Culture method of 3D neural immune organoid containing microglial cells

Abstract

The invention belongs to the field of stem cell biology, and relates to a culture method of a 3D neural immune organoid containing microglial cells, which comprises the following steps: S1, respectively inducing induced pluripotent stem cells in an ectodermal direction and a mesodermal direction to obtain a neural embryoid and a yolk sac embryoid; s2, culturing the neural embryoid to perform neural differentiation to obtain a neural garland structure, and re-digesting the neural garland structure to obtain a neural progenitor cell single cell; culturing the yolk sac embryoid to enable the yolk sac embryoid to generate macrophage progenitor cells; and S3, fusing the neural progenitor cell single cell and the macrophage progenitor cell, and continuously culturing to form the 3D neural immune organoid containing the microglial cells. The brain-like organ provided by the invention has the functions of exploring the control of microglial cells on the progenitor cell proportion, the change of phagocytic function, the control on the number of mature neurons and the influence on cell proliferation and apoptosis in the development stage, and meanwhile, the brain-like organ can accept external stimulation and make stress change of corresponding functions.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of stem cell biology, and relates to a culture method of a 3D neuro-immune organoid containing microglia. BACKGROUND

[0002] Organoids are three-dimensional micro-organ models constructed by the self-renewal and directional differentiation ability of stem cells, and the core value lies in the ability to reproduce the spatiotemporal dynamics and complex cell interaction network of human organ development in vitro. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have become an ideal tool for constructing organoid models due to their unique self-renewal ability and strong differentiation potential. By inhibiting bone morphogenetic protein (BMP) and transforming growth factor-β / NODAL signaling pathways to different degrees (i.e., the "double SMAD signaling pathway inhibition" strategy), hPSCs can be guided to differentiate into neural stem cells, cortical pyramidal neurons and other neural cell types. At present, researchers have developed various 3D human brain organoid models, such as whole brain, forebrain, midbrain, cerebellum and vascularized organoids. However, these models have a fundamental flaw in terms of cell composition: the lack of microglia, a key regulator of the neuro-immune microenvironment. As the only resident immune cells in the central nervous system, microglia have a role far beyond being an immune sentinel. It is actually a multi-dimensional regulator of the nervous system. Recent single-cell sequencing studies have revealed that there are at least 12 functional subtypes of microglia, and they play a core role in neural development. First, it is a dynamic architect of neural development. From the embryonic period to the early postnatal period, microglia shapes neural circuits. In terms of synaptic pruning, it adopts a "eat-tag" mechanism to identify redundant synapses and guide the pruning process. In terms of regulating neurogenesis, microglia maintains the stemness of neural progenitor cells in the ventricular zone through TGF-β signaling, and stimulates the proliferation of adjacent cells by phagocytosing sphingosine-1-phosphate (S1P) released by apoptotic cells. In the cortical plate region, it even forms a "cell bridge" and secretes matrix metalloproteinase 9 (MMP9) to degrade the extracellular matrix, providing navigation for the precise laminar positioning of neurons. Secondly, mature microglia continuously monitor the environment to maintain functional balance. The absence of microglia leads to significant distortion in organoids when simulating core physiological or pathological processes that are highly dependent on neuro-immune interactions, such as neuroinflammation and synaptic remodeling. Statistics in 2024 show that more than 80% of drug development failures related to brain diseases are due to the inability of preclinical models to effectively predict neuro-immune interactions. Therefore, it is crucial to construct brain models containing functional neuro-immune units.

[0003] Currently, there are mainly two technical routes to construct organoids containing microglia: 1. Late-stage implantation method: microglia cells (such as iPSC-derived or primary cells) induced in vitro are injected into a pre-matured brain organoid. The defects of this method are: the dense extracellular matrix in the mature organoid will seriously hinder the migration of the implanted cells, resulting in very low implantation efficiency; the implanted microglia cells often highly express pro-inflammatory markers (such as CD68) and lowly express steady-state markers (such as TMEM119 / P2RY12), showing a "pathologically activated" state, which makes them lose function; the implantation time is not appropriate, and the microglia cells miss the critical period of neurogenesis and cannot effectively participate in the synaptic pruning process. 2. Mixed differentiation method: cytokines such as M-SCF and IL-34 are added at the early stage of neural induction of iPSCs, trying to promote a part of stem cells to spontaneously differentiate into microglia-like cells. The defects of this method are: the final proportion of microglia cells is usually less than 2%, and the batch difference is large, making it difficult to stably control the proportion; the differentiation process of microglia cells often lags behind neurogenesis (electron microscopy observation shows that its synaptic phagocytosis activity is only about 30% of the normal level in vivo); due to non-directional differentiation, a large number of peripheral macrophages are produced, which interfere with the accurate evaluation of the specific functions of the central nervous system.

[0004] For example, Park, D. S et al. disclosed that mature neural spheroids were fused with iMac at day 26 to form 3D human brain organoids containing microglia cells. Although this method can obtain mature and functional microglia cells, during the fusion operation, the development stage of iMac does not match the process of macrophage progenitor cells migrating into the neural tube and gradually developing into microglia cells in the real human brain. In addition, the ratio of neural progenitor cells and microglia-like cells during the fusion process is difficult to accurately control; although the amount of iMac can be adjusted during fusion, due to the batch difference and individual difference of neural spheroids itself, the number of cells is difficult to accurately estimate, which leads to a significant gap between the proportion of microglia cells in the final brain organoid and the 5%-10% in the real human brain.

[0005] Existing technology CN118389433A discloses a method for inducing microglia differentiation from pluripotent stem cells sequentially using culture media 1, 2, 3, and 5III; and for inducing brain-like organisms from pluripotent stem cells sequentially using culture media IV, V, VI, and VII. The resulting microglia and brain-like organisms are then co-cultured sequentially using culture media VIII, IX, and X to obtain neuroimmune brain-like organisms. This method successfully induces pluripotent stem cells to differentiate into neuroimmune brain-like organisms and is simpler than existing technologies, allowing microglia to infiltrate into the brain-like organisms. However, the detected microglia proportion is only 2.39%, far lower than the proportion in the real human brain.

[0006] Prior art CN113924362A discloses a method for generating brain organoids with sufficient microglia, comprising the following steps: incubating primitive macrophages together with brain organoids aged 15 to 30 days in a brain organoid culture medium containing CSF-1 in a low-adsorption cell culture vessel to generate microglia, wherein a) the primitive macrophages are generated from a first stem cell population by the following steps: i) incubating the stem cells in a medium containing GSK3 inhibitor, BMP4, and VEGF to differentiate the stem cells into mesodermal cell lines; ii) incubating the mesodermal cell lines in a medium containing FGF-2 to differentiate the mesodermal cell lines into angiogenic cells; iii) incubating the angiogenic cells in a medium containing VEGF and FGF-2. However, the detected microglia rate is less than 5%, far lower than the actual human brain rate. Summary of the Invention

[0007] The purpose of this invention is to provide a method for culturing 3D neuroimmune organoids that can accurately simulate the co-development process of the human brain's neural-immune system and contain microglia in a controllable proportion.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for culturing 3D neuroimmune organoids containing microglia, comprising the following steps: S1. Induced pluripotent stem cells were induced in the ectoderm direction and mesoderm direction respectively to obtain neural embryoids and yolk sac embryoids; S2. Culture neural embryoids to induce neural differentiation, obtain neural rosette structures, and then digest them to obtain neural progenitor single cells; culture yolk sac embryoids to produce macrophage progenitor cells; S3, fused with neural progenitor single cells and macrophage progenitor cells, and continued to be cultured to form 3D neuroimmune organoids containing microglia.

[0010] The unique role of microglia in neural development is determined by its strict time window dependence and spatial specificity. During human embryonic development, macrophage progenitor cells originating from the yolk sac will migrate into the neural tube through the blood stream at 4-5 weeks, and finally differentiate into mature microglia under the induction of the local microenvironment. This special developmental origin means that the traditional, only containing neural lineage organoid model, is fundamentally impossible to autonomously produce fully functional microglia. Therefore, it is of great significance to develop a brain organoid model that can simulate neural development and integrate neural immune function, containing microglia.

