A midbrain organoid and a preparation method and application thereof

Abstract

The application provides a midbrain organoid and a preparation method and application thereof. The midbrain organoid is obtained in vitro by using pluripotent stem cells, can simulate the development and maturation rules of nerve cells in vivo, provides a solid foundation for later researches such as building a drug screening platform and modeling of diseases related to dopaminergic neurons, and opens up a new way for cell replacement therapy.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a midbrain organoid, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases are a group of diseases characterized by the progressive loss of brain nerve cells with age. Their causes are complex and their impact is widespread. The slow progress in mechanistic research and clinical trials is partly due to the research models currently used. Currently, research models for neurodegenerative diseases primarily rely on animal models. These models mainly amplify one or more influencing factors in the complex pathogenesis process in humans. However, due to the complexity of the causes and significant individual differences, animal models cannot fully simulate the disease progression. Furthermore, because animals and humans have different genetic backgrounds, some drugs or treatments that are effective in animal experiments have yielded unsatisfactory results in clinical trials.

[0003] For these reasons, numerous studies have used the differentiation of human pluripotent stem cells (hPSCs) to mimic the developmental patterns of nerve cells in vivo. This allows for the acquisition of nerve cells in vitro, which are then combined with animal experimental results to facilitate drug research, treatment methods, and the exploration of disease mechanisms. hPSCs include human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), possessing self-replication and multi-lineage differentiation potential. They can be directed to differentiate in vitro by inhibiting or activating specific signaling pathways, thereby obtaining different types of cells, tissues, and organs.

[0004] Parkinson's disease (PD) is the second most common neurodegenerative disease, with main symptoms including motor and non-motor symptoms, and has a significant impact on society and families. Research indicates that the progressive loss of dopaminergic neurons (DANs) in the substantia nigra of the midbrain is likely a direct cause of symptoms as PD progresses. Therefore, dopaminergic neurons obtained through in vitro differentiation can serve as a human in vitro research model for PD. However, monolayer two-dimensional (2D) differentiated neurons are of limited variety, lacking diverse neuronal types, glial cells, and other stromal cells, and cannot simulate the three-dimensional (3D) structure of brain tissue and the neuronal development process. Summary of the Invention

[0005] To address the problems existing in the prior art, this application simulates the developmental trajectory of dopaminergic neurons and constructs a midbrain organoid with functional dopaminergic neurons. The midbrain organoid better simulates in vivo tissues in terms of cells, structure, and function, providing a solid foundation for the subsequent research on drug screening platforms and modeling for dopaminergic neuron-related diseases, and opening up new avenues for cell replacement therapy.

[0006] Specifically, this application relates to the following aspects:

[0007] 1. A method for preparing a midbrain organoid, comprising:

[0008] Pluripotent stem cells are digested into single-cell suspensions and cultured for differentiation to obtain cells in the early stage of differentiation.

[0009] The cells in the early stage of differentiation were cultured to obtain the midbrain organoids.

[0010] In the step of digesting pluripotent stem cells into a single-cell suspension and performing differentiation culture to obtain cells in the early differentiation stage, the marker for obtaining the cells in the early differentiation stage is that the following conditions (1) and / or (2) are met:

[0011] Condition (1) Differentiated pluripotent stem cells were observed to be spherical with a diameter greater than 500 μm under an optical microscope;

[0012] Condition (2) Immunofluorescence staining of differentiated pluripotent stem cells revealed that they expressed markers of ventral lateral lamina cells of the midbrain.

[0013] 2. According to the preparation method described in item 1, wherein the marker for obtaining the cells in the early stage of differentiation is that conditions (1) and (2) are met.

[0014] 3. The preparation method according to item 1 or 2, wherein the midbrain ventral basal plate cell marker is LMX1A and / or FOXA2.

[0015] 4. The preparation method according to any one of items 1-3, wherein the stage of digesting pluripotent stem cells into a single-cell suspension and performing differentiation culture to obtain early-stage differentiation cells includes: performing differentiation culture using NIM basal medium to obtain the early-stage differentiation cells;

[0016] Preferably, the NIM basal culture medium contains SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators.

[0017] 5. The preparation method according to item 4, wherein the NIM basal medium is a medium containing 92-99% DMEM / F12 medium, 0.5-4% N-2 additive, 0.5-4% MEM NEAA and 1-5 μg / mL Heparin;

[0018] Preferably, the NIM basal culture medium contains 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM SB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021;

[0019] More preferably, the single-cell suspension is seeded into a low-adsorption plate and cultured for 3-5 days under conditions of 35°C to 39°C and 3% to 7% CO2, and cells in the early stage of differentiation are harvested.

[0020] More preferably, the single-cell suspension is subjected to a concentration of 1×10⁻⁶ cells / mL at 37°C and 5% CO₂. 3 -1×10 4 Cells were seeded in low-adsorption 96-well V plates for differentiation culture. Half of the culture medium was added on day 2, and cells in the early differentiation stage were harvested on day 4.

[0021] 6. The preparation method according to any one of items 1-5, wherein, in the step of differentiating and culturing the cells in the early differentiation stage to obtain the midbrain organoid, the indicator of obtaining the midbrain organoid is that any two or more of the following conditions are met:

[0022] Condition (1) The diameter of cells in the early stage of differentiation is greater than 1 mm as observed under an optical microscope;

[0023] Condition (2) Immunofluorescence staining of early-stage differentiated cells revealed that they expressed dopaminergic neuron markers;

[0024] Condition (3) The cells in the early stage of differentiation have dopaminergic neuronal calcium activity;

[0025] Condition (4) The cells in the early stage of differentiation have the electrophysiological function of dopaminergic neurons;

[0026] Condition (5) involves cutting early-stage differentiated cells, which have the ability to self-assemble.

[0027] 7. The preparation method according to item 6, wherein the stage of differentiating and culturing the cells in the early stage of differentiation to obtain the midbrain organoid includes: differentiating and culturing the cells in multiple sub-stages using different culture media to obtain the midbrain organoid;

[0028] Preferably, NDM medium is used in all sub-stages except the first sub-stage;

[0029] More preferably, the NDM medium is a medium containing 92-99% Neurobasal medium, 0.5-4% N-2 additive and 0.5-4% MEM NEAA.

[0030] 8. The preparation method according to item 7, wherein the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoid includes the following four sub-stages:

[0031] In the first sub-stage, NIM basal medium containing SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators was used.

[0032] In the second sub-stage, NDM medium containing GSK-3 inhibitor was used;

[0033] In the third sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, and GDNF was used.

[0034] In the fourth sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, GDNF, and AA was used.

[0035] Preferably, the NIM basal medium is a medium containing 92-99% DMEM / F12 medium, 0.5-4% N-2 additive, 0.5-4% MEM NEAA and 1-5 μg / mL Heparin;

[0036] More preferably, no matrix adhesive is added in any of the sub-stages.

[0037] 9. The preparation method according to item 8, wherein the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoid includes the following four sub-stages:

[0038] In the first sub-stage, NIM basal medium containing 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM MSB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021 was used.

[0039] In the second sub-stage, NDM medium containing 1-5 mM CHIR99021 was used;

[0040] In the third sub-stage, NDM medium containing 0.5–2 μM cAMP, 1–5 ng / mL TGFβ3, 1–20 ng / mL BDNF, and 1–20 ng / mL GDNF was used.

[0041] In the fourth sub-stage, NDM medium containing 0.5–2 μM cAMP, 1–5 ng / mL TGFβ3, 1–20 ng / mL BDNF, 1–20 ng / mL GDNF, and 100–200 μM AA was used.

[0042] 10. The preparation method according to item 9, wherein the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoid includes the following four sub-stages:

[0043] In the first sub-stage, the cells were differentiated and cultured for 2-3 days in NIM basal medium containing 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM MSB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021.

[0044] In the second sub-stage, the cells were cultured in NDM medium containing 1-5 mM CHIR99021 for 1-2 days for differentiation.

[0045] In the third sub-stage, the cells were differentiated and cultured for 3-7 days in NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, and 1-20 ng / mL GDNF.

