementioned method, and transplanting the retinal organoid disease model into the animal eyeball to obtain the animal disease model.

[0230] According to an embodiment of the present invention, the animal includes: mouse, rat, guinea pig, rabbit, cat, dog, monkey, pig or sheep.

[0231] Those skilled in the art will appreciate that the features and advantages described previously for the culture medium and methods for constructing retinal organoid disease models also apply to this method and will not be further elaborated here. The resulting animal disease models more accurately mimic the onset and progression of diseases in the human body, facilitating disease treatment and drug development.

[0232] In one aspect of the present invention, a method for constructing a retinal organoid library or a retinal organoid disease model library is proposed, the method comprising: obtaining retinal organoids according to the aforementioned method or obtaining retinal organoid disease models according to the aforementioned method, subculturing the retinal organoids or retinal organoid disease models to obtain subcultured retinal organoids or retinal organoid disease models, and freezing the subcultured retinal organoids or retinal organoid disease models to obtain the retinal organoid library or retinal organoid disease model library.

[0233] Those skilled in the art will appreciate that the features and advantages described above for the culture medium, the method for constructing retinal organoids or retinal organoid disease models are also applicable to this method and will not be repeated here.

[0234] In one aspect, the present invention provides a retinal organoid disease model. According to an embodiment of the present invention, the retinal organoid disease model is constructed according to the aforementioned method.

[0235] According to an embodiment of the present invention, the retinal organoid disease model is a retinoblastoma organoid disease model. The resulting retinoblastoma organoids have high biological consistency and tumor characteristics, and can serve as an effective model for studying the pathogenesis and progression of MYCN retinoblastoma, related drug screening, and treatment options.

[0236] Those skilled in the art will appreciate that the features and advantages described above for the culture medium, the method for constructing retinal organoids or retinal organoid disease models are also applicable to this method and will not be repeated here.

[0237] The sequences involved in the present invention are detailed in Table 1.

[0238] Table 1 Nucleotide sequence description

[0239] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions shall be followed. Among them, the manufacturers and product numbers of the main reagents used in the organoid and organoid disease model experiments are shown in Table 2. The reagents not included in Table 2 and the instruments without the manufacturer specified are all conventional products that can be purchased commercially.

[0240] Table 2

[0241] It should be noted that the "plasmid" and "vector" described in the following examples have the same meaning and can be used interchangeably.

[0242] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand 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 specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0243] Example 1: Optimization and screening of kidney organoid culture medium

[0244] In this example, based on the previous development of a large number of kidney organoid culture media, the inventors further optimized and screened 9 candidate kidney organoid culture media. The specific method is as follows:

[0245] 1. Culture medium preparation

[0246] Prepare M1, M2, M3, M4, M5, M6, M7, M8, and M9 kidney organoid culture media according to Table 3.

[0247] Table 3. Components of M1-M9 Kidney Organoid Culture Medium

[0248] 2. Acquisition of Human Kidney Tissue Samples

[0249] Fresh kidney surgical resection specimens are immersed in tissue preservation fluid and transferred from the hospital to the laboratory for organoid culture-related experiments.

[0250] Before conducting related experiments such as kidney surgical specimen collection and kidney organoid culture, informed consent was obtained from the patients, who voluntarily participated in the experiments and signed informed consent forms.

[0251] 3. Isolation of Human Kidney Tissue Cells and Culture of Kidney Organoids

[0252] (1) Tissue sample cleaning: Use forceps to grasp the kidney tissue and place it in 1× DPBS (containing penicillin at a final concentration of 50 U / mL and streptomycin at 0.05 mg / mL), 10 mL each time, for three washes;

[0253] (2) Transfer the cleaned kidney tissue to a sterile culture dish, remove all non-epithelial tissue (such as muscle or fat) as much as possible, and use sterile tissue scissors to cut the tissue into pieces of about 0.5-2 mm. 3 size;

[0254] (3) The minced kidney tissue was washed again with 1× DPBS (containing penicillin at a final concentration of 50 U / mL and streptomycin at 0.05 mg / mL), and then the tissue digestion solution (the formula is shown in Table 4) was added. The tissue was enzymatically digested at 37°C and 100 rpm for 1 to 1.5 hours. The tissue digestion suspension was pipetted using pipettes of different sizes (10 mL, 5 mL, and 1 mL, from large to small) until the kidney tissue was visibly dispersed. After sufficient digestion, the tissue digestion suspension was obtained.

[0255] Table 4 Tissue digestion solution formula

[0256] (4) Add fetal bovine serum to a final concentration of 10% to the digested tissue suspension to protect the cells;

[0257] (5) The tissue digestion suspension was passed through a 100 μm cell sieve to obtain a single-cell suspension. The cells were washed twice with basal culture medium (Advanced DMEM / F12) (mixed and centrifuged at 150 g for 3 min, and the supernatant was discarded). For the tissue pieces that did not pass through the filter, steps (3) to (5) were collected and repeated. If the obtained single-cell suspension contained a large number of red blood cells, red blood cell lysis solution (hypotonic ammonium chloride solution) could be added to the precipitate before washing to lyse the red blood cells.

[0258] (7) taking a small amount of the washed cell suspension for viable cell detection and counting, and adding Matrigel at low temperature according to a cell density of 100,000 to 500,000 cells / mL and mixing on ice to obtain Matrigel containing cells;

[0259] (8) Place the mixed cell-containing matrix gel on ice and inoculate 10 μL / well into a 48-well plate (or 30 μL / well into a 24-well plate);

[0260] (9) Place the 48-well plate (or 24-well plate) seeded with cells in a 37°C incubator and incubate for 20 minutes to solidify the matrix gel;

[0261] (10) After the matrigel solidifies, slowly add the above-mentioned M1-M9 culture medium (500 μL / well for 24-well plates and 250 μL / well for 48-well plates) along the wall of the cell culture plate for P0 culture. The culture medium is replaced every 3-4 days. The cells are passaged after about one week of culture and cultured for another 12 days. The growth of the kidney organoids is recorded by photographing (Figure 1).