[0011] The key point of the present application is that the present application realizes the different developmental lineage origins of neuroectoderm and mesoderm, and the precise regulation of cell proportion. The present application introduces lineage-purified macrophage progenitor cells at the initial stage of neural induction (day 0), thereby reconstructing the development dialogue between neuroectoderm (neural progenitor cells) and mesoderm (macrophage progenitor cells) at the source, accurately simulating the core event of “neural-immune co-development” during the embryonic period; at the same time, by accurately setting the initial inoculation proportion of neural progenitor cells and macrophage progenitor cells, it is ensured that the proportion of microglia in the finally formed organoid stably reaches 5%-10% of the real human brain. This optimization scheme will more truly simulate the interaction between neural progenitor cells and macrophage progenitor cells, the key regulatory function of microglia and the complete neural immune microenvironment during neural development, which is more in line with the development law of human brain. Studies have shown that the origin of microglia is a cell type that exists in the developing brain tissue before the neural plate forms, the neural tube closes and the blood-brain barrier closes. A series of previous methods for inducing brain organoids containing microglia all involve immersing mature microglia into brain organoids that have been developed for a period of time, which will miss the time node of the microglia itself to play a series of functions including “progenitor pool control”, “influence neuron differentiation and maturation” and the like. At the same time, more controllable cell types can be obtained, which are in line with the law and cell type characteristics of human brain development.

[0012] According to the embodiments of the present application, the present application can be further optimized, and the following is the technical scheme formed after optimization:

[0013] In one preferred embodiment, before step S1, the induced pluripotent stem cells are pre-cultured in mTeSR TM Plus culture medium, and the culture time is 3-4 days.

[0014] The pre-culture stop standard is that the cells cover 80%-90% of the bottom area of the six-well plate hole, form a clear boundary, the cell arrangement is compact, there is no obvious gap in the colony, and the cell morphology is uniform.

[0015] Differentiated cells account for less than 5% of the field of view. The criteria for determining differentiated cells are: the cell body is extended and the length of the protrusion is greater than twice the cell body diameter; or the morphology is irregular and significantly different from the clonal cluster.

[0016] In one preferred embodiment, step S1, which involves inducing ectoderm orientation in induced pluripotent stem cells, includes:

[0017] First, induced pluripotent stem cells are digested into single cells; neural induction medium is added to the single cells for induction, and they are cultured for 7-13 days to obtain neural embryos.

[0018] In one preferred embodiment, the digestive fluid used for digestion is Accutase digestive fluid.

[0019] In one preferred embodiment, the culture process uses AggreWell culture plates. TM 800 culture plate.

[0020] In one preferred embodiment, the neural induction medium is a STEMdiff medium containing 0.01-0.02 mM Y-27632. TM Neural induction culture medium.

[0021] In one preferred embodiment, the culture medium is changed periodically during the cultivation process; the replacement medium is STEMdiff. TM Neural induction culture medium.

[0022] In one preferred embodiment, step S1, which involves inducing the mesodermal orientation of induced pluripotent stem cells, includes:

[0023] Digest induced pluripotent stem cells to obtain single cells; add induction culture medium to the single cells and induce for 1-2 days to obtain yolk sac embryos.

[0024] In one preferred embodiment, the induction medium comprises a basal medium and additives; the additives include: bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and Y-27632; the basal medium is mTeSR. TM Plus medium.

[0025] In one preferred embodiment, the induction culture medium contains 40-50 ng / ml bone morphogenetic protein 4, 50-60 ng / ml vascular endothelial growth factor (VEGF), 10-20 ng / ml stem cell factor, and 1-2 μl / ml Y-27632.

[0026] In one preferred embodiment, the step of culturing the neural embryoid bodies to neural differentiation in step S2 comprises: The neural embryoid bodies are digested to obtain rosette structures; the rosette structures are further expanded for 15-21 days to obtain neural progenitor cells.

[0027] In one preferred embodiment, the reagent used for digesting the neural embryoid bodies is a neural rosette selection reagent.

[0028] In one preferred embodiment, the neural rosette selection reagent is STEMdiff TM neural rosette selection reagent.

[0029] In one preferred embodiment, the rosette structures are further expanded using a culture medium STEMdiff TM neural induction medium.

[0030] In one preferred embodiment, the step of culturing the yolk sac embryoid bodies to neural differentiation in step S2 comprises:

[0031] The yolk sac embryoid bodies are added with macrophage progenitor cell culture medium and cultured for 5-21 days to obtain macrophage progenitor cells.

[0032] In one preferred embodiment, the macrophage progenitor cell culture medium comprises a basic culture medium and an additive; the additive comprises interleukin-3 and macrophage colony-stimulating factor; and the basic culture medium is X-VIVO 15 culture medium.

[0033] In one preferred embodiment, the macrophage progenitor cell culture medium contains 25-35 ng / ml interleukin-3 and 90-100 ng / ml macrophage colony-stimulating factor.

[0034] In one preferred embodiment, the step of fusing in step S3 comprises: The neural progenitor cells are digested into neural progenitor cell single cells; and the neural progenitor cell single cells and the macrophage progenitor cells are fused in a fusion culture medium for 1-5 days.

[0035] In one preferred embodiment, the fusion culture medium contains macrophage progenitor cell culture medium and STEMdiff™ neural precursor cell culture medium in a volume ratio of 1-2:1-2.

[0036] In one preferred embodiment, the neural progenitor cell single cells and the macrophage progenitor cells are fused at a ratio of 5-9:3.

[0037] In one preferred embodiment, the neural progenitor cell single cells and the macrophage progenitor cells are fused at a ratio of 7:3.

[0038] In one preferred embodiment, after 25-30 days of continuous culture, the brain-like organoid is embedded and then induced to form a 3D neuro-immune organoid containing microglia cells by using a maturation medium.

[0039] In one preferred embodiment, the medium used for continuous culture is a fusion medium.

[0040] In one preferred embodiment, the embedding is performed using Matrigel.

[0041] In one preferred embodiment, the maturation medium comprises a basal medium and an additive; the additive comprises interleukin 34 and granulocyte-macrophage colony-stimulating factor; the basal medium is BrainPhys TM Neuronal Medium.

[0042] In one preferred embodiment, the maturation medium comprises 100-200 ng / ml interleukin 34 and 10-20 ng / ml granulocyte-macrophage colony-stimulating factor.

[0043] The following experiment is used to explain the present application: During the development of the embryonic central nervous system (CNS), myeloid progenitor cells derived from the yolk sac migrate to the developing neural tube and interact with neural progenitor cells (NPCs). These cells proliferate synergistically and eventually differentiate into various cell types that constitute the nervous system, including neurons, astrocytes, oligodendrocytes, and microglia.

[0044] The core method of the present invention starts with human induced pluripotent stem cells (hiPSCs). First, by inducing hiPSCs towards mesoderm, using a medium containing 50 ng / ml Bone Morphogenetic Protein 4 (BMP4), 50 ng / ml Vascular Endothelial Growth Factor (VEGF) and 20 ng / ml Stem Cell Factor (SCF), the differentiation of embryoid bodies (EBs) into a hematopoietic progenitor cell line with mesodermal characteristics and with the potential to differentiate into primitive myeloid lineage is promoted (i.e. yolk sac embryoid bodies, YS-EBs). At day 5 of differentiation, 100 ng / ml Macrophage Colony-Stimulating Factor (M-CSF) and 25 ng / ml Interleukin-3 (IL-3) are added to induce YS-EBs to produce myeloid progenitors and further differentiate into macrophage progenitors. These macrophage progenitors mimic their behavior in neural development, are able to cooperate with neural progenitors and finally differentiate into microglia.

[0045] Meanwhile, another set of hiPSCs is used to form neural embryoid bodies. This process employs a neural induction medium (NIM) containing a SMAD inhibitor and neural induction factors N2, effectively inhibiting mesoderm / endoderm differentiation and ensuring directional induction towards neural lineage. A key innovation of the present invention is the use of a neural progenitor proliferation medium (STEMdiff® Neural Progenitor Medium) instead of traditional differentiation or maturation medium at this stage. This strategy aims to maximize the maintenance of the stemness of neural progenitors and their multipotential differentiation potential, thus more accurately mimicking the microenvironment in which neural progenitors interact with subsequently migrated macrophage progenitors and cooperatively differentiate during in vivo development. TM Neural Progenitor Medium) instead of traditional differentiation or maturation medium at this stage. This strategy aims to maximize the maintenance of the stemness of neural progenitors and their multipotential differentiation potential, thus more accurately mimicking the microenvironment in which neural progenitors interact with subsequently migrated macrophage progenitors and cooperatively differentiate during in vivo development.