[0046] In the fourth sub-stage, the cells were differentiated and cultured for at least 7 days in NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, 1-20 ng / mL GDNF, and 100-200 μM AA.

[0047] 11. The preparation method according to item 10, wherein:

[0048] The first sub-stage of differentiation culture for the cells in the early differentiation stage is from day 4 to day 5 from the start of the pluripotent stem cell differentiation culture.

[0049] The second sub-stage of differentiation culture of the cells in the early differentiation stage is the 6th day from the start of the pluripotent stem cell differentiation culture;

[0050] The third sub-stage of differentiation culture of the cells in the early differentiation stage is from day 7 to day 11 from the start of the pluripotent stem cell differentiation culture.

[0051] The fourth sub-stage of differentiation culture of the cells in the early differentiation stage is any day from the 12th day to the 243rd day before the start of the pluripotent stem cell differentiation culture.

[0052] Preferably, each sub-stage is carried out under the conditions of 35°C to 39°C and 3% to 7% CO2 until the midbrain organoids are harvested from day 12 to day 242 from the start of the pluripotent stem cell differentiation culture. In the third sub-stage and thereafter, the medium is completely replaced every 2-3 days.

[0053] More preferably, each sub-stage is carried out in a shaker under conditions of 37°C and 5% CO2. In the first sub-stage, the cells in the early differentiation stage are seeded in a low-adsorption culture dish and cultured in a shaker until the midbrain organoids are harvested from day 19 to day 242 from the start of the pluripotent stem cell differentiation culture.

[0054] 12. The preparation method according to any one of items 1-11, wherein the pluripotent stem cells are human pluripotent stem cells, preferably human embryonic stem cells or human induced pluripotent stem cells, and more preferably human embryonic stem cells.

[0055] 13. A midbrain organoid prepared by any one of items 1-12.

[0056] 14. A midbrain organoid tissue block obtained by separating the midbrain organoid described in item 13.

[0057] 15. The midbrain organoid tissue block according to item 14, wherein separating the midbrain organoid is performed by cutting the midbrain organoid using a cutting method;

[0058] Preferably, the cutting diameter is 100μm-300μm, and the midbrain organoid tissue block obtained by cutting has a function similar to that of the midbrain organoid;

[0059] More preferably, the midbrain organoids are midbrain organoids harvested from day 20 to day 120 of the start of the pluripotent stem cell differentiation culture.

[0060] 16. The use of the midbrain organoid according to claim 13, or the midbrain organoid tissue block according to claim 14 or 15, in the preparation of drugs for the prevention and / or treatment of dopaminergic neuron-related diseases, the screening of drugs for the prevention and / or treatment of dopaminergic neuron-related diseases, or the construction of models for the prevention and / or treatment of dopaminergic neuron-related diseases.

[0061] 17. The application according to item 16, wherein the disease is Parkinson's disease.

[0062] Beneficial effects:

[0063] The midbrain organoids described in this application can be obtained through a single-step induction of pluripotent stem cells into spheres. The operation is simple, low-cost, and reduces intra-group and batch-to-batch heterogeneity, making them suitable for large-scale production and application.

[0064] The dopaminergic neurons in the midbrain organoids of this application mature earlier, significantly shortening the differentiation and culture cycle compared to existing technologies, and exhibiting a longer functional time window, possessing complete neuronal characteristics such as long-distance projection, calcium activity, and electrophysiological functions.

[0065] The midbrain organoids of this application are uniform in morphology, structurally complete, and non-polar. By cutting the midbrain organoids, tissue blocks of a certain size can be obtained. When the cutting diameter is 100μm-300μm, the midbrain organoid tissue blocks have self-assembly capabilities and have similar functions to the original midbrain organoids. The midbrain organoid tissue blocks are easy to culture and transplant, showing broad application prospects in drug screening and cell replacement therapy research. Attached Figure Description

[0066] Figure 1 Bright-field diagram of midbrain dopaminergic neuron organoids at different differentiation culture stages. Scale bar: 250 μm.

[0067] Figure 2 Results of immunofluorescence staining for dopaminergic neural progenitor-related markers. Scale bar: 100 μm.

[0068] Figure 3 Results of immunofluorescence staining for neuronal and glial cell-related markers. Scale bar: 100 μm.

[0069] Figure 4 Results of immunofluorescence staining for dopaminergic neuron-related markers. The medial and lateral views of the organoid structures were obtained by observing the portions outlined in white boxes under a laser confocal microscope. Scale bar: 100 μm.

[0070] Figures 5A-5E The results are from the detection of calcium activity in organoids of midbrain dopaminergic neurons. Among them, Figure 5A A schematic diagram of calcium imaging on day 19 (D19) of differentiation culture, with arrows indicating the calcium reaction area; Figure 5B A schematic diagram of calcium imaging on day 242 (D242) of differentiation culture, with arrows indicating the calcium reaction area; Figure 5C A schematic diagram of calcium trajectory on day 36 (D36) of differentiation culture; Figure 5D A schematic diagram of the giant depolarization event on day 36 (D36) of the culture for differentiation; Figure 5ESchematic diagram of calcium activity compared to baseline levels on day 36 (D36) of differentiation culture under the influence of dopaminergic neuron agonists and inhibitors. Scale bar: 100 μm.

[0071] Figures 6A-6D This represents the results of an assessment of the electrophysiological levels of organoids of midbrain dopaminergic neurons. Among them, Figure 6A The bright field diagram, voltage waveform diagram, amplitude diagram, and electrode array layout diagram for MEA detection are shown.

[0072] Figure 6B This is a schematic diagram showing the electrical signal intensity compared to baseline levels under the influence of dopaminergic neuron agonists. Figure 6C This is a schematic diagram of the amplitude distribution compared to baseline levels under the influence of dopaminergic neuron agonists. Figure 6D This is a schematic diagram of the frequency distribution compared to baseline levels under the influence of dopaminergic neuron agonists.

[0073] Figures 7A-7C The results of the screening criteria for the minimum functional modules and the identification of related biomarkers. Among them, Figure 7A This is a schematic diagram of the minimum functional module time screening criteria. From left to right, the results are shown on day 27 (D27), day 42 (D42), day 117 (D117), and day 121 (D121) of differentiation culture. Figure 7B A schematic diagram illustrating the selection criteria for the cutting diameter of the smallest functional module; Figure 7C Results of immunofluorescence staining for dopaminergic neuron-related markers. Scale bar: 100 μm ( Figure 7A and Figure 7C ), 500μm ( Figure 7B ).

[0074] Figure 8 Results of the identification of the exophytic capacity of the smallest functional module. Scale bar: 100 μm.

[0075] Figures 9A-9B This represents the results of co-culturing the minimum functional module with mouse midbrain slices in vitro. Among them, Figure 9A This is a schematic diagram showing the establishment of neural connections between the minimum functional module and a mouse midbrain slice under bright field conditions, with arrows indicating neural connections. Figure 9B The images on the right show the results of immunofluorescence staining, with the two images within the white boxes indicating the staining results. Scale bar: 500 μm.

[0076] Figure 10 Results of in vivo transplantation identification of the smallest functional module. Scale bar: 100 μm. Detailed Implementation

[0077] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0078] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0079] definition

[0080] As used herein, the term “about” is used to indicate a value that includes the inherent variation of the device’s error, the method used to determine that value, or the variation that exists between the subjects under study.

[0081] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0082] As used herein, the term "pluripotent stem cells (PSCs)" generally refers to cells with unlimited proliferative capacity and the ability to differentiate into multiple germ layers. Unlike adult stem cells, which differentiate into a single germ layer, pluripotent stem cells have the ability to differentiate into ectoderm, mesoderm, and endoderm cells. Specifically, pluripotent stem cells can include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are highly similar in function and structure, differing only in their origin.

[0083] As used herein, the term "DMEM / F12" refers to a mixture of two culture media, Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Medium, which are commercially available.

[0084] As used in this article, the term "MEM NEAA" refers to a minimum essential culture medium containing non-essential amino acids.