[0262] The results showed that after culturing renal tissue cells for 6 days using the M4 to M9 culture media of this example, morphologically complete human kidney organoids could be obtained (Figure 1). When the organoid formation rate was counted after 12 days of culture, it was found that the organoid formation rate of the M7 to M9 culture media exceeded 400%, and unexpectedly, the organoid formation rate of the M9 culture media was close to 800% (Figure 2).

[0263] The organoid formation rate is the percentage of isolated kidney tissue cells that successfully differentiated into kidney organoid spheroids. Specifically, it is determined by counting the number and diameter of organoids per well and analyzing them with software.

[0264] Furthermore, the inventors conducted gene expression profiling analysis to investigate whether the kidney organoids obtained after culturing kidney tissue cells for 12 days in M8-M9 culture medium, which has the highest organoid formation rate, have multi-lineage characteristics of nephrons and ureteric buds.

[0265] Gene expression profiling analyzed the mRNA expression abundance of EYA1, GATA3, ABCC4, SLC4A4, SLC12A1, SLC41A3, and NR3C2 genes in kidney organoid samples. Among them, EYA1-PC is a key gene for progenitor cell development, GATA3-UB is a key gene for ureteric bud development, ABCC4-Proximal is a key gene for proximal tubule development, SLC4A4-Proximal is a key gene for proximal tubule development, SLC12A1-LoH is a key gene for nephron loop development, SLC41A3-distal is a key gene for distal tubule development, and NR3C2-CD is a key gene for collecting duct development.

[0266] The gene expression profiling analysis method is as follows: by comparing the expression abundance of key genes for kidney development in organoids cultured under M8 and M9 culture medium conditions, the higher the mRNA transcription level, the higher the expression level of the protein encoded by the corresponding gene, and the more complete the kidney function development.

[0267] The results showed that the kidney organoids obtained after culturing kidney tissue cells in M9 medium for 12 days had multi-lineage characteristics of nephrons and ureteric buds (Figure 3).

[0268] Example 2: Construction of a Kidney Organoid Bank

[0269] Referring to Example 1, kidney organoids were obtained by culturing kidney tissue cells in M9 medium in a 48-well plate or a 24-well plate for 12 days, and then subcultured. Organoids with good growth status were selected, frozen, and stored in liquid nitrogen for a long time, thereby constructing a kidney organoid bank.

[0270] The steps for subculturing are as follows:

[0271] (1) Aspirate the culture medium from the 48-well plate and add 1 mL of pre-chilled 1× DPBS to each well. Place the plate on ice for 5 min.

[0272] (2) Use a 1 mL pipette tip to blow the Matrigel containing organoids in the 48-well plate to remove the Matrigel;

[0273] (3) Transfer the detached Matrigel containing organoids to a 15 mL centrifuge tube, wash the 48-well plate with pre-chilled 1× PBS, collect the wash solution and combine it into the above centrifuge tube;

[0274] (4) Place the 15 mL centrifuge tube on ice for 15 min to soften the Matrigel and obtain the organoid suspension.

[0275] (5) Use a 10 mL pipette to pipette up and down the organoid suspension obtained in step (4) 15 times to separate the organoids from the Matrigel;

[0276] (6) Centrifuge the organoid suspension treated in step (5) at 250g for 5 min at 4°C, remove the supernatant, add 10 mL of pre-chilled 1× DPBS, resuspend the organoids, and count;

[0277] (7) Centrifuge again at 250 g at 4°C for 5 min, remove the supernatant, and place on ice for 5 min;

[0278] (8) Aspirate the refluxed liquid (residual 1× DPBS) and Matrigel;

[0279] (9) Add new Matrigel to resuspend the organoids at a density of 8,000 to 10,000 cells per 10 μL of Matrigel.

[0280] (10) In a 24-well plate, add 30 μL / well of the organoid matrix gel suspension to the center of the well;

[0281] (11) Place the 24-well plate upside down in an incubator to solidify the Matrigel for 20 minutes;

[0282] (12) After the matrigel solidified, 500 μL of 37°C preheated M9 medium was added to each well and the cells were cultured under standard cell culture conditions (cell incubator, 37°C, 5% CO2);

[0283] (13) Replace the M9 medium every 3 to 4 days and culture for 6 to 12 days.

[0284] The steps for cryopreservation are as follows:

[0285] (1) Prepare a cell freezing program cooling box and place it at room temperature for equilibrium;

[0286] (2) Select a culture well where the organoids are growing well, discard the culture medium, gently scrape off the mixture of Matrigel and organoids with a micropipette tip, transfer it to a centrifuge tube, and mix thoroughly by pipetting. Add 1 mL of 1× DPBS to wash the organoids 1 to 2 times. After fully removing the residual Matrigel, centrifuge the organoids at 200 g for 5 minutes and remove the supernatant to obtain the organoids to be frozen.

[0287] (3) Add 500 μL to 1000 μL of pre-chilled organoid freezing solution to the organoid to be frozen, pipette to mix thoroughly, and quickly transfer to a cryogenic tube;

[0288] (4) Place the cryopreservation tube into a cell freezing program cooling box, then quickly place the cell freezing program cooling box into an ultra-low temperature refrigerator at -80°C. The next day, move the cryopreservation tube into liquid nitrogen for long-term storage.

[0289] To further verify the effectiveness of the kidney organoid bank, the inventors further resuscitated and subcultured the kidney organoids stored in liquid nitrogen.