[0046] To realize the physiological relevance of cell composition in the organoids, the induced neural progenitor cells are fused with the primitive macrophage progenitors (PMP) at an optimized ratio of 7:3. This ratio design ensures that the proportion of microglia in the mature brain organoids can be stabilized at about 8%, which is highly matched with the 5%-10% of microglia in the real human brain, effectively solving the significant defect that the proportion of microglia in the existing brain organoid model is generally insufficient and difficult to control. At the same time, this controllable proportion provides a solid operational basis for establishing a neuroimmunological disease research model.

[0047] In the early stage of fusion, a co-culture strategy is implemented on multiple organoids (5-8 organoids per well), and 20 μl of low-factor Matrigel is used for embedding subsequently. This combined method significantly reduces the size heterogeneity and structural dissociation risk of the organoids, effectively controls the batch-to-batch variation, and improves the repeatability of the model. The fusion culture uses MO-1 medium (mixed by NPC medium and PMP medium at a ratio of 1:1), which can simultaneously maintain the stemness and state of the two types of progenitor cells, and accurately simulate the early developmental events of the in-vivo myeloid progenitor cells migrating into the neural tube. On the 41st day of culture, the medium is replaced with BrainPhys TM Neuronal Medium (its basic components include N2, B27 supplement, Brain-Derived Neurotrophic Factor, BDNF, Glial Cell Line-Derived Neurotrophic Factor, GDNF, and Ascorbic acid). This medium effectively promotes the maturation of neurons while regulating the key biological behaviors of microglia in the organoids, including their proliferation, migration, and homeostasis maintenance, through the synergistic effect of IL-34 and GM-CSF.

[0048] Compared with the prior art, the beneficial effects of the present application are: The 3D human brain organoid cultured for 55 days according to the application has more complex dendritic structures and spine maturity of neurons, and the state of mutual fusion of NPCs and PMP cells in the early system of fusion, and contains a variety of neural lineage cells: including microglia (marker is transmembrane protein 119, TMEM119, CD11b, CD45 positive), neurons (marker is beta 3 tubulin, TUJ1 positive), astrocytes and neural progenitor cells (marker is nestin positive). This proves that the model successfully constructs a complex 3D human brain organoid containing functional microglia. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is a flowchart of the construction process of the brain organoid of the application.

[0050] Figure 2 It is an immunofluorescence staining diagram of progenitor cells in a brain organoid slice at 55 days (40x).

[0051] Figure 3 It is an immunofluorescence staining diagram of microglia in a brain organoid slice at 55 days (40x).

[0052] Figure 4 It is an immunofluorescence staining diagram of neurons in a brain organoid slice at 55 days (40x).

[0053] Figure 5 It is an immunofluorescence staining diagram of the co-localization of microglia and presynaptic membranes in a brain organoid slice at 55 days (40x).

[0054] Figure 6 It is a column chart of the changes of CD43, SOX2, SYNAPSIN I, SYNAPSIN I / CD11b and Ki67 in the brain organoid after being stimulated by inflammation.

[0055] Figure 7 It is a flow cytogram of the proportion of microglia in the brain organoids induced by two strains of iPSCs. DETAILED DESCRIPTION

[0056] The application is not limited to the following detailed description, and those skilled in the art can implement the application by using other various embodiments according to the disclosure of the application, or any simple changes or modifications made by using the design structure and ideas of the application, all fall within the protection scope of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] The reagents and culture media used in the following examples:

[0059] I. NPCs' Inducement

[0060] Phase 1: hiPS cell pretreatment and pre-induction status assessment (preparation phase) Figure 1 (0 days ago)

[0061] 1. hiPS cell culture: mTeSR was cultured in advance TM After incubating the Plus medium at room temperature for 30 minutes, take a sterile six-well plate and add 1×10⁻⁶ medium. 5 pcs / cm 2 hiPS cells (ACS1011, purchased from ATCC Cell Bank) were seeded at a density of [missing information], resuspended in 2 ml of culture medium, and placed in a 37°C, 5% CO2 incubator. Fresh mTeSR was replaced daily. TM Plus medium, culture continuously for 3-4 days.

[0062] 2. Pre-induction status check: NPC induction can be initiated by observing the following conditions using an inverted microscope (10× objective lens):

[0063] Cells cover 80%-90% of the bottom area of ​​the six-well plate, forming a clone cluster with clear boundaries and tightly packed cells (with no obvious gaps within the clone cluster and uniform cell morphology).

[0064] Differentiated cells account for less than 5% of the field of view (differentiated cells are defined as cells that are fully extended, with protrusions longer than twice the cell diameter, or with irregular shapes that are significantly different from the clonal clusters).

[0065] Phase 2: AggreWell TM 800 plate pretreatment and EB formation (days 0-1)

[0066] 1. AggreWell TM 800 plate pretreatment: Take a 24-well plate, add 1 mL of anti-adhesion rinsing solution to each target well, and incubate at room temperature (20-25℃) for 10 min. Do not shake the plate during this period to ensure that the solution evenly covers the bottom of the well.

[0067] 2. hiPS cell washing: Take out the hiPS cell six-well plate to be induced, and slowly suck the old culture medium along the well wall with a 1 mL pipette (avoid the cell clone group with a pipette tip); add 1 mL DPBS to each well, gently tilt the plate body to make the buffer cover the cell surface, and suck it off after 1 min of standing. Repeat the washing for 3 times.

[0068] 3. Cell digestion: Add 1 mL of Accutase digestion solution preheated at 37°C for 10 min to each well, and return to the incubator for timed digestion for 5 min; every 2 min, use a microscope to check, and when the clone group edge is loose and the cell gap is increased, immediately terminate the digestion.

[0069] 4. Cell collection and centrifugation: Add 2 mL of DMEM-F12 medium to each well to terminate the digestion, and use a 1 mL pipette with a cut tip (to avoid scratching the cells) to blow 3-5 times clockwise along the well wall to ensure that the cells are completely detached to form a single cell suspension; transfer the suspension to a 15 mL centrifuge tube, and centrifuge at 1500 rpm at room temperature for 5 min. After centrifugation, a white cell precipitate can be seen at the bottom of the tube.

[0070] 5. Cell resuspension and counting: Suck the supernatant, add 1 mL of STEMdiff TM neural induction medium to each tube, resuspend the cells by blowing 5-8 times, and take 10 μL of the suspension for counting under an inverted microscope to ensure that the cell viability is > 90%.

[0071] 6. EB induction system preparation: Take the suspension containing 3 million cells to a new 15 mL centrifuge tube, and add STEMdiff TM neural induction medium to a total volume of 1 mL; add 1 μL of 10 mM Y-27632, mix well by blowing 2-3 times (Y-27632 can inhibit apoptosis, and cannot be omitted).

[0072] 7. Cell inoculation and EB formation: Suck the AggreWell TM 800 plate hole anti-adhesion rinse solution, add 1 mL of the above cell suspension to each well (avoid air bubbles, and if there are air bubbles, use a pipette to remove them); place the 24-well plate in a centrifuge equipped with an adapter, and centrifuge at 100 x g (about 450 rpm) at room temperature for 3 min; after centrifugation, directly place it in the incubator, and after 24 h of culture (day 2), microscopic examination can see that the EBs are 100-150 μm in diameter and dense and uniform.

[0073] Stage 3: EB medium exchange culture (days 2-5)

[0074] 1. Medium exchange on day 2: Take out the 24-well plate containing EBs, and use a 1 mL pipette to suck 0.5 mL of old medium along the wall (gentle action to avoid taking out EBs); slowly add 1.5 mL of STEMdiff TM neural induction medium along the well wall, and return to the incubator.

[0075] 2. Medium change: Repeat the medium change procedure described above (discard 0.5 mL old medium, add 1.5 mL STEMdiff TM neural induction medium) every day. During the medium change, check the EBs under the microscope to ensure that the EBs are not disintegrated and do not have obvious dispersed cells.