[0085] As used in this article, the term "SHH" is equivalent to "sonic hedgehog," a secreted protein that plays an important role in the development of the hippocampus in animals. SHH signal transduction is crucial for neuronal patterning, axonal guidance, proliferation, survival, and differentiation in the hippocampus. Abnormal SHH signaling pathways can lead to various neurological disorders, such as depression and neurodegenerative diseases.

[0086] As used in this article, the term "fibroblast growth factor 8" is equivalent to "fibroblast growth factor 8" or "FGF8," and it is a member of the fibroblast growth factor (FGFs) family. It is expressed in various tissues during the human embryonic period and plays an important role in the formation of various organs.

[0087] As used herein, the term “SMAD” refers to an intracellular protein that transduces extracellular signals from transforming growth factor β (TGF-β) ligands to the cell nucleus. Therefore, as used herein, the term “SMAD inhibitor” refers to a molecular compound that can inhibit (i.e., suppress or downregulate) the activity of SMAD proteins.

[0088] As used herein, the term "SB431542" is a potent inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. The chemical structure of SB431542 is as follows:

[0089]

[0090] As used herein, the term "LDN193189" is a potent and selective BMP type I receptor inhibitor. The chemical structure of LDN193189 is as follows:

[0091]

[0092] As used herein, the term "CHIR99021" refers to a GSK-3α and GSK-3β inhibitor. The chemical structure of CHIR99021 is as follows:

[0093]

[0094] As used herein, the term "N-2 additive" and its non-commercially available formulations are referred to as "hormone mixtures," meaning a mixture of hormones containing transferrin, insulin, putrescine, selenium, and progesterone. For example, an N-2 additive may include 10 mg / ml transferrin, 2.5 mg / ml insulin, 1 mg / ml putrescine, 1 μL / ml 15% selenium, and 1 μL / ml progesterone. N-2 additives are commercially available from Gibco (Invitrogen / Thermo Scientific), Sigma, etc., or can be prepared in-house.

[0095] As used in this article, the term "Neurobasal medium" refers to a basic culture medium developed to meet the specific needs of neural cell culture.

[0096] As used in this article, the term “cAMP” is equivalent to “cyclic adenosine monophosphate”.

[0097] As used in this article, the term "brain-derived neurotrophic factor" is equivalent to "BDNF" and is one of the neurotrophic factors in the NGF-β family.

[0098] As used in this article, the term "glial cell line-derived neurotrophic factor" is equivalent to "GDNF" and is a neurotrophic factor belonging to the GDNF family of ligands (GFL).

[0099] As used in this article, the terms “midbrain organoids” and “midbrain dopaminergic neuron organoids” are used interchangeably and refer to miniature organs constructed in a 3D culture environment.

[0100] As used herein, the term "minimum functional module" refers to an organoid tissue block in an in vitro 3D culture system that can maintain the basic physiological functions of the original organoid. This module not only contains the key cell types of the original organoid but also performs specific biological functions associated with it and, to some extent, mimics cell-cell and cell-matrix interactions in the in vivo environment. Specifically, the term "minimum functional module" refers to a tissue block that can repair damaged sites through self-assembly, has similar marker expression and structure to the original organoid, and possesses similar functions to the original organoid.

[0101] In a first aspect, this application provides a method for preparing a midbrain organoid, the method comprising:

[0102] Pluripotent stem cells are digested into single-cell suspensions and cultured for differentiation to obtain cells in the early stage of differentiation.

[0103] The cells in the early stage of differentiation were cultured to obtain the midbrain organoids.

[0104] In the step of digesting pluripotent stem cells into a single-cell suspension and performing differentiation culture to obtain cells in the early differentiation stage, the marker for obtaining the cells in the early differentiation stage is that the following conditions (1) and / or (2) are met:

[0105] Condition (1) Differentiated pluripotent stem cells were observed to be spherical with a diameter greater than 500 μm under an optical microscope;

[0106] Condition (2) Immunofluorescence staining of differentiated pluripotent stem cells revealed that they expressed markers of ventral lateral lamina cells of the midbrain.

[0107] The cells in the early differentiation stage refer to the products of downward differentiation of a pluripotent stem cell, which can differentiate into various types of neural lineage cells (such as neural progenitor cells, neurons, and astrocytes). They are in a state between stem cells and adult cells and do not have the pluripotency of stem cells.

[0108] The pluripotent stem cells can be digested into a single-cell suspension using conventional methods in the art.

[0109] In some implementations, the sign that the cells in the early stage of differentiation have been obtained is that conditions (1) and (2) are met; that is, conditions (1) and (2) are met simultaneously.

[0110] In some embodiments, the midbrain ventral floor plate cell marker is LMX1A or FOXA2. In some embodiments, the midbrain ventral floor plate cell marker is both LMX1A and FOXA2.

[0111] Specifically, the expression of the midbrain ventral basal plate cell markers can be: expression of only LMX1A, expression of only FOXA2, or co-expression of LMX1A or FOXA2.

[0112] In some embodiments, the stage of digesting pluripotent stem cells into a single-cell suspension and then differentiating them to obtain early-differentiation cells includes: differentiating them using NIM basal medium to obtain the early-differentiation cells. In some embodiments, the NIM basal medium is infused with SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators.

[0113] In some embodiments, the SMAD inhibitor is a TGF-β inhibitor. Non-limiting examples of the TGF-β inhibitor include: A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB431542, SB505124, SD093, Sml6, SM305, SX-007, Antp-Sm2A, and LY2109761. For example, the TGF-β inhibitor is SB431542.

[0114] In some embodiments, the SMAD inhibitor is a BMP inhibitor. Non-limiting examples of BMP inhibitors include: dorsomorphin, dominant-inactivated BMP, truncated BMP receptor, soluble BMP receptor, BMP receptor-Fc chimera, noggin, LDN193189, folliculostatin, grimlin, cerberus / DAN family proteins, ventropin, high-dose activin, and amnionless proteins. For example, the BMP inhibitor is LDN193189.

[0115] In some embodiments, the SMAD inhibitor is a TGF-β inhibitor and a BMP inhibitor. For example, the SMAD inhibitor is SB431542 and LDN193189.

[0116] Non-limiting examples of the GSK-3 inhibitors include: NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR99021. For example, the GSK-3 inhibitor is CHIR99021.

[0117] Non-limiting examples of SHH signaling pathway activators include SHH C25II, SAG, GSA 10, and Purmorphamine. For example, the SHH signaling pathway activator is SHH C25II.

[0118] In some embodiments, the NIM basal medium is a medium containing 92-99% DMEM / F12 medium, 0.5-4% N-2 additive, 0.5-4% MEM NEAA, and 1-5 μg / mL Heparin. Examples include 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% DMEM / F12 medium; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% N-2 additive; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% MEM NEAA; and 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, and 5 μg / mL Heparin.

[0119] In some embodiments, the NIM basal medium is infused with 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM SB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021. For example, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 120ng / mL, 140ng / mL, 160ng / mL, 180ng / mL, 200ng / mL, 250ng / mL, 300ng / mL SHH C25II; 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 120ng / mL, 140ng / mL, 160ng / mL, 180ng / mL, 200ng / mL FGF8; 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM SB431524; 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 140nM, 160nM, 180nM, 200nM LDN193189; 1mM, 2mM, 3mM, 4mM, 5mM CHIR99021.

[0120] In some embodiments, the NIM basal medium is a medium containing 98% DMEM / F12 medium, 1% N-2 additive, 1% MEM NEAA and 2 μg / mL Heparin, and the NIM basal medium is infused with 100 ng / mL SHH C25II, 100 ng / mL FGF8, 10 μM MSB431524, 100 nM LDN193189 and 3 mM CHIR99021.

[0121] Using the aforementioned NIM basal medium, the single-cell suspension can be seeded into low-absorption plates and cultured for differentiation for 3-5 days at 35°C to 39°C and 3% to 7% CO2 to harvest cells in the early stages of differentiation. For example, differentiation culture for 3, 4, or 5 days. In some embodiments, the single-cell suspension is cultured at 1×10⁻⁶ cells / well at 37°C and 5% CO2. 3 -1×10 4 Cells were seeded in low-adsorption 96-well V plates for differentiation culture. Half of the culture medium was added on day 2, and cells in the early differentiation stage were harvested on day 4.