[0290] The steps for resuscitation are as follows:

[0291] (1) Prepare M9 culture medium, basal culture medium (Advanced DMEM / F12) and related reagents in advance, turn on the water bath, adjust the temperature to 37°C, and preheat the basal culture medium. Take out the organoid cryovial from the liquid nitrogen tank, quickly place the cryovial in a 37°C water bath, and shake it from time to time to melt it as quickly as possible. Stop the water bath before the ice is completely melted, transfer the organoid cryovial suspension to a 15mL centrifuge tube, and slowly add 5 times the volume of basal culture medium preheated at 37°C to dilute the cryovial suspension;

[0292] (2) Centrifuge the mixed suspension of the organoids obtained in step (1) and the cryopreservation suspension diluted with basal medium preheated at 37°C at 300g for 3 minutes, remove the supernatant, add 1 mL of basal medium to resuspend the organoids, transfer them to a 1.5 mL centrifuge tube, and centrifuge them. Repeat the washing 1 to 2 times to remove the residual cryopreservation solution;

[0293] (3) Centrifuge at 300 g for 3 minutes, remove the supernatant, add an appropriate amount of Matrigel to resuspend the organoid pellet, mix well on ice, and place on ice;

[0294] (4) Pipette the mixed suspension of extracellular matrix and cells into a cell culture well plate. For example, in a 24-well cell culture plate, apply 20-30 μL of the mixed suspension to each well. The mixed suspension must reach the bottom of the well.

[0295] (5) Place the 24-well cell culture plate in a 37°C, 5% carbon dioxide cell culture incubator for 15 minutes. After confirming that the extracellular matrix has completely solidified, add M9 culture medium.

[0296] (6) Place the cell culture plate in a 37°C, 5% CO2 incubator for culture. Observe and photograph the cells every 1-2 days, and replace the M9 medium every 2-4 days. After 6-12 days of culture, kidney organoids are obtained.

[0297] Generally, cells with a bright, smooth surface observed under an inverted microscope are live cells, while cells with a dull, opaque, and rough surface are dead cells or cells with low viability. Over time, live cells will gradually grow into 3D organoids.

[0298] FIG4 shows an inverted microscope photograph of kidney organoids obtained by the inventors after five consecutive resuscitation treatments and subcultures.

[0299] The results showed that the kidney organoids obtained by subculture in M9 medium had complete morphology and a high organoid formation rate; gene expression profile analysis of the kidney organoids cultured for the fifth time showed that they had multi-lineage characteristics of nephrons and ureteric buds (Figure 5).

[0300] The above results show that the kidney organoids obtained by subculturing the M9 medium of Example 1 have multi-lineage characteristics of nephrons and ureteric buds, and have complete morphology and a high organoid formation rate.

[0301] Therefore, M9 medium can be used to construct kidney organoid banks with a high number of passages.

[0302] Example 3: Construction of Wilms Tumor Model (Kidney Organoid Disease Model)

[0303] In this example, gene silencing technology was used to silence WT1, WTX, Trim28, and P53 in kidney cells. Then, the gene-silenced kidney cells were cultured as organoids using the M9 culture medium of Example 1, and the growth of the organoids was further investigated.

[0304] In this example, five groups were set up, including a control group, a WT1-KD group, a WTX-KD group, a Trim28-KD group, and a P53-KD group. The control group was infected with human kidney cells using a lentivirus expressing an empty vector, while the WT1-KD group, the WTX-KD group, the Trim28-KD group, and the P53-KD group were infected with human kidney cells using lentivirus expressing the corresponding shRNA.

[0305] The specific experimental methods are as follows:

[0306] Preparation of lentivirus containing shRNA and preparation for infection of human kidney cells:

[0307] (1) The shRNAs for the WT1-KD group, WTX-KD group, Trim28-KD group, and P53-KD group were designed and synthesized on the siDirect website; the synthesized shRNAs were cloned into the pLKO.1puro vector to obtain the core plasmid.

[0308] The shRNA targeting the WTX gene has a nucleotide sequence as shown in SEQ ID NO: 1,

[0309] 5'-ATTGCTGGTGAACTCTACCAG-3' (SEQ ID NO: 1);

[0310] The shRNA targeting the P53 gene has a nucleotide sequence as shown in SEQ ID NO: 2,

[0311] 5'-GACTCCAGTGGTAATCTAC-3' (SEQ ID NO: 2);

[0312] Among them, the shRNA targeting the WT1 gene has a nucleotide sequence as shown in SEQ ID NO: 3,

[0313] 5'-ATGCTTGAATGAGTGGTTGGG-3' (SEQ ID NO: 3);

[0314] The shRNA targeting the Trim28 gene has a nucleotide sequence as shown in SEQ ID NO: 4,

[0315] 5'-TAAGCACAGGTTTTGGTCTCAG-3' (SEQ ID NO: 4).

[0316] (2) Resuscitate a well-conditioned 293T cell line and culture it in DMEM (containing 10% fetal bovine serum) until the cell density reaches 70% to 90% during transfection.

[0317] (3) 1 to 2 hours before transfection, the cells were replaced with antibiotic-free DMEM (catalog number: 12800017, company: Gibco) culture medium containing 10% fetal bovine serum (catalog number: F8318, company: Sigma);

[0318] (4) Take 5-8 μg DNA (initial amount 5 μg), add diluent (enzyme-free water) to a total volume of 100 μL, mix gently, and place at room temperature. The core plasmid: lentiviral packaging plasmid psPAX2 (hereinafter referred to as "PH1"): lentiviral packaging plasmid pMD2.G (hereinafter referred to as "PH2") is in a mass ratio of 7:5:2. 7 μg core plasmid, 5 μg PH1, and 2 μg PH2 are added to a 10 cm culture dish, and then the plasmid packaging is completed by 293T cells;

[0319] (5) Take 4 μL of eukaryotic transfection reagent (VigoFect) (initial amount 2 μL), add it to the diluent (enzyme-free water) to a total volume of 100 μL, mix gently, and let it stand at room temperature for 5 minutes. Add the diluted VigoFect dropwise to the diluted DNA solution and mix gently. The resulting transfection working solution is left at room temperature for 15 minutes, then gently and evenly dripped into the 293T cells. Finally, shake gently to mix, and place the 293T cells in the incubator for culture;

[0320] (6) After 16 to 20 hours of transfection, discard the culture medium and replace with fresh DMEM medium (containing FBS). Continue transfection for 36 hours. Collect the viral supernatant and centrifuge at 4°C, 1000 rpm for 10 minutes. Aliquot the supernatant into 1.5 mL centrifuge tubes. Store temporarily at 4°C or long-term at -80°C.