[0076] Stage 4: EB adhesion culture preparation (Day 6)

[0077] 1. Matrigel coating: Take a sterile six-well plate and add 1 mL of Matrigel to each well (thawed in advance at 4°C and operated on ice). Incubate the plate in a culture incubator at 37°C for 1 h to allow the Matrigel to evenly cover the bottom of the wells.

[0078] 2. EB screening and collection: Take a 40 pm cell filter and place it in a 50 mL centrifuge tube. Gently suck the EBs in the 24-well plate out with a wide-bore syringe tip (avoid squeezing the EBs). Slowly add the filter to the EBs. Rinse the filter with 1 mL of STEMdiff TM neural induction medium twice to remove single cells and old medium, and collect the EBs on the filter.

[0079] 3. EB adhesion culture: Discard the Matrigel in the six-well plate (a small amount of residual Matrigel will not affect the experiment). Add 2 mL of STEMdiff TM neural induction medium to each well. Uniformly inoculate the collected EBs into the six-well plate and place it in the culture incubator to allow the EBs to naturally adhere to the plate.

[0080] Stage 5: EB expansion (Day 7-13)

[0081] 1. Medium change: From Day 7, take out the six-well plate and discard all the old medium with a 1 mL pipette. Add 2 mL of STEMdiff TM neural induction medium to each well and return it to the culture incubator.

[0082] 2. Induction state observation: Observe the EBs under the microscope every day. You can see that the EBs gradually spread after adhering to the plate, and spindle-shaped or polygonal cells appear at the edges (initial differentiation into the neural lineage). Continue the medium change for 6 days (until Day 13).

[0083] Stage 6: Neural Rosette screening and NPC purification (Day 14)

[0084] 1. Matrigel coating: Coat a new six-well plate with Matrigel and incubate it at 37°C for 1 h for later use.

[0085] 2. Cell washing and digestion: Take out the six-well plate and discard the old medium. Add 1 mL of DPBS to each well and discard it after 1 min of standing. Repeat the washing once. Add 1 mL of STEMdiff TMNeural Rosette Selection Reagent, place in incubator at 37°C for 1.5 hours.

[0086] 3. Neural Rosette Collection: Aspirate the digestion reagent and quickly pipette 3-4 times with a 1 mL pipette tip against the area of the "rosette" structure (NPC-enriched area) visible under the microscope to dislodge the cells from this area. Add 2 mL of DMEM-F12 medium and transfer the suspension to a 15 mL centrifuge tube.

[0087] 4. Cell Centrifugation and Resuspension: Centrifuge at 350 x g (approximately 1200 rpm) for 5 minutes at room temperature, aspirate the supernatant; add 2 mL of STEMdiff™ Neural Progenitor Cell Medium per tube. TM Neural Induction Medium, resuspend the cells by pipetting 5 times.

[0088] 5. NPC Seeding: Aspirate the Matrigel in the reserve six-well plate, add 1 mL of the cell suspension per well; aspirate the old medium and wash once with 1 mL of DPBS, then add 2 mL of STEMdiff™ Neural Progenitor Cell Medium per well. TM Neural Induction Medium, place in incubator.

[0089] Stage 7: NPC Expansion and Harvest (Days 15-21)

[0090] Medium Change: From day 15 to day 20, aspirate the old medium daily and add 2 mL of fresh STEMdiff™ Neural Progenitor Cell Medium per well.

[0091] NPC (Neural Progenitor Cell) Harvest and Identification: On day 21, remove the culture plate and observe the uniform spindle-shaped cells (typical morphology of NPCs) under the microscope. Perform immunofluorescence staining detection by following the steps:

[0092] Cell Pretreatment

[0093] Remove the 6-well plate containing the NPCs (Neural Progenitor Cells) from the clean bench and aspirate the medium in the wells; wash the cells with DPBS buffer (Gibco #14190-144) for 3 times, 5 minutes each time (at room temperature, low-speed shaking at 50 rpm), to completely remove the residual medium and cell metabolic waste.

[0094] Cell Fixation

[0095] Add pre-cooled (4°C) 4% paraformaldehyde fixing solution (paraformaldehyde powder Sigma-Aldrich #P6148, prepared with DPBS buffer, pH 7.2-7.4) to each well to ensure complete coverage of the NPC cells on the coverslips; fix at room temperature for 15-20 minutes; aspirate the fixing solution and wash with DPBS buffer for 3 times, 5 minutes each time, to completely remove the residual fixing solution and avoid damage to the cell structure.

[0096] Cell Membrane Permeabilization

[0097] Add 0.3% Triton X-100 permeabilization solution (Thermo Fisher #HFH10, 1% stock diluted with DPBS) to each well, incubate at room temperature for 10-15 minutes to create micropores in the cell membrane for antibody penetration; aspirate the permeabilization solution and wash 3 times with DPBS buffer for 5 minutes each to remove residual permeabilization solution.

[0098] Blocking

[0099] Aspirate the DPBS buffer from the wells and add 5% BSA blocking solution (Bovine Serum Albumin Sigma-Aldrich #A7906, prepared in DPBS buffer) to the surface of the NPC cells on the coverslips, 50-100 μL per coverslip, ensuring complete coverage of the cell surface; transfer the coverslips to a humidified chamber and block at room temperature for 30-60 minutes to block non-specific sites on the cell surface for secondary antibody binding.

[0100] Primary Antibody Incubation

[0101] Aspirate the blocking solution (no wash step) and add the diluted NPC-specific primary antibody solution directly to the cell surface (50-80 μL per coverslip); select Nestin and Sox2 dual-label combination (Nestin (10C2) Mouse Monoclonal Antibody CST #33475, Sox2 (D6D9) Rabbit Monoclonal Antibody CST #3579) at 1:100-1:500 dilution in 5% BSA blocking solution, ensuring uniform coverage of the cells with the antibody solution; seal the humidified chamber and incubate overnight (12-16 hours) at 4°C; remove the humidified chamber the next day and allow to warm at room temperature for 30 minutes; wash 3 times with DPBS buffer for 10 minutes each to remove unbound free primary antibody.

[0102] Secondary Antibody Incubation

[0103] Drop 50-80 μL of diluted secondary antibody solution (1:200-1:1000 dilution with 5% BSA blocking solution) onto the cell surface; the secondary antibody is a fluorescently labeled antibody (Anti-rabbit IgG(H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) CST#4412, Anti-mouse IgG(H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) CST#4408) that matches the species of the primary antibody; place the wet box in a room temperature environment and incubate for 60 minutes in the dark; discard the secondary antibody solution and wash with DPBS buffer for 3 times, 10 minutes each time, in the dark to avoid background interference caused by residual fluorescein.

[0104] Nuclei staining

[0105] Drop 1 μg / mL DAPI staining solution (CST#4083, prepared with DPBS buffer) onto the cell surface and incubate for 5 minutes at room temperature in the dark; discard the staining solution and wash with DPBS buffer for 2 times, 5 minutes each time, in the dark to remove residual DAPI.

[0106] Mounting

[0107] Take a clean glass slide and drop 1-2 drops of anti-fluorescence quenching mounting medium (Thermo Fisher #P36961, ProLong™ Diamond) in the center; carefully remove the coverslip (cell side down) with tweezers and slowly cover it on the mounting medium on the glass slide to avoid air bubbles; use sterile filter paper to absorb the excess mounting medium on the edge of the coverslip and let it dry at room temperature for 10 minutes.

[0108] Observation and imaging

[0109] Place the mounted glass slide under the Zeiss LSM980 laser confocal microscope, select the corresponding fluorescence channel, and observe and collect the Nestin and Sox2 specific fluorescence signals and nuclei staining signals in the NPC cells, record the cell morphology and marker expression localization, and obtain the experimental results. The results show that the positive rate of NPC specific marker Nestin is ≥92%, the positive rate of Sox2 is ≥90%, and the cell viability is ≥88%, which is determined as qualified NPC. Then use STEMdiff TM Neural precursor cell medium to culture and amplify the NPCs.

[0110] II. PMP induction

[0111] Stage 1: hiPSC pretreatment and state determination (1 day before induction)

[0112] 1. hiPSC culture: Take the logarithmic growth phase of hiPSC, use mTeSR TM Plus culture medium, replace fresh culture medium every day, ensure that the cells are in the logarithmic growth phase, and avoid excessive confluence (confluence is controlled at 70%-80%).