[0122] During the differentiation culture process, the culture medium can be changed / replenished based on the culture status of the pluripotent stem cells (i.e., single-cell suspensions of pluripotent stem cells) and changes in the content of nutrients and cell secretions in the culture medium. Typically, the content of nutrients and / or cell secretions in the culture medium can be determined by observing color changes.

[0123] In the aforementioned stage of digesting pluripotent stem cells into a single-cell suspension and performing differentiation culture to obtain cells in the early differentiation stage, this application does not intend to limit the specific time of differentiation culture. In fact, when preparing cells in the early differentiation stage, the differentiation culture time can be determined according to the condition of the pluripotent stem cells themselves. Those skilled in the art will also understand that if it can be determined that the differentiated pluripotent stem cells meet the above conditions (1) and / or (2), it can be judged that the differentiation culture has been completed and cells in the early differentiation stage have been obtained.

[0124] In some embodiments, in the step of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoid, the determination that the midbrain organoid has been obtained is based on satisfying any two or more of the following conditions:

[0125] Condition (1) The diameter of cells in the early stage of differentiation is greater than 1 mm as observed under an optical microscope;

[0126] Condition (2) Immunofluorescence staining of early-stage differentiated cells revealed that they expressed dopaminergic neuron markers;

[0127] Condition (3) The cells in the early stage of differentiation have dopaminergic neuronal calcium activity;

[0128] Condition (4) The cells in the early stage of differentiation have the electrophysiological function of dopaminergic neurons;

[0129] Condition (5) involves cutting early-stage differentiated cells, which have the ability to self-assemble.

[0130] In some embodiments, the criteria for obtaining the midbrain organoid are determined by satisfying any three of the conditions (1), (2), (3), (4), and (5). In some embodiments, the criteria for obtaining the midbrain organoid are determined by satisfying any four of the conditions (1), (2), (3), (4), and (5). In some embodiments, the criteria for obtaining the midbrain organoid are determined by satisfying the conditions (1), (2), (3), (4), and (5); that is, conditions (1), (2), (3), (4), and (5) are satisfied simultaneously.

[0131] Among them, the dopaminergic neuron markers are, for example, TH, DAT, NURR1, FOXA2, EN1 / EN2.

[0132] The calcium activity and electrophysiological function can be detected by conventional methods in the art.

[0133] In some embodiments, the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes: performing differentiation and culture in multiple sub-stages using different culture media to obtain the midbrain organoids. In some embodiments, NDM medium is used in all sub-stages except for the first sub-stage.

[0134] In some embodiments, the NDM medium is a medium comprising 92-99% Neurobasal medium, 0.5-4% N⁻² additive, and 0.5-4% MEM NEAA. For example, it may be 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Neurobasal medium; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% N⁻² additive; or 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% MEM NEAA. In some embodiments, the NDM medium is a medium comprising 98% Neurobasal medium, 1% N⁻² additive, and 1% MEM NEAA.

[0135] In some embodiments, the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes the following four sub-stages:

[0136] In the first sub-stage, NIM basal medium containing SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators was used.

[0137] In the second sub-stage, NDM medium containing GSK-3 inhibitor was used;

[0138] In the third sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, and GDNF was used.

[0139] In the fourth sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, GDNF, and AA was used.

[0140] The NIM basal medium comprises 92-99% DMEM / F12 medium, 0.5-4% N⁻² additive, 0.5-4% MEM NEAA, and 1-5 μg / mL Heparin. Specifically, the NIM basal medium comprises 98% DMEM / F12 medium, 1% N⁻² additive, 1% MEM NEAA, and 2 μg / mL Heparin.

[0141] In some embodiments, no matrix gel is added during the four sub-stages of differentiating the early-stage cells to obtain the midbrain organoids.

[0142] The applicant of this application discovered that when constructing and culturing midbrain organoids using the preparation method provided in this application, exogenous additives such as matrix gel can be avoided. The process can be completed solely through one-step spheroidization induction, relying on cell free gravity sedimentation aggregation and self-assembly, which further reduces the culture cost. The obtained midbrain organoids exhibit good intra-group and batch-to-batch uniformity and high quality.

[0143] In some embodiments, NIM basal medium containing 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM SB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021 is used in the first sub-stage. For example, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 120ng / mL, 140ng / mL, 160ng / mL, 180ng / mL, 200ng / mL, 250ng / mL, 300ng / mL SHH C25II; 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 120ng / mL, 140ng / mL, 160ng / mL, 180ng / mL, 200ng / mlFGF8; 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM SB431524; 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 140nM, 160nM, 180nM, 200nM LDN193189; 1mM, 2mM, 3mM, 4mM, 5mM CHIR99021.

[0144] In some embodiments, NDM medium containing 1-5 mM CHIR99021 is used in the second sub-stage. For example, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM CHIR99021.

[0145] In some embodiments, NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, and 1-20 ng / mL GDNF is used in the third sub-stage. For example, 0.5μM, 1μM, 1.5μM, 2μM cAMP; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL TGFβ3; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 20ng / mL BDNF; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 20ng / mL GDNF.

[0146] In some embodiments, the fourth sub-stage uses NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, 1-20 ng / mL GDNF, and 100-200 μM AA. For example, 0.5μM, 1μM, 1.5μM, 2μM cAMP; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL TGFβ3; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 20ng / mL BDNF; 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 20ng / mL GDNF; 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190μM, 200μM AA.

[0147] In some embodiments, the stage of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes the following four sub-stages:

[0148] In the first sub-stage, the cells were differentiated and cultured in NIM basal medium containing 100 ng / mL SHH C25II, 100 ng / mL FGF8, 10 μM MSB431524, 100 nM LDN193189 and 3 mM CHIR99021 for 2-3 days, for example, 2 or 3 days of differentiation culture.

[0149] In the second sub-stage, the cells were cultured in NDM medium containing 3mM CHIR99021 for 1-2 days to differentiate, for example, for 1 day or 1 day.

[0150] In the third sub-stage, the cells were differentiated and cultured in NDM medium containing 1 μM cAMP, 1 ng / mL TGFβ3, 10 ng / mL BDNF and 10 ng / mL LGDNF for 3-7 days, for example, 3, 4, 5, 6 or 7 days.

[0151] In the fourth sub-stage, differentiation culture was performed for at least 7 days using NDM medium containing 1 μM cAMP, 1 ng / mL TGFβ3, 10 ng / mL BDNF, 10 ng / mL GDNF and 200 μM AA, for example, differentiation culture for 7, 8, 9, 14, 28, 56, 112 and 224 days.

[0152] In some embodiments, the first sub-stage of differentiation culture of the early-stage differentiation cells is from day 4 to day 5 from the start of the pluripotent stem cell differentiation culture;

[0153] The second sub-stage of differentiation culture of the cells in the early differentiation stage is the 6th day from the start of the pluripotent stem cell differentiation culture;

[0154] The third sub-stage of differentiation culture of the cells in the early differentiation stage is from day 7 to day 11 from the start of the pluripotent stem cell differentiation culture.

[0155] The fourth sub-stage of differentiation culture of the cells in the early differentiation stage is any day from day 12 to day 243 before the start of the pluripotent stem cell differentiation culture, for example, day 12 to day 13, day 12 to day 14, day 12 to day 15, day 12 to day 16, day 12 to day 17, day 12 to day 18, day 12 to day 19, day 12 to day 20, day 12 to day 40, day 12 to day 120, or day 12 to day 242.

[0156] In some embodiments, the early-stage differentiation cells are cultured to obtain the midbrain organoids in four sub-stages. Each sub-stage is cultured at 35°C to 39°C and 3% to 7% CO2 until the midbrain organoids are harvested from day 12 to day 242 from the start of the pluripotent stem cell differentiation culture. The medium is completely replaced every 2-3 days from the third sub-stage onwards.