[0321] (7) After collecting the virus, refer to the method of Example 1 and digest the kidney organoids cultured in M9 medium for 5-10 minutes. Filter with a 40 μm filter to obtain a single cell suspension, which is placed on ice for later use.

[0322] Prepare infection medium: Add 100 μL of virus solution and transfection reagent polybrene (10 μg / mL) to M9 medium to obtain a total volume of 250 μL of mixed solution.

[0323] Viral infection: Resuspend the cell pellet from the single-cell suspension, mix thoroughly, and add the pellet to a 48-well plate. Seal the plate with sealing film. Transfer the plate to a microcentrifuge and centrifuge at 600g for 60 minutes at 32°C. Then, transfer the plate to a 37°C incubator and continue incubation for 5-6 hours. Next, centrifuge the kidney cells 5-6 hours after infection at room temperature at 1000 rpm for 3 minutes, and discard the supernatant.

[0324] Organoid culture: Resuspend cells in 30 μL of Matrigel and plate the transfected cells into a 24-well plate. Add 500 μL of M9 medium and culture in a 37°C, 5% CO2 cell culture incubator. Change the medium every 3-4 days. After 7 days of culture, observe the growth of organoids in each group using a microscope (Figure 6). Image J was used to count and calculate the organoid expansion area (Figure 7).

[0325] The results showed that nephroblastoma organoids were cultured in all experimental groups. Among them, the organoid expansion area in the WTX-KD group was significantly higher than that in the control group (Figure 7), the expansion effect in the P53-KD group was the most obvious (Figure 6), and the organoid diameter in the WTX-KD group was significantly better than that in the WT1-KD group, Trim28-KD group, P53-KD group and control group (Figure 6).

[0326] Example 4: Establishment of Wilms' tumor xenograft animal model

[0327] To further verify whether the Wilms tumor organoids obtained by gene silencing in Example 3 possess tumor characteristics, the inventors orthotopically transplanted the Wilms tumor organoids from the WTX-KD group and the P53-KD group in Example 3 into the renal capsule of NSG mice and performed tissue identification and analysis. The specific steps are as follows:

[0328] Sixty severely immunodeficient mice of the NOD-Prkdcscid Il2rgem1 / Smoc(M-NSG) (M-NSG) strain, aged 4-7 weeks and weighing 18-24 g, were selected for the experiment, including a control group, a WTX-KD group, and a P53-KD group, with 20 mice in each group. The mice in the control group were treated by injecting kidney organoids infected with an empty lentivirus into the subrenal capsule of NSG mice. The mice in the WTX-KD and P53-KD groups were transplanted with Wilms tumors, and the kidney organoids infected with the shRNA lentivirus of the WTX-KD and P53-KD groups were injected into the subrenal capsule of NSG mice, respectively, and treated as follows;

[0329] (1) Before inoculation, collect organoids (about 2×10 5 cells), washed with culture medium, centrifuged to remove all supernatant, and resuspended in 15 μL of Matrigel and placed on ice until use;

[0330] (2) Prepare 1% sodium pentobarbital and administer 40 mg / kg of pentobarbital intraperitoneally to anesthetize the mouse. After the mouse is anesthetized and its breathing is stable, use sterile surgical instruments to cut open the mouse's abdominal cavity and fully expose the kidney.

[0331] (3) Inject the mixture of matrigel and organoids under the renal capsule of the mouse, suture the wound, and inject antibiotics into the mouse to prevent infection;

[0332] (4) After the experimental mice were kept for 30 days, the tumorigenicity was observed by dissection, and the origin of the transplanted cells in the WTX-KD group and the P53-KD group was analyzed by immunofluorescence staining using human maker Numa (Figure 9).

[0333] The results showed that: (1) Mice transplanted with kidney organoids from the WTX-KD and P53-KD groups in Example 3 had obvious cancer lesions in their kidneys, while the control group only had local tissue expansion at the injection site (Figure 8). (2) Immunofluorescence staining experiments showed that the transplants from both the WTX-KD and P53-KD groups expressed the human maker Numa (Figure 9), indicating that the kidney organoids in the WTX-KD shRNA and P53-KD shRNA lentiviruses successfully mimicked the characteristics of human nephroblastoma.

[0334] The above results show that the Wilms tumor organoids of Example 3 have tumor characteristics and can be used as an effective model for studying the occurrence and development mechanism of Wilms tumor disease, screening related drugs, and studying treatment options.

[0335] Example 5: Retinal Organoid Culture Medium and Human Retinal Organoid Culture

[0336] In this example, based on a large number of previous research and development of organoid culture media, the inventors screened and obtained a human retinal organoid culture medium, the components of which are shown in Table 5.

[0337] Table 5 Retinal organoid culture medium components

[0338] Furthermore, human retinal organoids were cultured as follows:

[0339] (1) Fresh eyeball surgical specimens were immersed in tissue preservation solution and transferred from the hospital to the laboratory at a low temperature of 0-4°C within 24 hours for organoid culture related experiments;

[0340] Before conducting related experiments such as eyeball surgical specimen collection and retinal organoid culture, informed consent was obtained from the patient, who voluntarily participated in the experiment and signed the informed consent form.

[0341] (2) After the tissue sample is transported to the laboratory, it is transferred to sterile phosphate buffer solution (tissue wash solution) in a biosafety cabinet and washed twice; the washed sample is transferred to a sterile culture dish to obtain retinal tissue;

[0342] (3) mincing the retinal tissue and mechanically blowing to collect human retinal cells;

[0343] (4) Centrifuge the human retinal cells at 150 g for 3 minutes, discard the supernatant, and retain the cell pellet;

[0344] (5) Take a small amount of the suspension for viable cell detection and counting, add extracellular matrix at a density of 100,000-500,000 cells / mL and mix on ice. After mixing, place on ice;

[0345] (6) Pipette the mixture of extracellular matrix and cells into a 24-well cell culture plate, and add 20-30 μL of the mixed suspension to each well;

[0346] (7) Place the culture plate in a 37°C, 5% CO2 cell culture incubator and incubate for 15 minutes. After the extracellular matrix has completely solidified, slowly add organoid culture medium (its composition is shown in Table 2) along the well wall. For a 24-well cell culture plate as an example, add 500 μL of organoid culture medium (the composition is shown in Table 2) to each well.

