A brain organoid with microglia, its culture method and application

Abstract

This invention relates to the fields of tissue engineering and neurobiology, and particularly to a brain organoid containing microglia, its culture method, and its applications. The method includes: isolating and digesting primary embryonic brain tissue to obtain a suspension containing neural cell clusters; culturing the digested neural cell clusters in three-dimensional suspension; and culturing them in a culture medium containing neurotrophic factors and survival-promoting factors to induce the cell clusters to self-reorganize and form an organoid with active neural networks. Furthermore, verification of this invention's model has shown that, in addition to different types of neurons, it also contains natural microglia. This model better preserves the cellular diversity and maturity of primary tissues, making it suitable for drug screening, disease modeling, and personalized medicine.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and neurobiology, and in particular to a brain organoid with microglia, its culture method and application. Background Technology

[0002] Neurological diseases seriously endanger human health, encompassing neurodevelopmental disorders, neuropsychiatric diseases, epilepsy, neurodegenerative diseases, cerebrovascular diseases, neuroinflammatory diseases, demyelinating diseases, motor neuron diseases, neuroinfectious diseases, hereditary neurological diseases, metabolic or toxic neurological diseases, and neurodegenerative diseases. Current research primarily relies on animal models and two-dimensional cell culture systems. However, animal models exhibit species differences, making it difficult to fully simulate the complexity and disease specificity of the human nervous system. Two-dimensional cell culture systems lack the in vivo three-dimensional microenvironment, complex intercellular interactions, and neural network structures, making it difficult to reproduce the pathogenesis and development of human neurological diseases. Therefore, establishing in vitro models capable of simulating the structure, function, and pathological characteristics of the human nervous system is of great significance. In recent years, brain organoid technology has provided a new platform for studying human brain development and diseases. However, existing brain organoid technologies have many limitations, such as: (1) random cell types and disordered structures within organoids, lacking reproducible, specific neural circuit structures; (2) large differences between different batches, resulting in low reproducibility; and (3) a lack of functionally mature neurons and glial cells, making it difficult to simulate higher brain functions and related disease phenotypes.

[0003] Current brain organoid models derived from induced pluripotent stem cells (iPSCs) and two-dimensional culture systems of primary neural system cells still fall short of fully meeting the needs of modeling neurological diseases. While iPSC-derived brain organoids can form a certain degree of three-dimensional neural tissue structure, their preparation cycle is long, cell maturity is limited, and the resulting neurons and neural networks often exhibit embryonic or fetal characteristics, failing to fully reflect the structure, function, and pathological state of the nervous system in adults, the elderly, or specific age groups. Although two-dimensional culture of primary neural system cells has the advantage of mature cell sources, it lacks the in vivo three-dimensional microenvironment, intercellular spatial interactions, and functional neural network structures, and has a short in vitro survival time and limited phenotypic maintenance ability. Therefore, existing models are not widely applicable to the pathological simulation and drug evaluation of various types of neurological diseases, including neurodevelopmental disorders, neuropsychiatric diseases, epilepsy, neurodegenerative diseases, cerebrovascular diseases, neuroinflammatory diseases, demyelinating diseases, motor neuron diseases, neuroinfectious diseases, genetic and metabolic neurological diseases, nerve injuries, and nervous system tumors. Therefore, there is an urgent need in this field for a brain organoid model that can highly simulate specific regions of the human brain, has controllable and functional neural circuits, and has high reproducibility in its preparation. Summary of the Invention

[0004] To address the limitations of existing technologies in maintaining the long-term activity and function of primary brain tissue and enabling it to self-organize into functional three-dimensional organoids, this invention provides a brain organoid with microglia, its culture method, and its applications. The organoid retains the cellular heterogeneity of primary embryonic brain tissue, notably including microglia, radial glial cells, intermediate progenitor cells, and neurons. Furthermore, the organoid self-organizes into a complex three-dimensional morphology with folded edges during culture. Verification has shown that the model of this invention, in addition to possessing different types of neurons, also contains natural microglia. This model better preserves the cellular diversity and maturity of primary tissue, making it suitable for drug screening, disease modeling, and personalized medicine.