[0157] In some embodiments, the differentiation and culture of the early-stage differentiated cells to obtain the midbrain organoids involves four sub-stages. Each sub-stage is conducted on a shaker at 37°C and 5% CO2. In the first sub-stage, the early-stage differentiated cells are seeded in a low-adsorption culture dish and cultured on a shaker until the midbrain organoids are harvested from day 19 to day 242, following the start of the pluripotent stem cell differentiation and culture. For example, the midbrain organoids are harvested from day 19 to day 23. Specifically, for example, the midbrain organoids are harvested from day 19 to day 20.

[0158] During the differentiation culture process, the culture medium can be changed / replenished based on the initial culture status of the cells and changes in the content of nutrients and cell secretions in the culture medium. Typically, the content of nutrients and / or cell secretions in the culture medium can be determined by observing color changes.

[0159] This application is not intended to limit the specific time of each of the above sub-stages. When preparing midbrain organoids through the above four sub-stages, the differentiation and culture time of each sub-stage can be determined according to the condition of the cells in the early stage of differentiation. Those skilled in the art will understand that if it can be determined that the cells in the early stage of differentiation meet any two or more of the above conditions (1), (2), (3), (4) and (5), it can be judged that the differentiation and culture has been completed and midbrain organoids have been obtained.

[0160] In this application, the pluripotent stem cells used can be any pluripotent stem cells in the art that can be used for organoid construction. For example, they can be pluripotent stem cells obtained directly through commercial channels, or they can be pluripotent stem cells prepared in accordance with existing technologies in the art. This application does not particularly limit the type of pluripotent stem cells; for example, they can be human pluripotent stem cells, such as human embryonic stem cells or induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are human embryonic stem cells.

[0161] The term "human embryonic stem cells" refers to embryonic stem cells isolated or obtained from human embryos that have not developed in vivo and are within 14 days of fertilization, or commercially available established human embryonic stem cell lines. Human embryonic stem cells can divide for a long time without differentiating during culture and develop into cells and tissues of the three primary germ layers. For example, human embryonic stem cell lines can be SA01(SA001), SA02(SA002), ES01(HES-1), ES02(HES-2), ES03(HES-3), ES04(HES-4), ES05(HES-5), ES06(HES-6), BG01(BGN-01), BG02(BGN-02), BG03(BGN-03), TE03(I3), TE04(I4), TE06(I6), UC01(HSF1), UC06(HSF6), WA01(H1), WA07(H7), WA09(H9), WA13(H13), WA14(H14), and Q-2.

[0162] Secondly, this application provides a midbrain organoid prepared using the above-described method.

[0163] Thirdly, this application provides a midbrain organoid tissue block, which is obtained by separating the aforementioned midbrain organoids.

[0164] In some embodiments, the midbrain organoid is separated by cutting the midbrain organoid.

[0165] Those skilled in the art can employ any cutting method to cut the midbrain organoid, such as manual mechanical cutting (e.g., using an ophthalmic scalpel), automated mechanical cutting, laser microsurgery, and other cutting methods discovered in the future. In some embodiments, the cutting method is mechanical cutting. In some embodiments, the cutting method is using an ophthalmic scalpel. Specifically, the midbrain organoid is cut using an ophthalmic scalpel in an average cutting manner.

[0166] In some embodiments, the cutting diameter is 100μm-300μm, and the resulting midbrain organoid tissue block has a function similar to that of the midbrain organoid. In some embodiments, the midbrain organoid is a midbrain organoid harvested from day 20 to day 120 of the start of the pluripotent stem cell differentiation culture.

[0167] The applicant of this application discovered that by cutting the midbrain organoids of this application, tissue blocks of a certain size can be obtained. When the cutting diameter is 100μm-300μm, the obtained midbrain organoid tissue blocks have self-assembly capabilities, and exhibit similar marker expression and structure to the original midbrain organoids, while maintaining similar functions. The midbrain organoid tissue blocks obtained by this application are of suitable and uniform size, have low heterogeneity, are easy to culture and transplant, and can be used for subsequent drug screening applications and cell replacement therapy research.

[0168] Fourthly, this application provides the use of the aforementioned midbrain organoids or midbrain organoid tissue blocks in the preparation of medicaments for the prevention and / or treatment of dopaminergic neuron-related diseases.

[0169] Fifthly, this application provides the use of the aforementioned midbrain organoids or midbrain organoid tissue blocks in screening drugs for the prevention and / or treatment of dopaminergic neuron-related diseases.

[0170] Sixthly, this application provides the application of the aforementioned midbrain organoids or the aforementioned midbrain organoid tissue blocks in constructing models for the prevention and / or treatment of dopaminergic neuron-related diseases.

[0171] Among these, dopaminergic neuron-related diseases include Parkinson's disease, Alzheimer's disease, cerebral hemorrhage, stroke, Huntington's disease, Pick's disease, Kreutzfeldt-Jacob's disease, and brain developmental disorders. Specifically, Parkinson's disease is one of the dopaminergic neuron-related diseases mentioned above.

[0172] This application utilizes pluripotent stem cells to obtain in vitro midbrain organoids through one-step spheroidization and differentiation. Compared with monolayer dopaminergic neurons obtained through 2D differentiation, it better simulates the development and maturation of nerve cells in vivo. The midbrain organoids of this application shorten the differentiation and culture cycle while greatly expanding the functional time window. They possess complete neuronal characteristics such as long-distance projection, calcium activity, and electrophysiological functions, and can serve as a basic research model for dopaminergic neuron-related diseases. In addition, the midbrain organoid tissue blocks obtained through this application are small in size, highly uniform, and morphologically and functionally complete, showing broad application prospects in drug screening and cell replacement therapy research.

[0173] Example

[0174] The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0175] Example 1: Preparation of in vitro dopaminergic neuron organoids in the midbrain

[0176] Functional midbrain dopaminergic neuron organoids were obtained by differentiating and culturing them according to the following steps:

[0177] Day 1 of differentiation culture (D1): Human embryonic stem cell line H9 ESCs (purchased from James A. Thomson, University of Wisconsin-Madison) were digested into single-cell morphology and seeded at a density of 3000 cells / well in low-adsorption 96-well V-type plates. 100 μL of the corresponding time point medium I (DMEM / F12 medium containing 1% N-2 additive, 1% MEMNEAA, and 2 μg / mL heparin, with the addition of 100 ng / mL SHH C25II, 100 ng / mL LFGF8, 10 μM SB431542, 100 nM LDN193189, and 3 mM CHIR99021) was added and the cells were incubated at 37°C in a 5% CO2 incubator.

[0178] Day 2 of differentiation culture (D2): Add 50 μL of medium I corresponding to the time point and continue culturing;

[0179] Day 4 of differentiation culture (D4): Place the well plate upright under an optical microscope, select organoids with a diameter >700μm (i.e., cells in the early stage of differentiation), transfer them to a 10cm low-adsorption culture dish, add 20mL of the corresponding time point medium I, and place them on a shaker for culture;

[0180] Day 6 of differentiation culture: After 48 hours, discard the culture medium and replace it with 20 mL of fresh medium II corresponding to the time point (add 3 mM CHIR99021 to NDM medium (Neurobasal medium containing 1% N-2 additive and 1% MEM NEAA)) and continue culturing;

[0181] Day 7 of differentiation culture (D7): Discard the medium and replace it with 20 mL of fresh medium III corresponding to the time point (Neurobasal medium containing 1% N-2 additive and 1% MEM NEAA in NDM medium, with 1 μM cAMP, 1 ng / ml TGFβ3, 10 ng / ml BDNF and 10 ng / ml GDNF added), and continue culturing;

[0182] Day 9 of differentiation culture: Discard the culture medium and replace it with 20 mL of fresh culture medium III corresponding to the time point and continue the culture;

[0183] Day 12 of differentiation culture: Place the culture dish upright under an optical microscope, reduce the roundness, select organoids with a diameter between 1-2 mm, transfer them to a new 10 cm low-adsorption culture dish, add the corresponding time point medium IV (Neurobasal medium containing 1% N-2 additive and 1% MEM NEAA, with 1 μM cAMP, 1 ng / ml TGFβ3, 10 ng / ml BDNF, 10 ng / ml GDNF and 200 μM AA added), and continue to culture on a shaker;

[0184] After 12 days of differentiation culture, 20 mL of culture medium IV was replaced every three days at the corresponding time points. During the differentiation culture process, the morphological changes of the organoids were observed using an optical microscope, and the results are as follows: Figure 1 As shown, with the increase of differentiation culture time, the volume of organoids increased, and obvious stratification appeared on their surface, showing a more complex internal structure.