[0347] (8) Continue culturing and take photos to record the growth of retinal organoids (Figure 10).

[0348] The results showed that human retinal organoids began to form after 7 days of culture of human retinal cells in the culture medium of this embodiment; after about 28 days of continuous culture, morphologically complete human retinal organoids were obtained; during the culture period, the human retinal organoids grew well and had a high organoid formation rate.

[0349] Example 6: Identification of cell types in human retinal organoid cultures

[0350] Furthermore, the inventors performed multi-cell type identification on the human retinal organoids obtained in Example 5 using immunofluorescence staining to evaluate the consistency of the human retinal organoids in Example 5 with clinical samples. The specific method is as follows:

[0351] (1) Embed human retinal organoids in paraffin in a tissue embedding box and perform tissue staining and sectioning;

[0352] (2) Dewax the organoid paraffin sections in xylene for 5 minutes; dewax in fresh xylene twice for 5 minutes; dewax in anhydrous ethanol twice for 5 minutes; 90% ethanol for 5 minutes; 70% ethanol for 5 minutes; 50% ethanol for 5 minutes; and distilled water twice for 5 minutes. Heat in a pressure cooker with 10mM sodium citrate antigen retrieval solution for 20 minutes, then slowly cool to room temperature.

[0353] (3) Block with blocking solution (1× PBS containing 5% BSA) for 60 min, discard the blocking solution, add diluted primary antibody, and incubate at 4°C overnight;

[0354] (4) Wash the sections in a glass jar with 1× PBS for 5 min with gentle shaking, then wash twice more with fresh 1× PBS.

[0355] (5) Dilute the fluorescently labeled secondary antibody with blocking solution in an appropriate ratio, incubate at room temperature in the dark for 1 h, and wash three times with 1× PBS;

[0356] (6) Add anti-fluorescence quencher containing DAPI, cover with a coverslip, and examine under a confocal microscope.

[0357] In this example, the primary antibodies were SOX2 (from Santa Cruz), PAX6 (from Biolegend), OTX2 (from R&D), CRX (from Abnova), RXRG (from Santa Curz), ARR3 (from NOVUS), CHX10 (from Santa Curz), and SOX9 (from Millipore), and the secondary antibodies were donkey anti-mouse IgG, donkey anti-rabbit IgG, and donkey anti-goat IgG (all from Abbkine).

[0358] The results, as shown in Figure 11, show that SOX2 and PAX6 double positivity are marker genes for retinal progenitor cells; PAX6 positivity is a marker gene for ganglion cells, horizontal cells, or amacrine cells; OTX2 positivity or CRX positivity is a marker gene for photoreceptor cells; RXRG positivity or ARR3 positivity is a marker gene for cone precursor cells; CHX10 is a marker gene for bipolar cells; and SOX9 is a marker gene for Müller cells. Staining results for these marker genes were consistent in both clinical retinal samples from Tissue and human retinal organoid samples from Organoid.

[0359] The above results show that the human retinal organoids obtained in Example 5 are consistent with the biological characteristics of clinical retinal samples, such as multiple cell types, marker gene expression, and tissue spatial structure. Therefore, the human retinal organoids have high biological consistency and can be used as a reliable research model for scientific, clinical and drug research.

[0360] Example 7: Construction of a MYCN retinoblastoma organoid model (human retinal organoid disease model)

[0361] Based on the fact that MYCN amplification can drive the occurrence of retinal tumors, the inventors creatively proposed to activate MYCN in human retinal organoids (causing overexpression of the MYCN gene) to drive their development into retinal tumor organoids, thereby obtaining a new retinal tumor model.

[0362] Therefore, in this example, human retinal cells were overexpressed with the MYCN gene through gene editing technology, and then the gene-edited human retinal cells were cultured as organoids using the culture medium of Example 5 to obtain a new retinal tumor model. The experimental method is as follows:

[0363] In this example, two groups were set up, including a control group (GFP group) and an experimental group (MYCN overexpression group). The control group was infected with human retinal cells using a lentivirus expressing GFP, and the MYCN group was infected with human retinal cells using a lentivirus expressing MYCN. The specific treatment method is as follows:

[0364] (1) The MYCN gene was cloned into the pLVX-P2A-EGFP (Addgene) vector to obtain the MYCN gene-expressing vector pLVX-MYCN-P2A-EGFP;

[0365] (2) In a 10 cm cell culture dish, revive a well-conditioned 293T cell line and culture it in DMEM medium (containing 10% fetal bovine serum) until the cell density reaches 70% to 90% at the time of transfection;

[0366] (3) 1 to 2 hours before transfection, the culture medium was replaced with antibiotic-free DMEM (catalog number: 12800017, company: Gibco) medium containing 10% fetal bovine serum (catalog number: F8318, company: Sigma);

[0367] (4) Core plasmid: lentiviral packaging plasmid psPAX2 (hereinafter referred to as "PH1"): lentiviral packaging plasmid pMD2.G (hereinafter referred to as "PH2") according to the mass ratio of 7:5:2, prepare an appropriate amount of plasmid and add physiological saline to a total volume of 400 μL, mix gently, and let it stand at room temperature; the core plasmid of the control group is pLVX-P2A-EGFP, and the core plasmid of the experimental group is pLVX-MYCN-P2A-EGFP;

[0368] (5) Take 8 μL of eukaryotic transfection reagent (VigoFect), add physiological saline to a total volume of 400 μL, mix gently, and let it stand at room temperature for 5 minutes. Add the diluted VigoFect dropwise to the diluted plasmid solution and mix gently. The resulting transfection working solution is left at room temperature for 15 minutes, gently and evenly dripped into the cells, and then gently shaken to mix. The cells are placed in an incubator for culture;

[0369] (6) 16–20 h after transfection, discard the culture medium and replace with fresh DMEM medium containing 2% FBS. Collect the viral supernatant 48 h after the medium replacement and centrifuge at 4°C and 1500 rpm for 15 min. Concentrate the viral supernatant to obtain a high-concentration virus.

