Disease cortex brain organ disease model and construction method and application thereof

Abstract

The invention relates to a diseased cortical brain organ disease model and a construction method and application thereof. The method comprises the following steps: (1) culturing cortical brain organs; (2) performing carbon black nanoparticle exposure on the cultured cortical brain organ; (3) performing immunofluorescence staining detection; (4) analyzing images and data; according to the method, carbon black nano-particle injury treatment is introduced into cortical brain organs cultured to the 35th day, and the pathological environment of ALS is simulated, so that an experimental platform which highly simulates the human brain environment is provided for studying the pathological mechanism of ALS.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a disease model of a lesioned cortical brain organoid, and a construction method and application thereof. Background Art

[0002] Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the central nervous system, primarily damaging neurons in the brain, brainstem, and spinal cord that control muscle movement. As motor neurons degenerate, patients gradually lose muscle control, leading to muscle atrophy and weakness, which may eventually affect breathing and swallowing functions. The cause of ALS is complex, involving genetic factors, environmental factors, and the accumulation of intracellular toxins. In ALS research, building disease models is crucial for understanding pathological mechanisms, discovering new biomarkers, and developing and testing treatments.

[0003] The existing ALS models mainly include: (1) Animal models: Commonly used animal models in ALS research include transgenic mice and rat models, which can simulate certain pathological characteristics of ALS. (2) Cell culture models: including primary neuron culture and induced pluripotent stem cell (iPSC)-derived motor neuron culture.

[0004] These models have played an important role in studying the molecular mechanisms of ALS, but due to the lack of three-dimensional structure and complex interactions between cells, there are some problems: (1) Existing ALS models often find it difficult to fully simulate the complex pathological changes in the cerebral cortex of ALS patients, such as synaptic connections between neurons, interactions between glial cells, and the formation of protein aggregates; (2) The cerebral cortex is composed of multiple types of cells, including neurons, astrocytes, etc., and existing models are usually unable to fully simulate the diversity of these cell types and their interactions in the ALS pathological process; (3) Existing ALS organoid models may show differences in stability and reproducibility between different experimental batches and different laboratories; (4) The use of animal models is subject to ethical restrictions and is costly, which limits their application in drug screening and large-scale pathological studies. Summary of the invention

[0005] Organoids are self-organizing three-dimensional cell structures that can simulate certain functions and structural features of the original organs. In the field of neuroscience, organoid technology has been used to simulate areas such as the cerebral cortex and hippocampus, providing a new platform for studying neural development and diseases.

[0006] In recent years, with the development of stem cell technology and three-dimensional culture technology, organoids have become a powerful tool for simulating human organs and disease states. Researchers have tried to use iPSCs from ALS patients to construct organoid models to simulate the pathological changes of ALS. These models have simulated the neurodegenerative changes of ALS to a certain extent, but there are still some limitations, such as the stability, repeatability and reproducibility of pathological characteristics of the model.

[0007] In order to solve the above technical problems, the present invention provides an improved method for constructing a cortical brain organoid disease model of ALS cortical-like lesions, which simulates the pathological environment of ALS by introducing carbon black nanoparticle damage treatment into cortical brain organoids cultured to the 35th day, thereby providing an experimental platform that highly simulates the human brain environment for studying the pathological mechanism of ALS.

[0008] In a first aspect, the present invention provides a method for constructing a disease model of a diseased cortical brain organoid, comprising the following steps:

[0009] (1) Cultivation of cortical brain organoids;

[0010] (2) Cultured cortical brain organoids were exposed to carbon black nanoparticles;

[0011] (3) Immunofluorescence staining;

[0012] (4) Image and data analysis.

[0013] Carbon black is a conventional nanomaterial with widespread applications. It is a product of incomplete combustion or cracking of hydrocarbons, mainly composed of carbon elements, with a carbon content of 90% to 99%, and also contains a small amount of oxygen, hydrogen, nitrogen and sulfur. These elements mainly come from the raw materials of carbon black, and are brought into the carbon black during the carbon black generation process and the oxidation process after generation. The use of carbon black nanoparticles in the present invention can cause damage to cortical brain organoids and simulate disease models.