[0185] Calcium activity was detectable on day 19 of differentiation culture (Figure 5), marking the initial realization of the organoid's function, from which mature dopaminergic neurons could be harvested. Furthermore, based on their functional characteristics, the organoids at different time points were divided into three maturation stages: early, middle, and late, as detailed below:

[0186] Early maturation (days 19 to 40 of differentiation culture): During this stage, the organoids initially exhibit significant calcium activity, indicating that dopaminergic neurons have begun to establish basic functional connections.

[0187] Mid-maturation (days 41 to 120 of differentiation culture): Organoids are more functionally active at this stage, with more calcium-responsive regions and large depolarization events compared to early maturation;

[0188] Late maturity (after 120 days of differentiation culture): At this stage, the calcium response of organoids is further reduced.

[0189] Identification and analysis of the molecular composition of brain dopaminergic neuron organoids in Example 2

[0190] 2.1 Identification of the precursor stage of dopaminergic neurons

[0191] Organoids from day 4 (D4) and day 19 (D19) of differentiation culture in Example 1 were selected for immunofluorescence staining detection according to the following steps. Organoids from day 4 of differentiation culture were incubated with the following primary and secondary antibodies: LMX1A (ab139726, dilution 1:200), FOXA2 (AF2400, dilution 1:200), 488 donkey anti-rabbit, and 594 donkey anti-goat; organoids from day 9 of differentiation culture were incubated with the following primary and secondary antibodies: EN1 (4G11, dilution 1:100) and 488 donkey anti-mouse.

[0192] Organoids were removed and placed in 1.5 mL centrifuge tubes. 4% paraformaldehyde was immediately added, and the tubes were fixed at 4°C for at least 24 hours. The tubes were then soaked in PBS for 3 hours and blocked in blocking buffer (containing 1% Triton X-100 and 5% BSA) for 12 hours. Diluted primary antibody was added, and the tubes were incubated at 4°C for 48 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). Diluted secondary antibody (1:1000) and Hoechst (HOE, 2 μg / mL) were added, and the tubes were incubated at 4°C in the dark for 6 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). The tubes were observed under a laser confocal microscope and fluorescence imaging was performed.

[0193] The results are as follows Figure 2 As shown, on day 4 of differentiation culture, the organoids expressed the midbrain ventral basal plate cell markers LMX1A and FOXA2; on day 19 of differentiation culture, the basal plate cells continued to differentiate into dopaminergic neuronal progenitors and expressed the major marker EN1, reflecting the specific midbrain dopaminergic neuron fate of the organoids.

[0194] 2.2 Identification of neurons and glial cells

[0195] Organoids from the differentiation culture at days 15 (D15), 74 (D74), and 85 (D85) in Example 1 were selected and subjected to immunofluorescence staining detection according to the following steps. The organoids cultured on day 15 of differentiation were incubated with the following primary and secondary antibodies: SYP (ab32127, dilution 1:500), MAP2 (NB300-213, dilution 1:1500), 488goat anti-chicken, and 594donkey anti-rabbit. The organoids cultured on days 74 and 85 of differentiation were incubated with the following primary and secondary antibodies: MAP2 (NB300-213, dilution 1:1500), S100B (ab52642, dilution 1:500), GFAP (13-0300, dilution 1:500), 488goat anti-chicken, 594donkey anti-rabbit, and 650donkey anti-rat.

[0196] Organoids were removed and placed in 1.5 mL centrifuge tubes. 4% paraformaldehyde was immediately added, and the tubes were fixed at 4°C for at least 24 hours. The tubes were then soaked in PBS for 3 hours and blocked in blocking buffer (containing 1% Triton X-100 and 5% BSA) for 12 hours. Diluted primary antibody was added, and the tubes were incubated at 4°C for 48 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). Diluted secondary antibody (1:1000) and Hoechst (2 μg / mL) were added, and the tubes were incubated at 4°C in the dark for 6 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). The tubes were then observed and fluorescence imaged under a laser confocal microscope.

[0197] The results are as follows Figure 3 As shown, from day 15 of differentiation culture, organoids began to express MAP2, a marker of mature neurons, and SYP, a marker of synaptic proteins. As differentiation progressed, the number of MAP2-positive mature neurons increased, while the number of S100B-positive astrocytes was relatively small. After further maturation, the number of S100B-positive and GFAP-positive astrocytes increased, and a large number of neurites were formed. This indicates that astrocytes promote the extension of nerve fibers and the maturation of neurons in the later stages. The results show that organoids contain both neurons and astrocytes.

[0198] 2.3 Identification of dopaminergic neurons

[0199] Organoids from differentiation cultured on days 16 (D16), 36 (D36), and 81 (D81) in Example 1 were selected for immunofluorescence staining detection according to the following steps. The following primary and secondary antibodies were used for incubation: TUJ1 (801201, dilution 1:1000), TH (ab112, dilution 1:200), SOX2 (AF2018, dilution 1:200), 488 donkey anti-rabbit, 594 donkey anti-mouse, and 647 donkey anti-goat.

[0200] Organoids were removed and placed in 1.5 mL centrifuge tubes. 4% paraformaldehyde was immediately added, and the tubes were fixed at 4°C for at least 24 hours. The tubes were then soaked in PBS for 3 hours and blocked in blocking buffer (containing 1% Triton X-100 and 5% BSA) for 12 hours. Diluted primary antibody was added, and the tubes were incubated at 4°C for 48 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). Diluted secondary antibody (1:1000) and Hoechst (2 μg / mL) were added, and the tubes were incubated at 4°C in the dark for 6 hours. The tubes were then washed three times with PBS (containing 0.1% Triton X-100) (2 hours each time). The tubes were then observed and fluorescence imaged under a laser confocal microscope.

[0201] The results are as follows Figure 4 As shown, on day 16 of differentiation culture, a large number of TUJ1 positive neurons were generated, and some cells were positive for the dopaminergic neuron marker TH. As differentiation progressed, the distribution of nerve fibers became more uniform, and structurally, the neuronal cell bodies were mainly located on the inner side of the organoid, while the neurites wrapped around and entangled on the outer side of the organoid.

[0202] Identification and analysis of organoid calcium activity of brain dopaminergic neurons in Example 3

[0203] Organoids from Example 1 at differentiation and culture days 19 (D19), 36 (D36), and 242 (D242) were selected for neuronal calcium activity detection according to the following steps.

[0204] Organoids were removed and placed in 1.5 mL centrifuge tubes, and culture medium IV (containing 2 μM Fluo-4AM) was added. The tubes were incubated at 37°C with 5% CO2 for 2 hours, followed by replacement with fresh culture medium. The organoids were then transferred to confocal cell culture dishes and observed using a laser confocal microscope at 488 nm wavelength. Images were acquired using ZEN software, with an interval of 5–10 seconds between each acquisition. Dopaminergic neuron activity was activated using 20 μM dopamine hydrochloride solution, and inhibited using 10 μM quetiapine and 10 μM corydaline. The cells were washed twice with fresh culture medium (20 minutes each time) between each stimulation. The ROI value was calculated, and neuronal calcium activity in the organoids was assessed based on the relative fluorescence intensity ΔF / F value.