[0113] 2. State check: observe by inverted microscope, confirm that hiPSC forms a dense clonal group, there are no obvious differentiated cells (differentiated cells are determined: cell body stretching, increased processes, and significant difference in morphology with clonal group), and cell viability is ≥95%, which can enter the subsequent YS-EB induction.

[0114] Stage 2: Induction of YS-EB formation (0-4 days)

[0115] 1. AggreWell TM 800 plate pretreatment: take a 24-well plate, add 1 mL of anti-adhesion rinse solution to each target well, incubate at room temperature (20-25°C) for 10 min, do not shake the plate during the incubation period, ensure that the solution covers the well bottom evenly, discard the anti-adhesion rinse solution after incubation, and reserve for use.

[0116] 2. YS-EB induction medium preparation: take an appropriate amount of mTeSR TM Plus medium, add BMP4 (50 ng / mL), SCF (20 ng / mL), and VEGF (50 ng / mL) to the final concentration, mix gently by inverting the centrifuge tube 3-5 times, filter sterilize with a 0.22 μm filter, and avoid generating air bubbles by repeated blowing.

[0117] 3. hiPSC digestion and inoculation:

[0118] 1) Take the pretreated hiPSC, aspirate the old medium, and wash once with 1 mL of DPBS; add 1 mL of Accutase digestion solution to each well, digest at 37°C for 5 min, and observe under a microscope until the clonal group edge is loose.

[0119] 2) Add 2 mL of DMEM-F12 medium to terminate digestion, gently blow with a wide-bore syringe to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, centrifuge at 1500 rpm at room temperature for 5 min, and discard the supernatant.

[0120] 3) Resuspend the cells with the YS-EB induction medium prepared above, adjust the cell concentration to 1×10 6 6 / mL, add 1 mL of cell suspension (containing 1×10 TM 6 hiPSC) to each well of the pretreated AggreWell 6 800 well.

[0121] 4) YS-EB formation culture: Place the 24-well plate into a suitable centrifuge and centrifuge at 100×g (about 450rpm) for 3min at room temperature to promote cell aggregation; after centrifugation, place it directly into a 37℃, 5% CO2 incubator and statically culture for 48h without changing the culture medium.

[0122] 5) YS-EB formation determination: After 48 hours of culture, observe with an inverted microscope (10× objective lens). If 180-220 YS-EBs are formed in each well, with a diameter of 120-180μm, and the morphology is round, the boundary is clear, and the cytoplasm is uniform, it is determined to be qualified YS-EB.

[0123] 6) Change the medium daily from day 2 to day 4, gently aspirate the original culture medium and add 1-2 ml of YS-EB induction medium.

[0124] Phase 3: YS-EB transfer and hPMP (Primitive Macrophage Precursor) culture (days 5-13)

[0125] 1. Preparation of PMP medium: Take X-VIVO 15 medium, add M-CSF (100ng / mL) and IL-3 (25ng / mL) to the final concentration, mix gently, and filter through a 0.22μm filter membrane for sterilization.

[0126] 2. YS-EB inoculation and culture: Add 15 mL of PMP medium to a T75 culture flask, gently resuspend the YS-EB in the medium in the wells of the plate, and transfer it to the culture flask; place it in a 37℃, 5% CO2 incubator and culture using a half-medium replacement method.

[0127] Days 5-9: Every 2 days, discard 5 mL of old culture medium and add 5 mL of fresh PMP culture medium;

[0128] From day 10 onwards: every 3 days, discard 8 mL of old culture medium and add 8 mL of fresh PMP culture medium to maintain the stability of the culture system.

[0129] Phase 4: Collection and yield detection of hPMP (Primitive Macrophage Precursor) (Days 14-21)

[0130] 1. hPMP collection: After 2-3 weeks of culture (days 14-21), hPMP (morphology: round or oval, diameter 8-12μm) can be observed in the culture supernatant under an inverted microscope. The supernatant is aspirated with a sterile pipette and transferred to a 50mL centrifuge tube. The tube is centrifuged at 1200rpm for 8min at room temperature. The precipitate is collected as crude hPMP. The supernatant can be cultured again (hPMP continues to be produced for about 3 months).

[0131] 2. hPMP production detection: cell counting plate counting combined with immunofluorescence detection of hPMP specific markers (CD235 + , CD43 + ) positive rate, calculate the cumulative production, immunofluorescence staining detection steps are:

[0132] Cell pretreatment

[0133] Take out the culture bottle with PMP, and suck the PMP cell suspension in the clean bench, and inoculate it in the 6-well plate coated with 0.1% Matrigel (Corning # 354234) (inoculation density about 5 x 10 4 Per hole) in 37℃, 5% CO2 cell incubator for 24 hours, then discard the culture medium in the hole; wash the cells with DPBS buffer (Gibco # 14190-144) for 3 times, 5 minutes each time (room temperature, low speed shaking of shaking table 50 rpm), completely remove the residual culture medium and cell metabolic waste.

[0134] Cell fixation

[0135] Add pre-cooled (4℃) 4% paraformaldehyde fixing solution (paraformaldehyde powder Sigma-Aldrich # P6148, DPBS buffer preparation, pH 7.2-7.4) to each hole, make sure to completely cover the PMP cells in the hole; Room temperature standing for 15-20 minutes; Discard the fixing solution, wash with DPBS buffer for 3 times, 5 minutes each time, completely remove the residual fixing solution, avoid cell structure damage.

[0136] Cell membrane permeation

[0137] Add 0.3% Triton X-100 permeation liquid (Thermo Fisher # HFH10, 1% mother liquor diluted with DPBS buffer) to each hole, incubate at room temperature for 10-15 minutes, make the cell membrane form micropores to facilitate antibody penetration; Discard the permeation liquid, wash with DPBS buffer for 3 times, 5 minutes each time, remove the residual permeation liquid.

[0138] Blocking

[0139] Suck the DPBS liquid in the hole, add 5% BSA blocking liquid (bovine serum albumin Sigma-Aldrich # A7906, DPBS buffer preparation) to the surface of PMP cells, 50-100 μL per hole, make sure to completely cover the cell surface; Put the 6-well plate into the wet box, block at room temperature for 30-60 minutes, block the binding of secondary antibody with non-specific sites on the cell surface.

[0140] Primary antibody incubation

[0141] Aspirate the blocking solution (no need to wash), directly add diluted PMP-specific primary antibody solution (50-80 μL per well) to the cell surface; the primary antibody is selected from CD235 and CD43 double-label combination (CD235a (JC159) Rabbit Monoclonal Antibody CST#75126, CD43 (MT1) Mouse Monoclonal Antibody CST#38428), each diluted with 5% BSA blocking solution at a ratio of 1:100-1:500, to ensure that the antibody solution uniformly covers the cells; after sealing the wet box, incubate it in a 4°C refrigerator overnight (12-16 hours); the next day, take out the wet box, warm it at room temperature for 30 minutes, wash it with DPBS buffer for 3 times, 10 minutes each time, to completely remove the unbound free primary antibody.

[0142] Secondary antibody incubation

[0143] Add diluted fluorescent secondary antibody solution (50-80 μL per well) to the cell surface; the secondary antibody is selected from fluorescently labeled antibodies (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) CST#4412, Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) CST#4408) matched with the species of the primary antibody, diluted with 5% BSA blocking solution at a ratio of 1:200-1:1000; place the wet box in a room temperature condition, avoid light, and incubate for 60 minutes; aspirate the secondary antibody solution, wash it with DPBS buffer for 3 times, 10 minutes each time (low-speed shaking on a shaking table), to avoid background interference caused by residual fluorescein.

[0144] Nucleus staining

[0145] Add 1 μg / mL DAPI nucleus staining solution (CST#4083, prepared with DPBS buffer) to the cell surface, incubate at room temperature for 5 minutes in the dark; aspirate the nucleus staining solution, wash it with DPBS buffer for 2 times, 5 minutes each time, to remove residual DAPI.