[0014] The carbon black used in the present invention is self-made and / or purchased. In addition, the carbon black can be removed and modified before use.

[0015] In the present invention, the concentration of the carbon black nanoparticles is 1 to 100 μg / mL.

[0016] In some specific embodiments of the present invention, the concentration of the carbon black nanoparticles is 10-50 μg / mL.

[0017] In the present invention, the exposure to the carbon black nanoparticles is performed at the early stage of maturation of cortical organoids.

[0018] In the present invention, the exposure time is 12 to 48 hours.

[0019] In some specific embodiments of the invention, the exposure time is 24 h.

[0020] In the present invention, the carbon black nanoparticles can reduce the expression of at least one protein among TDP-43 and TOMM20.

[0021] In a second aspect, the present invention provides a method for culturing the above-mentioned cortical brain organoids, comprising the following steps:

[0022] (1) hIPSCs or hESCs were cultured in six-well plates coated with Matrigel, PGM1 medium was added, and 1-5 mL of medium was replaced every day. After the cell density reached 80-90%, the cells were passaged.

[0023] (2) Embryoid body formation: After removing the PGM1 medium in step (1), add accutase, PGM1 medium, and EB formation medium to prepare a cell suspension with a cell concentration of 9,000-12,000 cells / well;

[0024] (3) Neuroectoderm induction: replace EB formation medium with Induction medium;

[0025] (4) Discard the Induction medium and add Maturation medium 1 for culture. Change the medium every 4 to 6 days and culture for 15 to 20 days to obtain mature cortical brain organoids.

[0026] In the present invention, the PGM1 culture medium components include PGM1 and supplements;

[0027] And / or, the EB formation medium comprises: DF12 and Supplement A;

[0028] and / or, the induction medium comprises DF12, Neurobasal, Glutamax, NEAA and B27;

[0029] And / or, the ingredients of the maturation medium include: DF12, Neurobasal, Glutamax, NEAA and BDNF.

[0030] In the present invention, the culture conditions of step (1) to step (4) are 4-6CO2, 35°C to 40°C.

[0031] In the present invention, the diseases that cause cerebral cortical lesions include: acute cerebrovascular disease, cerebral hemorrhage, cerebral infarction, inflammatory disease encephalitis, autoimmune encephalitis, amyotrophic lateral sclerosis, Alzheimer's disease, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Parkinson's disease dementia, neuromyelitis optica spectrum disorder, glioma, meningioma, brain parasitic disease, and epilepsy.

[0032] In some specific embodiments of the invention, the disease is amyotrophic lateral sclerosis.

[0033] In a third aspect, the present invention provides a cortical brain organoid prepared by the above-mentioned culture method.

[0034] In a fourth aspect, the present invention provides a disease model of a diseased cortical brain organoid, characterized in that it is obtained by any of the above-mentioned methods.

[0035] In a fifth aspect, the present invention provides an application of the above-mentioned cortical brain organoid or the above-mentioned diseased cortical brain organoid disease model in brain development research, nervous system disease research or drug screening.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention simulates the pathological environment of ALS by introducing carbon black nanoparticles into cortical brain organoids cultured to day 35, thereby providing an experimental platform that highly simulates the human brain environment for studying the pathological mechanism of ALS;

[0038] (2) The present invention uses immunofluorescence staining technology to verify the constructed model to ensure the accuracy and reliability of the model.

[0039] (3) The model constructed by the present invention can promote in-depth research on the pathological mechanism of ALS and can observe ALS-related pathological changes in detail in a controlled environment, including nerve cell damage and abnormal protein aggregation, providing a new perspective for understanding the development of the disease;

[0040] (4) The model can be used for drug screening and evaluation, accelerating the discovery and development of potential therapeutic drugs for ALS. By using human embryonic stem cells to construct homogeneous and reproducible cortical brain organoids, it will help promote the development of medical care. At the same time, the establishment and application of this model will reduce dependence on animal models and meet the requirements of modern scientific research ethics.