[0005] To achieve the above objectives, the present invention provides a method for culturing brain organoids containing microglia, comprising the following steps: (1) Select human embryonic brain tissue with an embryonic age of 8 to 15 weeks and a weight of less than 500g, and separate and digest the brain tissue into small tissue pieces with a diameter of 0.8-1.5 mm; (2) The tissue fragments were inoculated into a culture container, amplification medium was added, and three-dimensional suspension culture was carried out at 37°C and 5% CO2 environment with constant rotation speed to obtain organoid-like structures; The amplification medium consisted of a 1:1 mixture of Advanced DMEM / F12 and Neurobasal medium, supplemented with penicillin-streptomycin, GlutaMax and β-mercaptoethanol, and the following exogenous factors were added: vitamin A-free B27 additive, N2 additive, MEM non-essential amino acids, (human fibroblast growth factor 10) hFGF10, (human fibroblast growth factor 2) hFGF-2, (human epidermal growth factor) hEGF, IL-34, CSF1, and LAA; (3) After culturing for 4-6 days, when the organoid structure grows to a diameter of 2-3 mm, cut the organoid structure into 2-3 tissue blocks and transfer them to the same culture environment as in step (2) to continue culturing and complete the amplification; (4) The expanded organoids were transferred to a maturation culture medium for further culture to obtain brain organoids with microglia; The maturation medium consists of a 1:1 mixture of Advanced DMEM / F12 and Neurobasal medium, supplemented with penicillin-streptomycin, GlutaMax and β-mercaptoethanol, and fortified with the following exogenous factors: N2 additive, MEM non-essential amino acids, vitamin A-containing B27 additive, and LAA.

[0006] Further, in step (1), the human embryonic brain tissue with an embryonic age of 8 to 15 weeks and a weight of less than 500g is preserved and transported under sterile and low temperature conditions; before culture, the tissue is thoroughly cleaned with a buffer containing antibiotics and necrotic areas are removed.

[0007] Further, in step (1), the buffer solution is Advanced DMEM / F12; the antibiotic is penicillin-streptomycin.

[0008] Further, in step (2), the final concentrations of each component in the amplification medium are as follows: penicillin-streptomycin 1×, GlutaMax 1×, β-mercaptoethanol 50μM, vitamin A-free B27 additive 1×, N2 additive 1×, MEM non-essential amino acids 1×, hFGF10 concentration of 50ng / mL, hFGF-2 concentration of 40ng / mL, hFGF concentration of 50ng / mL, IL-34 of 100ng / mL, CSF1 of 50ng / mL, and LAA of 100ng / mL.

[0009] Furthermore, in step (2), the exogenous factor Primocin is added during the culture of primary tissues, but not when the culture medium is changed subsequently.

[0010] Furthermore, in step (3), the culture medium is changed every 2-3 days during the culture period.

[0011] Further, in step (4), the final concentrations of each component in the mature culture medium are: penicillin-streptomycin 1×, GlutaMax 1×, β-mercaptoethanol 50μM, N2 additive 1×, MEM non-essential amino acids 1×, vitamin A-containing B27 additive 1×, and LAA at a concentration of 100ng / mL.

[0012] A second aspect of the present invention provides a brain organoid with microglia, obtained by the method described in the present invention.

[0013] A third aspect of this invention provides the application of the brain organoids described herein in the preparation of pathological models of nervous system diseases, wherein the nervous system diseases include one or more of the following: neurodegenerative diseases, neurodevelopmental disorders, neuroinflammatory diseases, cerebrovascular diseases, demyelinating diseases, motor neuron diseases, epilepsy, psychoneurotic diseases, neuroinfectious diseases, hereditary nervous system diseases, metabolic nervous system diseases, and neuroinjury diseases; the effectiveness of the model is verified by detecting the expression levels of characteristic pathological markers in the organoids, neuronal dysfunction, glial cell activation, neuroinflammatory responses, synaptic damage, abnormal neural network activity, and / or cell death levels.

[0014] The fourth aspect of this invention provides the application of the brain organoids described herein in the screening of drugs for the central nervous system, the application including detecting the regulatory effects of drugs on neuronal activity, synaptic connection function and electrophysiological activity in organoids, and evaluating the efficacy and safety of drugs.

[0015] In other respects, the present invention provides applications of the above-mentioned human embryonic brain tissue organoids in the following aspects: 1. To study the early development process of the human brain.

[0016] 2. As a platform for studying the effects of specific genes or environmental factors on fetal brain development.