[0146] Mounting

[0147] Take a clean glass slide, add 1-2 drops of anti-fluorescence quenching mounting medium (Thermo Fisher #P36961, ProLong TM Diamond) in the center; carefully take out the coverslips in the 6-well plate with sterile forceps (if a coverslip is not used, directly transfer the cell crawl sheet to the glass slide with a pipette), with the cell surface facing down, slowly cover it on the mounting medium, to avoid air bubbles; use sterile filter paper to absorb the excess mounting medium on the edge of the coverslip, and air dry it at room temperature for 10 minutes.

[0148] Observation imaging

[0149] After mounting, the slides were observed under a Zeiss LSM980 laser confocal microscope, and the corresponding fluorescence channels (FITC: 488 nm excitation wavelength, DAPI: 350 nm excitation wavelength) were selected to observe and collect the CD235 and CD43 specific fluorescence signals and the nucleus staining signals in the PMP cells. The cell morphology and marker expression localization were recorded, and the experimental results were obtained. The results showed that the initial hiPSC inoculation amount was 3 x 10^6; within 3 months of culture, the cumulative yield of hPMPs reached about 1.2 x 10^8, which was about 40 times the number of initial hiPSCs. The identification was performed by immunofluorescence staining detection, and the CD235+ / CD43+ double positive rate was ≥ 85%.

[0150] III. Induction of brain organoids containing microglia

[0151] Stage 1: AggreWell TM 800-well fusion culture (days 21-24)

[0152] 1. NPC digestion: Take the NPCs cultured for 21 days, discard the old culture medium, and wash once with 1 mL of DPBS; add 1 mL of 37°C preheated Accutase digestion solution to each well, and place it in the incubator for 5 min of digestion. Microscopy showed that the intercellular space was enlarged and the adhesion was loose.

[0153] 2. MO-1 induction medium preparation: Take PMP medium and STEMdiff TM Neural precursor cell medium, prepared according to a volume ratio of 1:1, gently mixed and filtered with a 0.22 μm filter to remove bacteria, and ready for use.

[0154] 3. Termination and resuspension: Add 2 mL of DMEM-F12 medium to terminate digestion, and gently blow 8-10 times with a 1 mL syringe to prepare a single cell suspension; transfer to a 15 mL centrifuge tube, centrifuge at 1500 rpm at room temperature for 5 min, discard the supernatant and resuspend with 1 mL of MO-1 medium, count the cells and adjust the concentration to 2.1 x 10 6 cells / mL.

[0155] 4. PMP suspension preparation: Take the PMPs in the logarithmic growth phase, centrifuge at 800 rpm for 3 min at room temperature, discard the supernatant and resuspend with MO-1 medium, adjust the concentration to 0.9 x 10 6 cells / mL.

[0156] 5. Cell ratio mixing: take 1 mL NPC single cell suspension (2.1 million cells) and 1 mL PMP suspension (0.9 million cells) at a ratio of NPC:PMP = 7:3, mix gently for 3 times, and obtain 1 mL of fusion cell suspension (total cell number 3 x 10 6

[0157] 6. Culture plate pretreatment: take AggreWell TM 800 24-well plate, add 1 mL anti-adhesion rinse solution to each well, incubate at room temperature for 10 min, and discard after aspiration.

[0158] 7. Inoculation and centrifugation: add 1 mL of fusion cell suspension (containing 3 million mixed cells) to the pretreated well to avoid air bubbles; place the plate in a centrifuge equipped with an adapter, centrifuge at 100 x g (about 450 rpm) at room temperature for 3 min to promote cell aggregation.

[0159] 8. Fusion culture and medium change: after centrifugation, place in the incubator for continuous culture for 5 days, perform full medium change daily: gently aspirate the old culture medium in the well with a wide-bore gun head (avoid touching the organoid rudiment), slowly add 1 mL of fresh MO-1 medium along the well wall, and on the 25th day, 200-250 μm in diameter, dense in shape, brain-like organoid rudiments can be seen.

[0160] Stage 2: Ultra-low adsorption 96-well plate expansion culture (25-30 days)

[0161] 1. Brain-like organ transfer: gently aspirate the brain-like organ on the 25th day with a wide-bore gun head, transfer to a 15 mL centrifuge tube, centrifuge at 800 rpm at room temperature for 3 min, and discard the supernatant.

[0162] 2. Inoculation and centrifugation: take an ultra-low adsorption 96-well plate, add 200 μL of fresh MO-1 medium to each well, inoculate at a density of 5-8 brain-like organs per well; place the plate in a centrifuge, centrifuge at 100 rpm at room temperature for 3 min to make the brain-like organs sink to the bottom of the well.

[0163] 3. Culture and medium change: place in the incubator for 5 days, use the every-other-day full medium change method: aspirate 150 μL of old medium in the well, add 150 μL of fresh MO-1 medium, avoid drying of the brain-like organ; on the 30th day, microscopic examination shows that the diameter of the brain-like organ increases to 300-350 μm, with clear boundaries.

[0164] Stage 3: Matrigel embedding and shaking culture (31-40 days)

[0165] 1. Matrigel preparation: low-factor matrigel is thawed in advance at 4°C overnight, and is operated on ice to avoid solidification; take 20 μL of low-factor matrigel and drop it into a sterile culture dish for standby.

[0166] ​2. Brain organoid embedding: Take out the 30-day-old brain organoids with a wide-bore gun head, gently put them into a low-factor Matrigel drop, and make sure that each gel drop contains one brain organoid; place it in the incubator at 37°C for 30 min, and wait for the low-factor Matrigel to completely solidify (transparent gel state).

[0167] 3. Shaking culture setup: Take an ultra-low-attachment 6-well plate, and add 3 mL of MO-1 medium to each well; gently transfer the solidified low-factor Matrigel-brain organoid complex to the well, and place it in a constant-temperature shaker (60 rpm) in the incubator for culture.

[0168] 4. Medium replacement operation: Perform semi-replacement every 4 days: discard 1.5 mL of old medium in the well, slowly add 1.5 mL of fresh MO-1 medium along the well wall, and the diameter of the brain organoid can reach 400-450 μm on day 40, and fine nerve sprouts can be observed on the surface.

[0169] Stage 4: Microglia induction and maturation culture (days 41-55)

[0170] 1. Preparation of maturation medium: Take BrainPhys TM neuron medium, add IL-34 (100 ng / mL) and GM-CSF (10 ng / mL) at the final concentration, gently mix, filter-sterilize with a 0.22-μm filter membrane, and use it immediately after preparation.

[0171] 2. Medium replacement: Discard the old MO-1 medium in the 6-well plate, gently wash it once with 2 mL of DPBS, add 3 mL of maturation medium to each well, and continue shaking culture.

[0172] 3. Medium replacement and culture: Perform full replacement every 4 days: carefully aspirate the old medium with a wide-bore gun head (avoid touching the Matrigel complex), and add 3 mL of fresh maturation medium; continue culture until day 55 to obtain brain organoids containing microglia.

[0173] Stage 5: Extended culture (after day 55)

[0174] Continue to culture the brain organoids on day 55 with BrainPhys TM neuron medium (maturation medium) containing IL-34 (50 ng / mL) and GM-CSF (5 ng / mL), and perform full replacement every 6 days; extended culture for 2-4 weeks can significantly improve the maturity of the brain organoids, which is manifested in a more dense network of nerve sprouts and enhanced expression of synaptic markers.

[0175] The organoids tend to mature around day 55, and the specific sampling time depends on the experimental requirements. The sampling time in the early development stage is about 35 days, and the sampling time in the maturation stage is day 55 and later. The changes in the morphology and size of the final organoids are shown in Figure 1 .

[0176] The obtained brain organoids are subjected to immunofluorescence staining detection, and the specific steps are as follows:

[0177] I. Frozen section

[0178] Sample pretreatment: Take the brain organoids cultured to the target stage (diameter 500-1000 μm), gently rinse with DPBS buffer (Gibco #14190-144) for 3 times, 5 minutes each time (room temperature, low speed shaking of shaking table 50 rpm), and completely remove the residual medium.

[0179] Sample fixation: Transfer the brain organoids to a centrifuge tube containing 4% paraformaldehyde fixing solution (paraformaldehyde powder Sigma-Aldrich #P6148, pH 7.2-7.4), the volume of the fixing solution is more than 10 times the volume of the sample; stand still in the refrigerator at 4°C for 24 hours, gently invert the centrifuge tube every 6 hours during the period to ensure that the fixing solution penetrates fully; after fixation, wash with DPBS buffer for 3 times, 10 minutes each time, to remove the residual fixing solution.