[0041] (5) The implementation of the model of the present invention will also promote the development of the field of neuroscience and enhance the understanding of the relationship between brain development, function and disease. The technical solution of the present invention may provide new ideas and methods for the clinical treatment of ALS and other neurodegenerative diseases, which has important scientific and clinical significance.

[0042] The following abbreviations represent the following meanings:

[0043] ALS stands for amyotrophic lateral sclerosis;

[0044] hIPSCs stands for human pluripotent stem cells;

[0045] hESCs stands for human embryonic stem cells;

[0046] CBNPs stands for carbon black nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Differentiation of human pluripotent stem cells into cortical brain organoids, maturation process and exposure to carbon black nanoparticles;

[0048] Figure 2 Effects of carbon black nanoparticle treatment on TDP-43 and TOMM20 expression (human pluripotent stem cells);

[0049] Figure 3 Correlation analysis between TDP-43 protein and TOMM20 protein expression and distance (human pluripotent stem cells);

[0050] Figure 4 Differentiation of human embryonic stem cells into cortical brain organoids, maturation process and exposure to carbon black nanoparticles;

[0051] Figure 5 Effects of carbon black nanoparticle treatment on the expression of TDP-43 and TOMM20 (human embryonic stem cells);

[0052] Figure 6 Correlation analysis between TDP-43 protein and TOMM20 protein expression and distance (human embryonic stem cells). DETAILED DESCRIPTION

[0053] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Example 1

[0055] A method for constructing a cortical brain organoid disease model that simulates amyotrophic lateral sclerosis cerebral cortical lesions comprises the following steps:

[0056] 1. Cultivation of cortical brain organoids

[0057] (1) Human pluripotent stem cells (hIPSCs)

[0058] 2 hours in advance, a six-well plate was coated with matrix gel and placed in a 37°C, 5% CO2 incubator. After human pluripotent stem cells were revived, they were cultured in the six-well plate coated with matrix gel. PGM1 medium was added and the medium was replaced by 2 mL every day. The cells were passaged after the density reached 80-90%.

[0059] (2) Embryoid body formation

[0060] The culture medium in the six-well plate was aspirated, and the plate was washed once with PBS. 1 mL of accutase was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for digestion for 10 min. The cell suspension in the six-well plate was transferred to a 15 mL centrifuge tube, centrifuged at 1500×g for 3 min, the supernatant was discarded, and PGM1 culture medium was added to resuspend the cells; EB formation medium was added and added to a low-adsorption U-shaped 96-well plate to dilute the final cell concentration to 9000-12000 cells / well, and the plate was placed in a 37°C, 5% CO2 incubator for culture, and the medium was changed every two days; wherein the components of the PGM1 culture medium are PGM1 and supplements; and the components of the EB formation medium are DF12 and Supplement A.

[0061] (3) Neural ectoderm induction

[0062] On the 10th day, the culture medium in step (2) was discarded, and the embryoid bodies were transferred to a low-adsorption six-well plate using a pipette, 8 per well, and Induction medium was added. The plates were cultured on an orbital shaker in an incubator at 37°C and 5% CO2, and the medium was changed every two days; wherein the Induction medium contains DF12, Neurobasal, Glutamax, NEAA and B27.

[0063] (4) Cortical organoid maturation

[0064] On the 18th day, the Induction medium in the six-well plate was replaced with Maturation medium 1, and the medium was changed every 4 days; the Maturation medium contained DF12, Neurobasal, Glutamax, NEAA and BDNF.