[0205] The results are as follows Figures 5A-5E As shown, calcium signals could be detected within 7 days of the addition of terminal differentiation medium (medium IV). Figure 5A ), which can be detected no later than day 242 of differentiation culture. Figure 5B ); calcium imaging revealed different patterns of calcium activity in organoids cultured for 36 days after differentiation. Figure 5C And it has a resting-state giant depolarization event ( Figure 5D ), and the study of organoids revealed that dopaminergic neuron agonists and inhibitors could respectively induce or inhibit neuronal calcium activity. Figure 5E ).

[0206] Identification and analysis of the electrophysiological function of brain dopaminergic neuron organoids in Example 4

[0207] Extracellular spontaneous field potentials of organoids were detected using a microelectrode array (MEA) electrophysiological detection system, with 256MEA electrodes used. Organoids cultured on day 42 (D42) from Example 1 were selected and resuspended in Matrigel. Depending on the size of the organoid, 20-80 μL of suspension was added to the center of the electrode plate, and culture medium IV was slowly added. After stabilization for 1 hour (no exophysis), 48 hours (exophysis occurred), or drug treatment (adding 20 μM dopamine hydrochloride solution to the organoid to activate dopaminergic neurons), the samples were analyzed. The organoids were maintained at 37°C using a temperature control device, and the data was recorded and analyzed using Multi Channel Experimenter version 2.15.0 software, with each recording lasting 2 minutes and a 2-minute interval between recordings.

[0208] The results are as follows Figures 6A-6D As shown, organoid electrophysiological activity can be detected by MEA (electrophysiological activity detection). Figures 6A-6B ); After the addition of dopaminergic neuron agonists, the number of activated regions increased ( Figure 6C And the frequency increases ( Figure 6DThe results showed that dopaminergic neurons in organoids are electrophysiologically active and can be activated by dopaminergic neuron agonists.

[0209] Example 5: Establishment of the Minimum Functional Module and Identification Analysis of its Functions

[0210] 5.1 Establishment of the Minimum Functional Module

[0211] (1) Selection of time window

[0212] Organoids at the early, middle (day 27 of differentiation culture in Example 1), mid (day 42 and day 117 of differentiation culture in Example 1), and late (day 121 of differentiation culture in Example 1) stages of dopaminergic neuron maturation were selected. Organoid tissue blocks (approximately 200 μm in diameter) were obtained by mechanically cutting them with an ophthalmic scalpel under a stereomicroscope. These blocks were then placed on a shaker for self-reconstruction and repair. The repair was observed under a microscope after 7 days. The results are as follows: Figure 7A As shown, organoid tissue blocks obtained in the early and middle stages of neuronal maturation can repair damaged sites through self-assembly, while organoid tissue blocks obtained in the late stage of maturation cannot repair damaged sites through self-assembly. The results indicate that organoids in the early and middle stages of dopaminergic neuron maturation should be preferentially selected when establishing the minimum functional module.

[0213] (2) Selection of cutting diameter

[0214] Organoids in the early stage of dopaminergic neuron maturation (day 27 of differentiation culture in Example 1 (D27)) were selected and divided into two groups according to their cutting diameter (one group with a cutting diameter <100μm, and the other group with a cutting diameter between 100μm and 300μm). Organoid tissue blocks were obtained by uniform cutting with an ophthalmic scalpel under a stereomicroscope and placed on a shaker for self-reconstruction and repair. The repair status was observed under a microscope after 7 days, and the results are as follows: Figure 7B As shown, when the cutting diameter is between 100μm and 300μm, the obtained organoid tissue blocks can repair the damaged area through self-assembly. However, when the cutting diameter is <100μm, the obtained organoid tissue blocks cannot repair the damaged area through self-assembly. The results indicate that a cutting diameter of 100μm-300μm is preferred when establishing the minimum functional module.

[0215] (3) Identification of dopaminergic neurons

[0216] Organoids at the early stage of dopaminergic neuron maturation (day 27 of differentiation culture in Example 1 (D27)) were selected. The smallest functional modules (approximately 200 μm in diameter) were obtained by average cutting with an ophthalmic scalpel under a stereomicroscope. These modules were then placed on a shaker for self-reconstruction and repair. Immunofluorescence staining was performed 7 days later, using the same method as in Example 2.3. The following primary and secondary antibodies were used for incubation: TUJ1 (801201, dilution 1:1000), SOX2 (AF2018, dilution 1:200), 488 donkey anti-mouse, and 594 donkey anti-goat.

[0217] The results are as follows Figure 7C As shown, the obtained minimum functional module has the expression and structure of markers similar to those of the original organoid.

[0218] 5.2 Identification of the exophytic ability of the minimum functional module neurites

[0219] Organoids at the early stage of dopaminergic neuron maturation (day 27 of differentiation culture in Example 1 (D27)) were selected. The smallest functional modules (approximately 200 μm in diameter) were obtained by average cutting with an ophthalmic scalpel under a stereomicroscope. These modules were then placed on a shaker for self-reconstruction and repair. After 7 days, they were transferred to 1.5 mL centrifuge tubes, resuspended in pre-chilled Matrigel, and 20 μL of the suspension was dropped onto a cell slide. Then, 500 μL of culture medium IV was gently added, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 days. The cells were fixed with 4% paraformaldehyde and immunofluorescence staining was performed using the same method as in Example 2.3. The following primary and secondary antibodies were used for incubation: TH (ab112, dilution 1:200), NF68KD (ab78159, dilution 1:200), 488 donkey anti-rabbit, and 594 donkey anti-mouse.

[0220] The results are as follows Figure 8 As shown, the neurites of dopaminergic neurons have a physiological morphology of outward radiation, indicating that differentiated dopaminergic neurons have the ability to project outward.

[0221] 5.3 Identification of Minimum Functional Modules for In Vitro Cell Transplantation

[0222] Newborn C57BL / 6J mice (within 48 hours of birth) were dissected under a stereomicroscope to obtain midbrain coronal tissue sections, which were then immersed in cold HBSS for later use.

[0223] Organoids at the early stage of dopaminergic neuron maturation (day 27 of differentiation culture in Example 1 (D27)) were selected. The smallest functional modules (approximately 200 μm in diameter) were obtained by average cutting with an ophthalmic scalpel under a stereomicroscope. These modules were then placed on a shaker for self-reconstruction and repair. After 7 days, they were co-embedded with the obtained midbrain slices in 70 μL of Matrigel. The relative positions of each part were adjusted with sterile forceps, and the modules were placed in culture medium IV and incubated statically for 7 days. Immunofluorescence staining was performed using 4% paraformaldehyde, following the same detection method as in Example 2.3. The following primary and secondary antibodies were used for incubation: HuNu (ab191181, dilution 1:100), TH (ab152, dilution 1:200), MAP2 (NB300-213, dilution 1:1000), 488 donkey anti-mouse, 594 donkey anti-rabbit, and 647 goat anti-chicken.

[0224] The results are as follows Figures 9A-9B As shown, the neurites extending from the smallest functional module can establish neural connections with the mouse midbrain.

[0225] 5.4 Identification of Minimum Functional Module Transplantation

[0226] Organoids in the early stage of dopaminergic neuron maturation (day 30 of differentiation culture in Example 1 (D30)) were selected. The smallest functional modules (approximately 200 μm in diameter) were obtained by average cutting with an ophthalmic scalpel under a stereomicroscope. They were placed on a shaker for self-reconstruction and repair. After 7 days, they were resuspended in culture medium IV (with 50% Matrigel added). Each 10 μL of culture medium contained 1-3 smallest functional modules. They were transplanted into the unilateral striatum of immunodeficient mice using stereotactic injection technique, with a single injection of 10-20 μL. Four weeks after injection, mouse brain tissue was collected, and 20-30 μm frozen sections were prepared for immunofluorescence staining verification. The following primary and secondary antibodies were used for incubation: STEM121 (AB-121-U-050, dilution 1:100), TH (ab152, dilution 1:200), MAP2 (NB300-213, dilution 1:1000), 488donkey anti-mouse, 594donkey anti-rabbit, and 647goat anti-chicken.

[0227] The results are as follows Figure 10 As shown, observations revealed that the graft survived in mice and expressed markers of human dopaminergic neurons.