[0017] 3. Used to study the developmental processes of the human nervous system, including neurogenesis, neuronal migration, glial cell differentiation, brain region formation, synapse formation, and neural network establishment. 4. Used to construct models of nervous system diseases, including neurodevelopmental disorders, neuropsychiatric diseases, epilepsy, neurodegenerative diseases, cerebrovascular diseases, neuroinflammatory diseases, demyelinating diseases, motor neuron diseases, neuroinfectious diseases, genetic or metabolic nervous system diseases, nerve injuries, and nervous system tumors. 5. Used to study the mechanisms of development and progression of nervous system diseases, including neuronal dysfunction, synaptic damage, neural network abnormalities, protein aggregation, neuroinflammation, glial cell activation, myelin damage, cell death, and tumor-related pathological processes. 6. Used for drug screening, efficacy evaluation, and neurotoxicity testing to evaluate the effects of drugs or compounds on nerve cell survival, differentiation, synaptic function, neural network activity, inflammatory response, and disease-related pathological phenotypes.

[0018] 7. Used for personalized disease modeling, precision medicine research, disease biomarker screening, target validation, and high-throughput drug screening.

[0019] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages: 1. High fidelity: It uses embryonic tissue directly, perfectly preserving the original cell composition, spatial information and molecular characteristics of the human brain at a specific gestational week, avoiding deviations in the iPSC differentiation process.

[0020] 2. Short cycle and rapid function: Without the need for a long reprogramming and targeted differentiation stage, organoids can be formed in a few days and can quickly exhibit functional characteristics close to their developmental age.

[0021] 3. Long-term expansion capability: This organoid model can be stably expanded and maintained for several months, providing the possibility for long-term research.

[0022] 4. Highly targeted application: It is particularly suitable for studying the critical period of cerebral cortex formation and rapid expansion, which is the origin window for many neurodevelopmental disorders.

[0023] 5. Complex tissue structure: rich in various nerve cell types, including microglia. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments are further described below. It should be understood that the accompanying drawings only show some embodiments of the present invention. For those skilled in the art, other forms of drawings or equivalent content can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of a brain tissue-derived organoid cultured in 3D according to one embodiment of the present invention; Figure 2 This refers to microglia from organoids derived from brain tissue, cultured in 3D according to one embodiment of the present invention. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] In this invention, the acquisition of the brain tissue was approved by the ethics review committee (IRB), complied with the "Ethical Guidelines for Human Organoid Research" and relevant regulations on the use of human embryo biomaterials, and there was no deliberate destruction of human embryos or commercial use.

[0028] The following description of some steps of the present invention is provided by way of example and is not intended to limit the scope of the invention.

[0029] In this invention, the organoid structure is cut using microscissors.

[0030] In this invention, the culture plate is placed on a horizontal shaker during cultivation, and the cultivation conditions are: 37°C, 5% CO2 environment, and continuous cultivation at a constant speed of 90 rpm.

[0031] In this invention, the cultured brain organoids have a clear cortical layered structure and mature neurons. The culture period is ≤35 days. Immunofluorescence staining shows MAPT and TUJ1 positivity, and electrical signals can be detected.

[0032] The embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the invention. For those skilled in the art, other equivalent implementation methods can be obtained based on the content of this application without creative effort, and all such methods should be considered to fall within the protection scope of the present invention.

[0033] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0034] Ethical Statement: The human embryonic brain tissue sampling involved in this invention strictly follows the "Ethical Guidelines for Human Embryonic Stem Cell Research" and relevant national laws and regulations. It has been approved by the ethics committee of the research institution and informed consent has been obtained from the embryo donor. It is used only for scientific research and does not involve reproductive cloning or other illegal operations.

[0035] Experimental materials and reagents Experimental samples: brain tissue from human embryos aged 8 to 15 weeks with an embryonic age that has been ethically approved, with an embryo weight of less than 500g; Basic culture media: Advanced DMEM / F12 medium (Gibco, catalog number 12634010), Neurobasal medium (Gibco, catalog number 21103049). Core reagents: Penicillin-Streptomycin (100×, Gibco), GlutaMax (100×, Gibco), β-mercaptoethanol (Sigma), Vitamin A-free B27 additive (50×, Gibco), Vitamin A-containing B27 additive (50×, Gibco), N2 additive (100×, Gibco), MEM non-essential amino acids (100×, Gibco), recombinant human hFGF10 (PeproTech), recombinant human hFGF-2 (PeproTech), recombinant human hEGF (PeproTech), Primocin (Invivogen); Experimental consumables and instruments: low-adsorption 6-well culture plate, stereomicroscope, sterile microscissors, CO2 constant temperature incubator, constant temperature shaker, laser confocal microscope.