[0065] 2. Damage treatment of carbon black nanoparticles (CBNPs)

[0066] The cortical brain organoids on the 35th day of culture were treated with carbon black nanoparticles for 24 hours and cultured in a 37°C, 5% CO2 incubator; the concentration of carbon black nanoparticles used in the amyotrophic lateral sclerosis cortical lesion cortical brain organoid disease model was 10 μg / mL, and the incubation time was 24 hours.

[0067] 3. Immunofluorescence staining

[0068] (1) Use a pipette to transfer the cortical brain organoids from the low-adhesion six-well plate to a 24-well plate, placing one organoid in each well;

[0069] (2) Remove the residual culture medium from the 24-well plate, wash three times with PBS, add 1.5 ml of 4% paraformaldehyde to each well, and place in a 4°C refrigerator for fixation for 24 h;

[0070] (3) Wash three times with PBS, add 1.5 ml of 30% sucrose solution to each well, and dehydrate in a 4°C refrigerator for 24 h;

[0071] (4) Aspirate the sucrose solution, add OCT, and equilibrate at room temperature for 30 min;

[0072] (5) Place half the volume of OCT in the embedding box and freeze it in a -20°C refrigerator until the OCT turns from transparent to white.

[0073] (6) Place the cortical brain organoids in an embedding box, pour OCT until the cortical brain organoids are completely covered, and store in a -80°C refrigerator;

[0074] (7) Using a cryosectioner, the cortical brain organoids embedded in OCT were cut into tissue slices with a thickness of 15 μm and adsorbed onto glass slides;

[0075] (8) Place the slides in a washing box and wash with PBS for 5 min;

[0076] (9) Dry the PBS and draw a circle around the slice with an immunohistochemistry pen;

[0077] (10) 0.3% Triton-100X permeabilization for 15 min;

[0078] (11) Discard Triton-100X and use ready-to-use goat serum to block at room temperature for 1 h;

[0079] (12) Washing with PBS three times, incubating with primary antibody and ready-to-use goat serum according to the antibody ratio, and incubating at 4°C for 8-16 hours; wherein the primary antibody includes: TDP-43, TOMM20;

[0080] (13) Washed with PBS three times, incubated with secondary antibody and ready-to-use goat serum at room temperature for 2 h;

[0081] (14) Wash with PBS three times, stain the cell nuclei with DAPI dye, and incubate at room temperature for 15 min;

[0082] (15) The sections were washed three times with PBS and dried, and the sections were sealed with anti-fluorescence attenuation sealing agent. The fluorescence signal was detected using a confocal laser scanning microscope, and the subsequent image quantitative analysis was performed using MATLAB and ImageJ.

[0083] Example 2

[0084] A method for constructing a cortical brain organoid disease model that simulates amyotrophic lateral sclerosis cerebral cortical lesions comprises the following steps:

[0085] 1. Cultivation of cortical brain organoids

[0086] (1) Human pluripotent stem cells (hIPSCs)

[0087] 2 hours in advance, a six-well plate was coated with matrix gel and placed in a 37°C, 5% CO2 incubator. After human pluripotent stem cells were revived, they were cultured in the six-well plate coated with matrix gel. PGM1 medium was added and the medium was replaced by 2 mL every day. The cells were passaged after the density reached 80-90%.

[0088] (2) Embryoid body formation

[0089] The culture medium in the six-well plate was aspirated, and the plate was washed once with PBS. 1 mL of accutase was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for digestion for 10 min. The cell suspension in the six-well plate was transferred to a 15 mL centrifuge tube, centrifuged at 1500×g for 3 min, the supernatant was discarded, and PGM1 culture medium was added to resuspend the cells; EB formation medium was added and added to a low-adsorption U-shaped 96-well plate to dilute the final cell concentration to 9000-12000 cells / well, and the plate was placed in a 37°C, 5% CO2 incubator for culture, and the medium was changed every two days; wherein the components of the PGM1 culture medium are PGM1 and supplements; and the components of the EB formation medium are DF12 and Supplement A.