[0228] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A method for preparing a midbrain organoid, comprising: Pluripotent stem cells are digested into single-cell suspensions and cultured for differentiation to obtain cells in the early stage of differentiation. The cells in the early stage of differentiation were cultured to obtain the midbrain organoids. In the step of digesting pluripotent stem cells into a single-cell suspension and performing differentiation culture to obtain cells in the early differentiation stage, the marker for obtaining the cells in the early differentiation stage is that the following conditions (1) and / or (2) are met: Condition (1) Differentiated pluripotent stem cells were observed to be spherical with a diameter greater than 500 μm under an optical microscope; Condition (2) Immunofluorescence staining of differentiated pluripotent stem cells revealed that they expressed markers of midbrain ventral basal plate cells; Preferably, the marker for obtaining the cells in the early stage of differentiation is that conditions (1) and (2) are met; More preferably, the midbrain ventral basal plate cell markers are LMX1A and / or FOXA2.

2. The preparation method according to claim 1, wherein, The stage of digesting pluripotent stem cells into a single-cell suspension and then differentiating them to obtain early-differentiation cells includes: using NIM basal medium for differentiation culture to obtain the early-differentiation cells; Preferably, the NIM basal culture medium contains SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators.

3. The preparation method according to claim 2, wherein, The NIM basal medium is a medium containing 92-99% DMEM / F12 medium, 0.5-4% N-2 additive, 0.5-4% MEM NEAA and 1-5 μg / mL Heparin; Preferably, the NIM basal culture medium contains 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM SB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021; More preferably, the single-cell suspension is seeded into a low-adsorption plate and cultured for 3-5 days under conditions of 35°C to 39°C and 3% to 7% CO2, and cells in the early stage of differentiation are harvested. More preferably, the single-cell suspension is subjected to a concentration of 1×10⁻⁶ cells / mL at 37°C and 5% CO₂. 3 -1×10 4 Cells were seeded in low-adsorption 96-well V plates for differentiation culture. Half of the culture medium was added on day 2, and cells in the early differentiation stage were harvested on day 4.

4. The preparation method according to any one of claims 1-3, wherein, In the step of differentiating and culturing the cells in the early stage of differentiation to obtain the midbrain organoid, the determination that the midbrain organoid has been obtained is characterized by satisfying any two or more of the following conditions: Condition (1) The diameter of cells in the early stage of differentiation is greater than 1 mm as observed under an optical microscope; Condition (2) Immunofluorescence staining of early-stage differentiated cells revealed that they expressed dopaminergic neuron markers; Condition (3) The cells in the early stage of differentiation have dopaminergic neuronal calcium activity; Condition (4) The cells in the early stage of differentiation have the electrophysiological function of dopaminergic neurons; Condition (5) involves cutting early-stage differentiated cells, which have the ability to self-assemble.

5. The preparation method according to claim 4, wherein, The stage of differentiating and culturing the cells in the early stage of differentiation to obtain the midbrain organoids includes: differentiating and culturing the cells in multiple sub-stages using different culture media to obtain the midbrain organoids; Preferably, NDM medium is used in all sub-stages except the first sub-stage; More preferably, the NDM medium is a medium containing 92-99% Neurobasal medium, 0.5-4% N-2 additive, and 0.5-4% MEM NEAA.

6. The preparation method according to claim 5, wherein, The process of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes the following four sub-stages: In the first sub-stage, NIM basal medium containing SMAD inhibitors, GSK-3 inhibitors, and SHH signaling pathway activators was used. In the second sub-stage, NDM medium containing GSK-3 inhibitor was used; In the third sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, and GDNF was used. In the fourth sub-stage, NDM medium containing cAMP, TGFβ3, BDNF, GDNF, and AA was used. Preferably, the NIM basal medium is a medium containing 92-99% DMEM / F12 medium, 0.5-4% N-2 additive, 0.5-4% MEM NEAA and 1-5 μg / mL Heparin; More preferably, no matrix adhesive is added in any of the sub-stages.

7. The preparation method according to claim 6, wherein, The process of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes the following four sub-stages: In the first sub-stage, NIM basal medium containing 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM MSB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021 was used. In the second sub-stage, NDM medium containing 1-5 mM CHIR99021 was used; In the third sub-stage, NDM medium containing 0.5–2 μM cAMP, 1–5 ng / mL TGFβ3, 1–20 ng / mL BDNF, and 1–20 ng / mL GDNF was used. In the fourth sub-stage, NDM medium containing 0.5–2 μM cAMP, 1–5 ng / mL TGFβ3, 1–20 ng / mL BDNF, 1–20 ng / mL LGDNF, and 100–200 μM AA was used.

8. The preparation method according to claim 7, wherein, The process of differentiating and culturing the early-stage differentiated cells to obtain the midbrain organoids includes the following four sub-stages: In the first sub-stage, the cells were differentiated and cultured for 2-3 days in NIM basal medium containing 50-300 ng / mL SHH C25II, 50-200 ng / mL FGF8, 10-20 μM MSB431524, 50-200 nM LDN193189 and 1-5 mM CHIR99021. In the second sub-stage, the cells were cultured in NDM medium containing 1-5 mM CHIR99021 for 1-2 days for differentiation. In the third sub-stage, the cells were differentiated and cultured for 3-7 days in NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, and 1-20 ng / mL GDNF. In the fourth sub-stage, the cells were differentiated and cultured for at least 7 days in NDM medium containing 0.5-2 μM cAMP, 1-5 ng / mL TGFβ3, 1-20 ng / mL BDNF, 1-20 ng / mL LGDNF, and 100-200 μM AA.

9. The preparation method according to claim 8, wherein: The first sub-stage of differentiation culture for the cells in the early differentiation stage is from day 4 to day 5 from the start of the pluripotent stem cell differentiation culture. The second sub-stage of differentiation culture of the cells in the early differentiation stage is the 6th day from the start of the pluripotent stem cell differentiation culture; The third sub-stage of differentiation culture of the cells in the early differentiation stage is from day 7 to day 11 from the start of the pluripotent stem cell differentiation culture. The fourth sub-stage of differentiation culture of the cells in the early differentiation stage is any day from the 12th day to the 243rd day before the start of the pluripotent stem cell differentiation culture. Preferably, each sub-stage is carried out under the conditions of 35°C to 39°C and 3% to 7% CO2 until the midbrain organoids are harvested from day 12 to day 242 from the start of the pluripotent stem cell differentiation culture. In the third sub-stage and thereafter, the medium is completely replaced every 2-3 days. More preferably, each sub-stage is carried out in a shaker under conditions of 37°C and 5% CO2. In the first sub-stage, the cells in the early differentiation stage are seeded in a low-adsorption culture dish and cultured on a shaker until the midbrain organoids are harvested from day 19 to day 242 from the start of the pluripotent stem cell differentiation culture.

10. The preparation method according to any one of claims 1-9, wherein, The pluripotent stem cells are human pluripotent stem cells, preferably human embryonic stem cells or human induced pluripotent stem cells, and more preferably human embryonic stem cells.

11. Midbrain organoids prepared by the method according to any one of claims 1-10.

12. A midbrain organoid tissue block obtained by separating the midbrain organoid of claim 11.

13. The midbrain organoid tissue block according to claim 12, wherein, The midbrain organoid was separated by cutting the midbrain organoid. Preferably, the cutting diameter is 100μm-300μm, and the midbrain organoid tissue block obtained by cutting has a function similar to that of the midbrain organoid; More preferably, the midbrain organoids are midbrain organoids harvested from day 20 to day 120 of the start of the pluripotent stem cell differentiation culture.

14. The use of the midbrain organoid according to claim 11, or the midbrain organoid tissue block according to claim 12 or 13, in the preparation of drugs for the prevention and / or treatment of dopaminergic neuron-related diseases, the screening of drugs for the prevention and / or treatment of dopaminergic neuron-related diseases, or the construction of models for the prevention and / or treatment of dopaminergic neuron-related diseases; Preferably, the disease is Parkinson's disease.

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