[0036] Culture medium preparation (1) Amplification medium: Advanced DMEM / F12 and Neurobasal medium were mixed at a volume ratio of 1:1. 1× penicillin-streptomycin, 1× GlutaMax, 50 μM β-mercaptoethanol were added to the final concentration. 1× vitamin A-free B27 additive, 1× N2 additive, 1× MEM non-essential amino acids, and 50 ng / mL hFGF10, 40 ng / mL hFGF-2, 50 ng / mL hEGF, 100 ng / mL IL-34, and 50 ng / mL CSF1 were added. At the same time, 100 ng / mL LAA was added. The mixture was filtered through a 0.22 μm sterile filter membrane for sterilization and stored at 4℃ in the dark for 7 days.

[0037] (2) Mature culture medium: Based on the mixture of Advanced DMEM / F12 and Neurobasal medium at a volume ratio of 1:1, add 1× penicillin-streptomycin, 1× GlutaMax, 50 μM β-mercaptoethanol, 1× N2 additive, 1× MEM non-essential amino acids, 1× vitamin A-containing B27 additive, and LAA at a concentration of 100 ng / mL. Sterilize by filtration through a 0.22 μm sterile filter membrane, store at 4℃ in the dark, and have a shelf life of 7 days.

[0038] Example 1: Establishment and long-term culture of embryonic brain tissue organoids (FBOs) 1. Source of tissue: Human embryonic brain tissue weighing less than 500g was used after obtaining informed consent and ethical approval.

[0039] 2. Tissue processing: The tissue was thoroughly washed in cold Advanced DMEM / F12 (containing antibiotics). After removing necrotic areas, it was separated into small pieces with a diameter of approximately 1 mm using microscopic tools.

[0040] 3. Initial culture: Seed 10-15 tissue fragments into a 6cm cell culture plate and add amplification medium. Place the culture plate in a 37℃, 5% CO2 incubator and perform suspension culture on a track shaker at 100 rpm.

[0041] 4. Amplification culture: After about 5 days of culture, when the organoid-like structure grows to a diameter of 2-3 mm, cut it into 2-3 small pieces with micro-scissors, wash it with buffer, and transfer it to a new 6 cm cell culture plate for further culture to complete the amplification.

[0042] 5. Maturation culture: For specific experiments, individual organoids can be transferred to 6cm cell culture plates and replaced with maturation medium (containing BME and B27 containing vitamin A) and cultured for 3 to 10 days.

[0043] Example 2: Growth and Characterization of Organoids 1. Growth Assessment: Microscopic images were taken periodically, and the surface area and volume of the organoids were calculated using ImageJ software. The calculated volume growth of the organoids confirmed the stable expansion of organoid biomass.

[0044] Histological identification: Organoids were fixed overnight in 4% formaldehyde, embedded in low-melting-point agarose, and then sectioned into 40-micrometer-thick sections using a vibratory microtome. Immunofluorescence staining confirmed the positive expression of key brain development markers such as PAX6 (radial glial cell marker), TBR2 (intermediate progenitor cell marker), microglia (IBA1), and CTIP2 (deep neuron marker), indicating that the organoids successfully preserved the cellular hierarchical structure of the fetal brain.

[0045] Example 3: Microglia Culture and Identification 1. Organoid Culture and Microglia Induction: Human brain organoids were prepared according to the method in Example 1. On day 30 of organoid culture (i.e., the initiation point of neurogenesis), the culture medium was replaced with maturation medium. To enrich endogenous microglia in the organoids, the following cytokine combination was added to the maturation medium: IL-34 (interleukin-34): final concentration 100 ng / mL; CSF1 (macrophage colony-stimulating factor): final concentration 50 ng / mL; TGF-β1 (transforming growth factor-β1): final concentration 10 ng / mL (optional, for maintaining microglia homeostasis). The above cytokines were freshly added every 2-3 days with half-volume medium replacement, and cultured continuously until day 60-90.

[0046] 2. Morphological observation: Under an inverted microscope, cells with typical amoebic (round / elliptical) or branched morphology can be observed within and at the edges of the organoid parenchyma. These cells have small cell bodies (approximately 5-10 μm in diameter), slender processes, and surround neuronal cell bodies or synaptic junctions, consistent with the characteristic morphology of microglia.