[0090] (3) Neural ectoderm induction

[0091] On the 10th day, the culture medium in step (2) was discarded, and the embryoid bodies were transferred to a low-adsorption six-well plate using a pipette, 8 per well, and Induction medium was added. The plates were cultured on an orbital shaker in an incubator at 37°C and 5% CO2, and the medium was changed every two days; wherein the Induction medium contains DF12, Neurobasal, Glutamax, NEAA and B27.

[0092] (4) Cortical organoid maturation

[0093] On the 18th day, the Induction medium in the six-well plate was replaced with Maturation medium 1, and the medium was changed every 4 days; the Maturation medium contained DF12, Neurobasal, Glutamax, NEAA and BDNF.

[0094] 2. Damage treatment of carbon black nanoparticles (CBNPs)

[0095] The cortical brain organoids on the 35th day of culture were treated with carbon black nanoparticles for 24 hours and cultured in a 37°C, 5% CO2 incubator; the concentration of carbon black nanoparticles used in the amyotrophic lateral sclerosis cortical lesion cortical brain organoid disease model was 50μg / mL, and the incubation time was 24 hours.

[0096] 3. Immunofluorescence staining

[0097] (1) Use a pipette to transfer the cortical brain organoids from the low-adhesion six-well plate to a 24-well plate, placing one organoid in each well;

[0098] (2) Remove the residual culture medium from the 24-well plate, wash three times with PBS, add 1.5 ml of 4% paraformaldehyde to each well, and place in a 4°C refrigerator for fixation for 24 h;

[0099] (3) Wash three times with PBS, add 1.5 ml of 30% sucrose solution to each well, and dehydrate in a 4°C refrigerator for 24 h;

[0100] (4) Aspirate the sucrose solution, add OCT, and equilibrate at room temperature for 30 min;

[0101] (5) Place half the volume of OCT in the embedding box and freeze it in a -20°C refrigerator until the OCT turns from transparent to white.

[0102] (6) Place the cortical brain organoids in an embedding box, pour OCT until the cortical brain organoids are completely covered, and store in a -80°C refrigerator;

[0103] (7) Using a cryosectioner, the cortical brain organoids embedded in OCT were cut into tissue slices with a thickness of 15 μm and adsorbed onto glass slides;

[0104] (8) Wash in PBS for 5 min in a slide washing box;

[0105] (9) Dry the PBS and draw a circle around the slice with an immunohistochemistry pen;

[0106] (10) 0.3% Triton-100X permeabilization for 15 min;

[0107] (11) Discard Triton-100X and use ready-to-use goat serum to block at room temperature for 1 h;

[0108] (12) Washing with PBS three times, incubating with primary antibody and ready-to-use goat serum according to the antibody ratio, and incubating at 4°C for 8-16 hours; wherein the primary antibody includes: TDP-43, TOMM20;

[0109] (13) Washed with PBS three times, incubated with secondary antibody and ready-to-use goat serum at room temperature for 2 h;

[0110] (14) Wash with PBS three times, stain the cell nuclei with DAPI dye, and incubate at room temperature for 15 min;

[0111] (15) The sections were washed three times with PBS and dried, and the sections were sealed with anti-fluorescence attenuation sealing agent. The fluorescence signal was detected using a confocal laser scanning microscope, and the subsequent image quantitative analysis was performed using MATLAB and ImageJ.

[0112] Example 3

[0113] A method for constructing a cortical brain organoid disease model that simulates amyotrophic lateral sclerosis cerebral cortical lesions comprises the following steps:

[0114] 1. Cultivation of cortical brain organoids

[0115] (1) Human embryonic stem cells (hESC)

[0116] 2 hours in advance, a six-well plate was coated with matrix gel and placed in a 37°C, 5% CO2 incubator. Human embryonic stem cells were revived and cultured in the six-well plate coated with matrix gel. PGM1 medium was added and the medium was replaced by 2 ml every day. The cells were passaged after the density reached 80-90%.