[0180] Trypan blue staining: Dilute 0.4% trypan blue solution (Sigma-Aldrich #T8154) by 30 times to prepare trypan blue staining solution; transfer the brain organoids to a 6-well cell culture plate with a wide-bore gun head, add appropriate amount of trypan blue staining solution (ensure complete coverage of the sample); place the culture plate on a horizontal shaking table, 80 rpm room temperature staining for 10 minutes; aspirate the trypan blue staining solution, wash with DPBS buffer for 3 times, 5 minutes each time.

[0181] Embedding agent preparation: Mix OCT embedding agent (Sakura #4583) and 20% sucrose solution (sucrose Sigma-Aldrich #S9378) at a volume ratio of 2:1, mix thoroughly to prepare brain organoid special embedding agent.

[0182] Sample embedding: Take a sterile embedding mold, add 200-300 μL of prepared embedding agent to the bottom of the mold, carefully transfer the brain organoids to the center of the embedding mold with a wide-bore gun head, and then add embedding agent to completely cover the sample; quickly place the embedding mold in liquid nitrogen for quick freezing for 5-10 minutes until the embedding agent completely solidifies.

[0183] Frozen section: Take the solidified embedding block from the mold, install it on the sample stage of the frozen section machine (Leica CM1950), set the section thickness to 15-20 μm, and perform continuous sectioning; gently adhere the cut brain organoid sections to the polylysine-coated glass slides (Fisher Scientific #12-550-15) and dry at room temperature for standby.

[0184] II. Immunofluorescence staining

[0185] Baking: Place the glass slide with the brain organotypic slice attached onto a slide baker and bake at 60°C for 1 hour to enhance the adhesion of the slice to the glass slide.

[0186] Circle: Use a hydrophobic pen (Vector Laboratories #H-4000) to circle the brain organotypic slice on the glass slide to define the staining area and avoid reagent loss.

[0187] Secondary fixation: Add 1 mL of 4% paraformaldehyde fixative (Sigma-Aldrich #P6148) to the circled area and let it sit at room temperature for 20 minutes for fixation; aspirate the fixative and wash with DPBS buffer (Gibco #14190-144) for 3 times, 5 minutes each.

[0188] Cell membrane permeation: Add 1 mL of 1% Triton X-100 permeabilization solution (Thermo Fisher #HFH10) to the circled area and incubate at room temperature for 15 minutes to make micropores on the cell membrane for antibody penetration; aspirate the permeabilization solution and wash with DPBS buffer for 3 times, 5 minutes each.

[0189] Blocking: Aspirate the DPBS solution and add 100-150 μL of 5% BSA blocking solution (Bovine Serum Albumin Sigma-Aldrich #A7906, prepared in DPBS) to the surface of the slice to ensure complete coverage; place the glass slide into a humidified chamber and block at room temperature for 1 hour to block non-specific binding sites.

[0190] Primary antibody incubation: Dilute brain organotypic specific primary antibodies to 1:200 (recommended primary antibodies: CD11b / ITGAM (E3J2F) Rabbit Monoclonal Antibody CST #48893, CD45 (D3F8Q) Rabbit Monoclonal Antibody CST #70257, TMEM119 (E4B9S) Mouse Monoclonal Antibody CST #98778, Synapsin-1 (D12G5) Rabbit Monoclonal Antibody CST #5297, beta3-Tubulin (D71G9) Rabbit Monoclonal Antibody CST #5568) with 1% BSA + 0.3% Triton X-100 mix (BSA #A7906, Triton X-100 #HFH10); aspirate blocking solution, do not wash, directly add 200 pL of diluted primary antibody solution to the surface of the sections, ensuring even coverage of the sections; place the humidified chamber in the 4°C refrigerator for overnight incubation (12-16 hours); the next day, remove the humidified chamber, allow to warm to room temperature for 30 minutes, then wash 3 times with DPBS buffer for 10 minutes each, thoroughly removing unbound free primary antibody.

[0191] Secondary antibody and nuclear staining: Dilute fluorescent secondary antibodies and DAPI nuclear stain to 1:400 (recommended secondary antibodies: Anti-rabbit IgG (Alexa Fluor® 488 Conjugate) CST #4412, Anti-mouse IgG (Alexa Fluor® 594 Conjugate) CST #8890; DAPI CST #4083) with 1% BSA + 0.3% Triton X-100 mix; add 200 pL of diluted secondary antibody and DAPI mix to the surface of the sections, place in the humidified chamber, and incubate at room temperature for 1 hour in the dark; aspirate the mix, wash 3 times with DPBS buffer for 10 minutes each in the dark.

[0192] Mounting: Take a clean coverslip, add 1-2 drops of antifluorescence quenching mounting medium (Thermo Fisher #P36961) in the center; carefully pick up the coverslip with forceps, cell side down, slowly place over the sections, avoiding air bubbles; seal the edges of the coverslip with nail polish (Sigma-Aldrich #Z665850); store at 4°C in the dark.

[0193] Observation of imaging: using Zeiss LSM980 laser confocal microscope, selecting the corresponding fluorescence channel (FITC: 488 nm, Cy3 / 594: 550 nm, DAPI: 350 nm) for observation, collecting the fluorescence signal image of the section and recording the results. The experimental results obtained are shown in Figures 2-5 .

[0194] Figure 2 To induce the brain-like organ at day 35 (15 days after fusion), which is at the initial stage of neurogenesis, the distribution of progenitor cells in the microglia-containing human brain organoid was identified by immunofluorescence. NESTIN is a specific marker for neural progenitor cells (NPCs), and CD235 is a specific marker for macrophage progenitor cells (PMPs). It can be observed that both types of progenitor cells are uniformly distributed within the organoid, indicating that the model can simulate the development process of the neural and macrophage lineages and can continuously observe the entire process of neural development, which is not available in other models.

[0195] Figure 3 , Figure 4 , Figure 5 To induce the brain-like organ at day 55 (35 days after fusion), Figure 3 is the result of identifying microglia in human brain organoids by immunofluorescence. TMETM119 is a specific marker for microglia, and CD11b is a marker for the macrophage lineage. At this stage, which is in the middle and late stages of neural development, most macrophage lineage cells have begun or have differentiated into microglia. Therefore, it can be observed that TMEM119, as a specific marker for mature microglia, is uniformly distributed in the organoid. CD11b, as a marker for the macrophage lineage, can label all cells that develop and differentiate from PMPs. By comparing the ratio of TMEM119 and CD11b, it can be observed that some mature microglia have appeared 35 days after fusion, and some macrophage lineage cells are still in the development stage. This indicates that the model has the ability to induce mature microglia and can simulate the entire development process from macrophage progenitor cells to mature microglia in the model.

[0196] Figure 4 is the result of immunofluorescence to identify neurons in microglia-containing human brain organoids. TUJ1 is a specific marker for mature neurons, and SOX2 is a marker for neural progenitor cells. As with the mature microglia obtained, mature neurons are also labeled in the model at this stage, and the results are similar to those of the macrophage lineage. Some neural progenitor cells have differentiated into mature neurons TUJ1, and a large number of progenitor cells are still in the differentiation stage, and the overall distribution is consistent with the distribution characteristics of the cerebral cortex, with peripheral distribution of neurons and distribution of neural progenitor cells within the neurons.

[0197] Figure 5is the result of co-localization of microglia and presynaptic membrane in microglia-containing human brain organoids identified by immunofluorescence. The core way of microglia to participate in the process of shaping neural circuits is to prune synapses, identify redundant synapses, and guide the pruning process. SYNAPSIN I is used to locate the presynaptic membrane of neurons, and the co-localization of the presynaptic membrane and CD11b microglia is observed. As can be seen from the figure, there are a large number of green and red co-stained areas (orange areas), which indicates that the presynaptic membrane and CD11b microglia have obvious co-localization, thereby confirming that microglia in the organoid play an important function of pruning synapses, and it is proved that the microglia-containing human brain organoids induced by the method contain mature microglia with function.

[0198] In summary, the above immunofluorescence results show that the microglia-containing human brain organoids induced by the method of the application not only can specifically reflect the development and differentiation process of neural lineage and macrophage lineage cells, but also have controllable proportions, and can obtain mature and normal microglia and neurons in morphology and function, and can be used as a model that conforms to the normal human brain for subsequent experiments.