[0047] 3. Immunofluorescence identification: Frozen sections of organoids cultured to day 60 were prepared and stained with immunofluorescence. The results were interpreted as follows: Positive marker: These cells co-expressed the Iba1 microglia cell marker.

[0048] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for culturing brain organoids containing microglia, characterized in that, Includes the following steps: (1) Select human embryonic brain tissue with an embryonic age of 8 to 15 weeks and a weight of less than 500g, and separate and digest the brain tissue into small tissue pieces with a diameter of 0.8-1.5 mm; (2) The tissue fragments were inoculated into a culture container, amplification medium was added, and three-dimensional suspension culture was carried out at 37°C and 5% CO2 environment with constant rotation speed to obtain organoid-like structures; The amplification medium consisted of a 1:1 mixture of Advanced DMEM / F12 and Neurobasal medium, supplemented with penicillin-streptomycin, GlutaMax and β-mercaptoethanol, and the following exogenous factors were added: B27 additive, N2 additive, MEM non-essential amino acids, hFGF10, hFGF-2, hEGF, IL-34, CSF1, and LAA. (3) After culturing for 4-6 days, when the organoid structure grows to a diameter of 2-3 mm, cut the organoid structure into 2-3 tissue blocks and transfer them to the same culture environment as in step (2) to continue culturing and complete the amplification; (4) The expanded organoids were transferred to a maturation culture medium for further culture to obtain brain organoids with microglia; The maturation medium consists of a 1:1 mixture of Advanced DMEM / F12 and Neurobasal medium, supplemented with penicillin-streptomycin, GlutaMax and β-mercaptoethanol, and fortified with the following exogenous factors: N2 additive, MEM non-essential amino acids, vitamin A-containing B27 additive, and LAA.

2. The method according to claim 1, characterized in that, In step (1), the human embryonic brain tissue with an embryonic age of 8 to 15 weeks and a weight of less than 500g is preserved and transported under sterile and low temperature conditions; before culture, the tissue is thoroughly cleaned with a buffer containing antibiotics and necrotic areas are removed.

3. The method according to claim 2, characterized in that, In step (1), the buffer solution is Advanced DMEM / F12; the antibiotic is penicillin-streptomycin.

4. The method according to claim 1, characterized in that, In step (2), the final concentrations of each component in the amplification medium are as follows: penicillin-streptomycin 1×, GlutaMax 1×, β-mercaptoethanol 50μM, B27 additive 1×, N2 additive 1×, MEM non-essential amino acids 1×, hFGF10 concentration of 50ng / mL, hFGF-2 concentration of 40ng / mL, hFGF concentration of 50ng / mL, IL-34 of 100ng / mL, CSF1 of 50ng / mL, and LAA of 100ng / mL.

5. The method according to claim 1, characterized in that, In step (2), the exogenous factor Primocin is added when culturing primary tissues, but not when changing the culture medium.

6. The method according to claim 1, characterized in that, In step (3), the culture medium is changed every 2-3 days during the culture period.

7. The method according to claim 1, characterized in that, In step (4), the final concentrations of each component in the mature culture medium are: penicillin-streptomycin 1×, GlutaMax 1×, β-mercaptoethanol 50μM, N2 additive 1×, MEM non-essential amino acids 1×, B27 additive 1×, and LAA at a concentration of 100ng / mL.

8. A brain organoid possessing microglia, characterized in that, It is obtained by the method described in any one of claims 1-7.

9. The application of the brain organoids of claim 8 in the preparation of pathological models of nervous system diseases, characterized in that, The neurological diseases mentioned include one or more of the following: neurodegenerative diseases, neurodevelopmental disorders, neuroinflammatory diseases, cerebrovascular diseases, demyelinating diseases, motor neuron diseases, epilepsy, neuropsychiatric diseases, neuroinfectious diseases, hereditary neurological diseases, metabolic neurological diseases, and neurodegenerative diseases. The effectiveness of the model is verified by detecting the expression levels of characteristic pathological markers in organoids, neuronal dysfunction, glial cell activation, neuroinflammatory responses, synaptic damage, abnormal neural network activity, and / or cell death levels.

10. The application of the brain organoids of claim 8 in drug screening for the central nervous system, characterized in that, The applications include detecting the regulatory effects of drugs on neuronal activity, synaptic connectivity, and electrophysiological activity in organoids, and evaluating drug efficacy and safety.

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