[0117] (2) Embryoid body formation

[0118] The culture medium in the six-well plate was aspirated, and the plate was washed once with PBS. 1 mL of accutase was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for digestion for 10 min. The cell suspension in the six-well plate was transferred to a 15 mL centrifuge tube, centrifuged at 1500×g for 3 min, the supernatant was discarded, and PGM1 culture medium was added to resuspend the cells; EB formation medium was added and added to a low-adsorption U-shaped 96-well plate to dilute the final cell concentration to 9000-12000 cells / well, and the plate was placed in a 37°C, 5% CO2 incubator for culture, and the medium was changed every two days; wherein the components of the PGM1 culture medium are PGM1 and supplements; and the components of the EB formation medium are DF12 and Supplement A.

[0119] (3) Neural ectoderm induction

[0120] On the 10th day, the culture medium in step (2) was discarded, and the embryoid bodies were transferred to a low-adsorption six-well plate using a pipette, 8 per well, and Induction medium was added. The plates were cultured on an orbital shaker in an incubator at 37°C and 5% CO2, and the medium was changed every two days; wherein the Induction medium contains DF12, Neurobasal, Glutamax, NEAA and B27.

[0121] (4) Cortical organoid maturation

[0122] On the 18th day, the Induction medium in the six-well plate was replaced with Maturation medium 1, and the medium was changed every 4 days; the Maturation medium contained DF12, Neurobasal, Glutamax, NEAA and BDNF.

[0123] 2. Damage treatment of carbon black nanoparticles (CBNPs)

[0124] The cortical brain organoids on the 35th day of culture were treated with carbon black nanoparticles for 24 hours and cultured in a 37°C, 5% CO2 incubator; the concentration of carbon black nanoparticles used in the amyotrophic lateral sclerosis cortical lesion cortical brain organoid disease model was 10 μg / mL, and the incubation time was 24 hours.

[0125] 3. Immunofluorescence staining

[0126] (1) Use a pipette to transfer the cortical brain organoids from the low-adhesion six-well plate to a 24-well plate, placing one organoid in each well;

[0127] (2) Remove the residual culture medium from the 24-well plate, wash three times with PBS, add 1.5 ml of 4% paraformaldehyde to each well, and place in a 4°C refrigerator for fixation for 24 h;

[0128] (3) Wash three times with PBS, add 1.5 ml of 30% sucrose solution to each well, and dehydrate in a 4°C refrigerator for 24 h;

[0129] (4) Aspirate the sucrose solution, add OCT, and equilibrate at room temperature for 30 min;

[0130] (5) Place half the volume of OCT in the embedding box and freeze it in a -20°C refrigerator until the OCT turns from transparent to white.

[0131] (6) Place the cortical brain organoids in an embedding box, pour OCT until the cortical brain organoids are completely covered, and store in a -80°C refrigerator;

[0132] (7) Using a cryosectioner, the cortical brain organoids embedded in OCT were cut into tissue slices with a thickness of 15 μm and adsorbed onto glass slides;

[0133] (8) Place the slides in a washing box and wash with PBS for 5 min;

[0134] (9) Dry the PBS and draw a circle around the slice with an immunohistochemistry pen;

[0135] (10) 0.3% Triton-100X permeabilization for 15 min;

[0136] (11) Discard Triton-100X and use ready-to-use goat serum to block at room temperature for 1 h;

[0137] (12) Washing with PBS three times, incubating with primary antibody and ready-to-use goat serum according to the antibody ratio, and incubating at 4°C for 8-16 hours; wherein the primary antibody includes: TDP-43, TOMM20;

[0138] (13) Washed with PBS three times, incubated with secondary antibody and ready-to-use goat serum at room temperature for 2 h;

[0139] (14) Wash with PBS three times, stain the cell nuclei with DAPI dye, and incubate at room temperature for 15 min;

[0140] (15) The sections were washed three times with PBS and dried, and the sections were sealed with anti-fluorescence attenuation sealing agent. The fluorescence signal was detected using a confocal laser scanning microscope, and the subsequent image quantitative analysis was performed using MATLAB and ImageJ.