[0370] (7) Referring to Example 5, the human retinal cells separated from fresh human retinal tissue were centrifuged to collect the precipitate, the virus solution was added to the culture medium and mixed evenly, the total volume was 250 μL, the cell precipitate was resuspended with the mixed solution and mixed evenly, added to a 48-well plate, and the well plate was sealed with a sealing film. Move to a well plate centrifuge and centrifuge at 32°C and 600g for 60 minutes, then transfer to a 37°C incubator for overnight culture. The next day, the human retinal cells infected overnight were centrifuged at room temperature and 150g for 3 minutes, and the supernatant was discarded. The cells were resuspended with 20 μL of extracellular matrix and plated into a 24-well plate. 500 μL of complete culture medium was added and cultured at 37°C;

[0371] (8) Continue culturing for 31 days to obtain opticoblastoma organoids. Photographs were taken to record the growth of the opticoblastoma organoids ( FIG12 ).

[0372] The nucleotide sequence of the MYCN gene is shown in SEQ ID NO: 5.

[0373] The results showed that organoids were cultured in all experimental groups. Compared with the control group, organoids derived from overexpressing the MYCN gene were significantly larger and exhibited tumor characteristics. These results indicate that organoids derived from overexpressing the MYCN gene are opticoblastoma organoids and can serve as an effective model for studying the pathogenesis and progression of MYCN opticoblastoma, screening related drugs, and developing treatment options.

[0374] Example 8: Subculture of MYCN opticoblastoma organoids and construction of an organoid bank

[0375] In this example, referring to Example 5, the MYCN optic blastoma organoids of Example 7 were subcultured in a 48-well plate or a 24-well plate using the culture medium of Example 5, and organoids with good growth status were selected. After cryopreservation, they were stored in liquid nitrogen for a long time, thereby constructing a MYCN optic blastoma organoid bank.

[0376] The steps for subculturing are as follows:

[0377] (1) Discard the complete culture medium, add an equal volume of 500 μL of pre-chilled PBS, and let it stand on ice for 5 min;

[0378] (2) Use a 1 mL pipette tip to gently scrape the extracellular matrix and organoid mixture from step (8) of Example 7, transfer it to a 1.5 mL centrifuge tube, pipette 5 to 10 times to separate the organoids and extracellular matrix, and centrifuge at 150 g for 3 min;

[0379] (2) Discard the supernatant and mechanically pipette until the organoids are broken into small cell clumps;

[0380] (3) Centrifuge at 150 g for 3 min and rinse once with PBS;

[0381] (4) Add the required volume of extracellular matrix to resuspend the organoids, mix thoroughly, and transfer the organoid and extracellular matrix mixture to a cell culture plate;

[0382] (5) The culture plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 15 minutes. After the extracellular matrix was completely solidified, the organoid culture medium of Example 5 (the components are shown in Table 5) was slowly added along the well wall. For example, 500 μL of the organoid culture medium with the components shown in Table 5 was added to the 24-well plate.

[0383] (6) Continue the culture for 14 days.

[0384] Organoids with good growth status were selected, frozen, and stored in liquid nitrogen for a long time to construct a MYCN opticoblastoma organoid library.

[0385] To further verify the effectiveness of the MYCN retinoblastoma organoids in the organoid bank, the inventors performed multiple resuscitation and subculture on the MYCN retinoblastoma organoids stored in liquid nitrogen.

[0386] Generally, cells with a bright, smooth surface observed under an inverted microscope are live cells, while cells with a dull, opaque, and rough surface are dead cells or cells with low viability. Over time, live cells will gradually grow into 3D organoids.

[0387] Figure 13 shows inverted microscope images of MYCN opticoblastoma organoids obtained by the inventors after four consecutive resuscitation and subculture. These images demonstrate that the organoid culture medium of Example 5, when used to subculture MYCN opticoblastoma organoids, produces organoids with intact morphology and a high organoid formation rate. Therefore, the organoid culture medium of Example 5 can be used to construct a MYCN opticoblastoma organoid bank with a high number of passages.

[0388] Example 9: Application of MYCN Optoblastoma Model

[0389] Referring to the process flow in Figure 15 , a drug to be screened is exposed to opticoblastoma organoids, and the volume change of the opticoblastoma organoids before and after exposure is compared to identify a target drug. If the volume of the opticoblastoma organoids is smaller than that before exposure, the drug to be screened can inhibit the growth of or kill the opticoblastoma organoids, thus identifying it as a target drug. In some embodiments, the drug to be screened is identified as a target drug when the viability of the opticoblastoma organoids is reduced by at least 50% compared to the viability of the opticoblastoma organoids before exposure.

[0390] The specific method is as follows:

[0391] 1. Establishment of human retinal organoids and MYCN opticoblastoma organoid models

[0392] Refer to Examples 5 and 7.

[0393] 2. In vitro drug testing using the opticoblastoma model

[0394] Based on the 3D cultured retinoblastoma model, a high-content screening system was used to screen small molecule drugs that can inhibit the growth of retinoblastoma organoids.

[0395] The specific experimental operations are as follows:

[0396] (1) Collect organoids from the culture well plate, break them into small cell clusters by mechanical pipetting, measure the number of cell clusters in the suspension, and resuspend them in extracellular matrix at a density of 10,000 cell clusters / mL;

[0397] (2) 3 μL of the mixture of cell clusters and extracellular matrix was added to each well of a 96-well plate, and 100 μL of culture medium was added to each well and cultured in a 37°C incubator;

[0398] (3) After 7 days of culture, each well was imaged using the PerkinElmer Opera Phenix high-content multislice scanning system, and then different concentrations of drug groups were added and cultured in a 37°C incubator;

[0399] (4) After 7 days of drug addition and culture, the liquid in the culture wells was discarded, and an organoid live cell fluorescence viability analysis kit was added to each well. After incubation in a 37°C incubator for 30 minutes, the fluorescence intensity was detected (excitation / emission wavelength: 560 / 590), and the organoid viability was calculated. At the same time, each well was imaged using the PerkinElmer Opera Phenix high-content multi-layer scanning system.