[0141] Example 4

[0142] A method for constructing a cortical brain organoid disease model that simulates amyotrophic lateral sclerosis cerebral cortical lesions comprises the following steps:

[0143] 1. Cultivation of cortical brain organoids

[0144] (1) Human embryonic stem cells (hESC)

[0145] 2 hours in advance, a six-well plate was coated with matrix gel and placed in a 37°C, 5% CO2 incubator. Human embryonic stem cells were revived and cultured in the six-well plate coated with matrix gel. PGM1 medium was added and the medium was replaced by 2 ml every day. The cells were passaged after the density reached 80-90%.

[0146] (2) Embryoid body formation

[0147] The culture medium in the six-well plate was aspirated, and the plate was washed once with PBS. 1 mL of accutase was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for digestion for 10 min. The cell suspension in the six-well plate was transferred to a 15 mL centrifuge tube, centrifuged at 1500×g for 3 min, the supernatant was discarded, and PGM1 culture medium was added to resuspend the cells; EB formation medium was added and added to a low-adsorption U-shaped 96-well plate to dilute the final cell concentration to 9000-12000 cells / well, and the plate was placed in a 37°C, 5% CO2 incubator for culture, and the medium was changed every two days; wherein the components of the PGM1 culture medium are PGM1 and supplements; and the components of the EB formation medium are DF12 and Supplement A.

[0148] (3) Neural ectoderm induction

[0149] On the 10th day, the culture medium in step (2) was discarded, and the embryoid bodies were transferred to a low-adsorption six-well plate using a pipette, 8 per well, and Induction medium was added. The plates were cultured on an orbital shaker in an incubator at 37°C and 5% CO2, and the medium was changed every two days; wherein the Induction medium contains DF12, Neurobasal, Glutamax, NEAA and B27.

[0150] (4) Cortical organoid maturation

[0151] On the 18th day, the Induction medium in the six-well plate was replaced with Maturation medium 1, and the medium was changed every 4 days; the Maturation medium contained DF12, Neurobasal, Glutamax, NEAA and BDNF.

[0152] 2. Damage treatment of carbon black nanoparticles (CBNPs)

[0153] The cortical brain organoids on the 35th day of culture were treated with carbon black nanoparticles for 24 hours and cultured in a 37°C, 5% CO2 incubator; the concentration of carbon black nanoparticles used in the amyotrophic lateral sclerosis cortical lesion cortical brain organoid disease model was 50μg / mL, and the incubation time was 24 hours.

[0154] 3. Immunofluorescence staining

[0155] (1) Use a pipette to transfer the cortical brain organoids from the low-adhesion six-well plate to a 24-well plate, placing one organoid in each well;

[0156] (2) Remove the residual culture medium from the 24-well plate, wash three times with PBS, add 1.5 ml of 4% paraformaldehyde to each well, and place in a 4°C refrigerator for fixation for 24 h;

[0157] (3) Wash three times with PBS, add 1.5 ml of 30% sucrose solution to each well, and dehydrate in a 4°C refrigerator for 24 h;

[0158] (4) Aspirate the sucrose solution, add OCT, and equilibrate at room temperature for 30 min;

[0159] (5) Place half the volume of OCT in the embedding box and freeze it in a -20°C refrigerator until the OCT turns from transparent to white.