[0400] Furthermore, the inventors orthotopically transplanted the MYCN retinoblastoma organoids from Example 7 into the eyeballs of nude mice. As shown in Figure 14, continued breeding resulted in mice with obvious tumor lesions in the eyeballs. Immunofluorescence staining was used to detect the MYCN retinoblastoma tumor marker gene. The results showed that the MYCN retinoblastoma successfully mimicked the characteristics of human MYCN retinoblastoma.

[0401] Therefore, the MYCN retinoblastoma organoids of Example 7 have tumor characteristics and can be used as an effective model for studying the occurrence and development mechanism of MYCN retinoblastoma disease, screening related drugs, and studying treatment plans.

[0402] Using MYCN retinoblastoma organoids or experimental animals transplanted in situ with them as drug screening models can reflect the effects of drugs on tumors in vivo and in vitro, and can be used to build a platform for in vitro high-throughput drug screening, with high application value.

[0403] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean 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 expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0404] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kind of organoid culture medium, characterized in that, Comprising: A basal medium and specific addition factors, wherein the specific addition factors include: an ALK-5 inhibitor and an FGF10 protein.

2. The culture medium according to claim 1, wherein, The ALK-5 inhibitor is A83-01; Optionally, the concentration of A83-01 is 0.2 - 2 μM, preferably 0.25 - 1 μM; Optionally, the concentration of the FGF10 protein is 10 - 100 ng / mL; preferably 50 - 100 ng / mL.

3. The culture medium according to claim 1, characterized in that, The specific addition factors further include at least one of the following: N-acetylcysteine, nicotinamide, a p38 MAPK inhibitor, and an EGF protein; Optionally, the concentration of N-acetylcysteine is 0.5 - 2.5 mM, preferably 0.75 - 2.5 mM; Optionally, the concentration of nicotinamide is 5 - 15 mM; preferably 2.5 - 10 mM; Optionally, the p38 MAPK inhibitor is SB202190; Optionally, the concentration of SB202190 is 0.2 - 2 μM, preferably 0.25 - 1 μM; Optionally, the concentration of the EGF protein is 10 - 100 ng / mL, preferably 50 ng / mL.

4. The culture medium according to claim 1, wherein The specific addition factors include at least one of the following: Forskolin, Prostaglandin E2, an R-Spondin protein, and a Noggin protein; Optionally, the concentration of Forskolin is 0.5 - 2 μM; preferably 0.8 - 1.2 μM; more preferably 1 μM; Optionally, the concentration of Prostaglandin E2 is 0.5 - 2 μM; preferably 0.8 - 1.2 μM; more preferably 1 μM; Optionally, the concentration of the R-Spondin protein is 250 - 1000 ng / mL; preferably 400 - 600 ng / mL; more preferably 500 ng / mL; Optionally, the concentration of the Noggin protein is 50 - 200 ng / mL; preferably 75 - 125 ng / mL; more preferably 100 ng / mL.

5. The culture medium according to claim 1, characterized in that, The specific addition factors further include a GSK-3β inhibitor; Optionally, the GSK-3β inhibitor is CHIR-99021; Optionally, the concentration of CHIR-99021 is 1 - 5 μM; preferably 1 μM.

6. The culture medium according to claim 1, characterized in that, The specific addition factors further include: Y-27632; Optionally, the concentration of Y-27632 is 2.5 - 10 μM; preferably 4 - 6 μM; more preferably 5 μM; Optionally, the specific addition factors further include: L-glutamine amide; Optionally, the concentration of L-glutamine is 1 - 4 mM; preferably 1.5 - 2.5 mM; more preferably 2 mM.

7. The recombinant antibody according to claim 1, wherein The basal medium is a DMEM / F12 serum-free medium; Optionally, the basal medium further includes: a hydrogen ion buffer, penicillin, and streptomycin; Optionally, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Optionally, the concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 5 - 25 mM, preferably 5 - 15 mM, preferably 10 mM; Optionally, the concentration of penicillin is 10 to 150 U / mL, preferably 20 to 70 U / mL, and the concentration of streptomycin is 0.075 to 0.15 mg / mL; Optionally, the basal medium further comprises: B27 additive; Optionally, the concentration of the B27 additive is 0.5% to 2% by volume, preferably 0.75% to 1.5% by volume, more preferably 1% by volume.

8. An organoid culture medium, characterized in that, Comprising: A basal medium and specific addition factors, the specific addition factors comprising: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, FGF10 protein and R-Spondin protein; Wherein, the ALK-5 inhibitor is A83-01 and the p38 MAPK inhibitor is SB202190; The basal culture comprises: DMEM / F12 serum-free medium, a hydrogen ion buffer, penicillin and streptomycin, and B27 additive; Optionally, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Optionally, based on the total volume of the medium, the medium comprises the following concentrations of the basal medium and specific addition factors: 98% to 99% by volume of the DMEM / F12 serum-free medium, 5 to 15 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 to 100 U / mL of the penicillin, 0.1 to 0.2 mg / mL of the streptomycin, 1% to 2% by volume of the B27 additive, 0.5 to 2.5 mM of the N-acetylcysteine, 5 to 15 mM of the nicotinamide, 0.2 to 2 μM of the A83-01, 0.2 to 2 μM of the SB202190, 10 to 100 ng / mL of the FGF10 protein, 100 to 900 μg / mL of the R-Spondin protein.

9. The culture medium according to claim 8, wherein The specific addition factors further comprise: EGF protein and a GSK-3β inhibitor; Optionally, the GSK-3β inhibitor is CHIR-99021; Optionally, based on the total volume of the medium, the medium comprises the following concentrations of the specific addition factors: 10 to 100 ng / mL of the EGF protein, 1 to 5 μM of the CHIR-99021.

10. The culture medium according to claim 8, wherein The specific addition factors further comprise: Forskolin, Prostaglandin E2, Noggin protein, L-glutamine and Y-27632; Optionally, based on the total volume of the medium, the medium comprises the following concentrations of the specific addition factors: 1 μM of the Forskolin, 1 μM of the Prostaglandin E2, 100 ng / mL of the Noggin protein, 2 mM of the L-glutamine, 5 μM of the Y-27632.

11. The culture medium according to claim 8, wherein Based on the total volume of the medium, the medium comprises the following concentrations of the basal medium and specific addition factors: 99% by volume of the DMEM / F12 serum-free medium, 10 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 50 U / mL of the penicillin, 0.05 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1.25 mM of the N-acetylcysteine, 10 mM of the nicotinamide, 1 μM of the A83-01, 1 μM of the SB202190, 1 μM of the CHIR-99021, 50 ng / mL of the EGF protein, 100 ng / mL of the FGF10 protein, 500 ng / mL of the R-Spondin protein.

12. The culture medium according to claim 8, wherein, based on the total volume of the culture medium, the culture medium comprises the following concentrations of the basal medium and specific additive factors: 99% by volume of the DMEM / F12 serum-free medium, 10 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 U / mL of the penicillin, 0.1 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1 μM of the Forskolin, 1 μM of the Prostaglandin E2, 500 ng / mL of the R-Spondin protein, 100 ng / mL of the Noggin protein, 100 ng / mL of the FGF10 protein, 2 mM of the L-glutamine, 1.25 mM of the N-acetylcysteine, 5 mM of the nicotinamide, 5 μM of the Y-27632, 500 nM of the SB202190.

13. Use of the culture medium according to any one of claims 1 to 12 in constructing an organoid or an organoid disease model.

14. The use according to claim 13, wherein The organoid includes at least one of a kidney organoid and a retinal organoid; Optionally, the organoid disease model includes at least one of a kidney organoid disease model and a retinal organoid disease model.

15. A method for constructing an organoid, characterized in that, Comprising: Culturing tissue cells in the culture medium according to any one of claims 1 to 2 to obtain the organoid.

16. The method according to claim 15, wherein The organoid includes at least one of a kidney organoid and a retinal organoid; Optionally, the tissue cells include at least one of kidney tissue cells and retinal tissue cells; Optionally, the retinal tissue cells include: retinal progenitor cells and / or precursor cells.

17. A method for constructing an organoid disease model, characterized in that, Comprising: Obtaining an organoid according to the method of claim 15 or 16; Performing gene editing on the organoid to obtain the organoid disease model; Optionally, the gene editing includes: gene silencing or gene knockout or gene overexpression; Optionally, the gene includes: a disease-related gene; Optionally, the gene editing includes: Digesting the organoid to obtain organoid single cells, Introducing a nucleic acid having gene silencing or gene knockout activity or a nucleic acid construct containing the same into the organoid single cells to obtain gene-silenced or gene-knocked-out organoid single cells, and the nucleic acid targets the disease-related gene; Culturing the single cells of the organoids after gene silencing or gene knockout to obtain the organoid disease model; Optionally, culturing the single cells of the gene - edited organoids in the culture medium according to any one of claims 1 - 12 to obtain the organoid disease model; Optionally, the nucleic acid comprises at least one of shRNA, siRNA, and sgRNA; Optionally, the organoids include at least one of kidney organoids and retinal organoids; Optionally, the diseases include at least one of nephroblastoma and retinoblastoma; Optionally, the retinoblastoma is MYCN - amplified retinoblastoma; Optionally, the organoid disease model includes at least one of nephroblastoma organoid disease model and retinoblastoma organoid disease model; Optionally, the retinoblastoma organoid disease model is MYCN - amplified retinoblastoma organoid disease model.

18. The method according to claim 17, characterized in that Comprising: The gene editing treatment is gene silencing treatment, and the disease - related gene is at least one of WTX, P53, WT1, and Trim28; Preferably, the disease - related gene is WTX and / or P53; Optionally, the nucleic acid is shRNA; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:1 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WTX gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:2 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the P53 gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:3 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WT1 gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the Trim28 gene.

19. The method according to claim 17, wherein Comprising: The disease - related gene is the MYCN gene; Optionally, the gene editing treatment includes: introducing a nucleic acid or nucleic acid construct containing the MYCN gene sequence into the retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells, or retinal organoid tissue - digested cells to obtain the retinal tissue cells, human induced pluripotent stem cells, human embryonic stem cells, or retinal organoid tissue - digested cells with over - expressed MYCN gene; 20. The method according to claim 17, wherein Comprising: The nucleic acid construct is a viral vector; Optionally, the viral vector is a non - pathogenic viral vector; Optionally, the non - pathogenic viral vector is selected from one of retroviral vectors, poxviral vectors, herpes simplex virus I vectors, lentiviral vectors, adenoviral vectors, and adeno - associated viral vectors; Optionally, the non - pathogenic viral vector is a lentiviral vector; Optionally, the infection titer of the lentiviral vector is 10 7 to 10 9 TU / mL.

21. A method for constructing an animal disease model, characterized in that, Comprising: The organoid disease model obtained by the method according to any one of claims 17 - 20, Transplant the organoid disease model into the animal to obtain the animal disease model; Optionally, the animal includes: mouse, rat, guinea pig, rabbit, cat, dog, monkey, pig or sheep.

22. A method for constructing an organoid library or an organoid disease model library, characterized in that, Comprising: An organoid obtained by the method according to claim 15 or 16 or an organoid disease model obtained by the method according to claims 17 to 20, Subculture the organoid or the organoid disease model to obtain a subcultured organoid or organoid disease model, Cryopreserve the subcultured organoid or organoid disease model to obtain the organoid library or the organoid disease model library.

23. An organoid disease model, characterized in that, The organoid disease model is constructed by the method according to claims 17 to 20; Optionally, the organoid disease model includes at least one of a Wilms' tumor organoid disease model and a retinoblastoma organoid disease model; Optionally, the retinoblastoma organoid disease model is an MYCN - amplified retinoblastoma organoid disease model.

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