[0160] (6) Place the cortical brain organoids in an embedding box, pour OCT until the cortical brain organoids are completely covered, and store in a -80°C refrigerator;

[0161] (7) Using a cryosectioner, the cortical brain organoids embedded in OCT were cut into tissue slices with a thickness of 15 μm and adsorbed onto glass slides;

[0162] (8) Place the slides in a washing box and wash with PBS for 5 min;

[0163] (9) Dry the PBS and draw a circle around the slice with an immunohistochemistry pen;

[0164] (10) 0.3% Triton-100X permeabilization for 15 min;

[0165] (11) Discard Triton-100X and use ready-to-use goat serum to block at room temperature for 1 h;

[0166] (12) Washing with PBS three times, incubating with primary antibody and ready-to-use goat serum according to the antibody ratio, and incubating at 4°C for 8-16 hours; wherein the primary antibody includes: TDP-43, TOMM20;

[0167] (13) Washed with PBS three times, incubated with secondary antibody and ready-to-use goat serum at room temperature for 2 h;

[0168] (14) Wash with PBS three times, stain the cell nuclei with DAPI dye, and incubate at room temperature for 15 min;

[0169] (15) The sections were washed three times with PBS and dried, and the sections were sealed with anti-fluorescence attenuation sealing agent. The fluorescence signal was detected using a confocal laser scanning microscope, and the subsequent image quantitative analysis was performed using MATLAB and ImageJ.

[0170] In Examples 1 to 4, human pluripotent stem cells (hIPSCs) and human embryonic stem cells (hESCs) were used to culture cortical brain organoids at different concentrations (10 μg / mL, 50 μg / mL). In the control group, TDP-43 was mainly located in the cell nucleus, but when carbon black nanoparticles were added, TDP-43 was located in the mitochondria, which is consistent with the protein pathological changes of TDP-43 in ALS, indicating that the cortical brain organoid disease model of amyotrophic lateral sclerosis cortical lesions was successfully constructed.

[0171] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a disease model of a diseased cortical brain organoid, characterized in that: The steps include: (1) Cultivation of cortical brain organoids; (2) Cultured cortical brain organoids were exposed to carbon black nanoparticles; (3) Immunofluorescence staining; (4) Image and data analysis.

2. The construction method according to claim 1, characterized in that: The concentration of the carbon black nanoparticles is 1 to 100 μg / mL; further, the concentration of the carbon black nanoparticles is 10 to 50 μg / mL.

3. The construction method according to claim 1 or 2, characterized in that: The exposure to the carbon black nanoparticles was performed at an early stage of cortical organoid maturation.

4. The construction method according to claim 1, characterized in that: The exposure time is 12 to 48 hours; further, the exposure time is 24 hours.

5. The construction method according to claim 1, characterized in that: The carbon black nanoparticles can reduce the expression of at least one protein among TDP-43 and TOMM20.

6. The construction method according to claim 1, characterized in that: The diseases include acute cerebrovascular disease, cerebral hemorrhage, cerebral infarction, inflammatory disease encephalitis, autoimmune encephalitis, amyotrophic lateral sclerosis, Alzheimer's disease, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Parkinson's disease dementia, neuromyelitis optica spectrum disorder, glioma, meningioma, brain parasitic disease, epilepsy; further, the disease is amyotrophic lateral sclerosis.

7. The method for culturing cortical brain organoids according to claim 1, characterized in that: The steps include: (1) hIPSCs or hESCs were cultured in six-well plates coated with Matrigel, PGM1 medium was added, and 1-5 mL of medium was replaced every day. After the cell density reached 80-90%, the cells were passaged. (2) Embryoid body formation: After removing the PGM1 medium in step (1), add accutase, PGM1 medium, and EBformation medium to prepare a cell suspension with a cell concentration of 9,000-12,000 cells / well; (3) Neuroectoderm induction: replace EB formation medium with Induction medium; (4) Discard the Induction medium and add Maturation medium 1 for culture. Change the medium every 4 to 6 days and culture for 15 to 20 days to obtain mature cortical brain organoids.

8. A cortical brain organoid, characterized in that Prepared by the culture method according to claim 7.

9. A disease model of a diseased cortical brain organoid, characterized in that: Obtained by the method according to any one of claims 1 to 6.

10. Use of the cortical brain organoid according to claim 8 or the diseased cortical brain organoid disease model according to claim 9 in brain development research, nervous system disease research or drug screening.

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