[0199] In general, the model has a relatively uniform mixed distribution pattern of NPC and PMP cells at about 35 days in the early stage, as shown in Figure 2 It can be seen that the two cell types show a spatial distribution characteristic similar to that of neurons in the cortex of the human brain, with progenitor cells located in the interior, as shown in Figure 4 Using the co-localization of the microglia-specific marker CD11b and the presynaptic membrane-specific marker SYNAPSIN I, it can be known that the model not only has cell types similar to the development stage of the human brain, but also has similar spatial distribution and function to the real human brain, as shown in Figure 5 .

[0200] The obtained brain organoids are verified whether they can simulate the response of the real human brain to external stimuli during the development stage. Microglia, as the main immune cells in the brain, are also the only cell type in the brain with IL-6 membrane receptors. After stimulating the brain organoids with IL-6 at a concentration of 10 ng / mL for 10 days, the specific operation is as follows: select the brain organoids cultured for 55 days, add IL-6 at a concentration of 10 ng / mL in the mature culture medium, and continuously culture for 10 days, change the liquid every day, and collect the brain organoids at the 10th day (65 days). The sampled brain organoids are subjected to immunofluorescence detection, and the steps of immunofluorescence detection are the same as above. At the same time, ImageJ software is used to analyze and detect the function and cell type changes of the model. The results are as shown in Figure 6Synapsin I / CD11b represents the co-localization of the two, indicating that a part of the synapses of neurons are phagocytosed into microglia, indicating that microglia are performing phagocytosis. The ratio of Synapsin I / CD11b indicates the proportion of co-localization of the two. It can be seen from the results that the brain organoids obtained in Example 1 can respond to IL-6 stimulation, and at the same time can affect the main functions of microglia at the development stage under the stimulation, and in the first part of the results, CD43 is a specific marker of PMP cells, and SOX2 is a specific marker of NPC cells, which is an indicator of progenitor cells in the early stage of embryoid body formation. It can be seen that the brain organoid system constructed by the present application can explore the interaction and function between cells at the early stage of the fusion of the two kinds of progenitor cells, which is not possessed by other models. Secondly, Synapsin I marks the presynaptic membrane, and CD11b is a specific marker of microglia. The co-staining of the two markers represents that microglia are performing phagocytosis. The results show that microglia can accept external stimulation signals and change their phagocytosis function accordingly. This stress function is also unique to the brain organoids obtained by the present application. Finally, Ki67 as a broad-spectrum proliferation-related marker, it can be seen that the entire system of the present application will be affected by this event after the microglia cell type receives external stimulation, changing the proliferation state of the entire system. This result also shows that the present application is not a simple cell type fusion body, but a neural development system containing microglia with complex interactive functions.

[0201] In summary, the brain organoids of the present application can realize the functions including: exploring the control of microglia on the proportion of progenitor cells at the development stage, the change of phagocytosis function, the control of the number of mature neurons, the influence on cell proliferation and apoptosis, and can accept external stimulation and make corresponding functional stress changes.

[0202] Example 2

[0203] Two strains of iPS (ACS1011, purchased from ATCC cell bank; T-iPSC, refer to prior art Kuang H, Li Y, Wang Y, et al. A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder [J]. Cell Reports, 2023, 42(12):25. DOI:10.1016 / j.celrep.2023.113445) of different sources were used for experiments, and two brain organoids were obtained according to the method of Example 1.

[0204] Flow cytometry detection of the obtained brain organoids, the specific steps are as follows:

[0205] Single cell suspension preparation: take the brain organoids cultured to the target stage, transfer to a 15 mL centrifuge tube under sterile conditions; add papain-Accutase mixed digestion solution (papain Worthington #LS003119, Accutase Thermo Fisher #A1110501, volume ratio 1:1), the volume of the digestion solution is more than 5 times the volume of the organoids; place in a 37°C, 5% CO2 incubator for 45 minutes, during which every 10 minutes, take out the centrifuge tube and gently crush the organoids with a sterile disposable blade (avoid excessive force to damage the cells), observe the digestion state under a light microscope until most of the tissue is dispersed into single cells.

[0206] Filter purification: prepare a 0.45 μm sterile cell screen (Corning #352340) in advance and place it in a new 15 mL centrifuge tube; filter the digested cell suspension slowly through the cell screen, rinse the screen with 1 mL of PBS buffer (Gibco #14190-144) twice, and collect the filtered single cell suspension to remove undigested tissue fragments.

[0207] Cell counting and concentration adjustment: mix 20 μL of single cell suspension with 20 μL of trypan blue dye (Sigma-Aldrich #T8154, 0.4% concentration) and let it stand at room temperature for 5 minutes; use a hemocytometer or an automatic cell counter (ThermoFisher #C10227) to count the number of viable cells (trypan blue-excluded cells), and adjust the cell concentration to 1×10 6 -5×10 6 cells / mL according to the counting results.

[0208] Sample grouping and sample loading: take sterile flow tubes (BD #352052) and label them as experimental group, same type control group, and blank control group (only PBS), with 3 replicates for each group; add 100 μL of single cell suspension with adjusted concentration to each flow tube, and only add 100 μL of PBS buffer to the blank control group.

[0209] Blocking non-specific binding: add 5 μL of blocking solution (5% BSA solution, Sigma-Aldrich #A7906; or fetal bovine serum Gibco #10099-141) to each flow tube in the experimental group and the same type control group, and incubate at room temperature for 15 minutes in the dark to block the non-specific binding sites on the cell surface.

[0210] Fluorescent antibody incubation: Add fluorescently labeled specific antibodies to the experimental group flow tubes according to the recommended ratio of the antibody instructions (recommended antibody combination and parameters are as follows); the same type of control group adds the same type of control antibody of the corresponding species, and the blank control group does not add antibodies. The antibody name and model number are as follows:

[0211] IBA1-PE (IBA1(E4O4W) Rabbit Monoclonal Antibody, PE Conjugate BioLegend #690504, 2 μL / tube);

[0212] TMEM119-FITC (TMEM119(E4B9S) Mouse Monoclonal Antibody, FITC Conjugate CST #98778-FITC, 3 μL / tube);

[0213] CD45-APC (CD45(D3F8Q) Rabbit Monoclonal Antibody, APC Conjugate CST #70257-APC, 2 μL / tube);

[0214] CD11b-PerCP-Cy5.5 (CD11b(M1 / 70) Mouse Monoclonal Antibody, PerCP-Cy5.5 Conjugate BioLegend #101228, 2 μL / tube).

[0215] Gently mix with vortex oscillator (speed 500 rpm, time 5 seconds), incubate at 4°C in the dark for 30 minutes, avoid fluorescence antibody quenching and non-specific binding.

[0216] Wash to remove free antibodies: After incubation, add 2 mL of PBS buffer to each flow tube, centrifuge at 1000 rpm for 5 minutes (room temperature), carefully discard the supernatant; repeat the washing 2 times to completely remove the unbound free antibodies.

[0217] Flow cytometry detection and data analysis: Open the flow cytometry data acquisition software, establish the detection scheme, run the blank control group first, adjust the threshold to exclude debris and noise signals, set the door with FSC (forward scattering light) vs SSC (side scattering light), and select the target cell population.

[0218] Load the control group and experimental group samples in turn, collect 1×10^4-1×10^5 target cell events per tube, and record the fluorescence signal intensity data.

[0219] During the detection process, run a blank control every 10 samples to calibrate instrument drift.

[0220] Import raw data with flow analysis software (e.g. FlowJo, BD FACSDiva) and pre-process data.

[0221] Analyze the difference between groups by statistical methods (e.g. independent sample t test)

[0222] Generate flow scatter plot, histogram and statistical analysis report to get the results of brain organoid flow cytometry detection, such as shown in Figure 7

[0223] The results show the flow cytometry detection results of the microglia-containing brain organoids at day 70. Here, two strains of iPSCs of healthy Chinese adult males were used for the experiment, and IBA1 was used to sort microglia, and the proportion of microglia IBA1+ obtained was 10.60% and 9.72% respectively. This result is consistent with the proportion of microglia in the real human brain, and the batch difference is very small, and the repeatability is extremely high. This feature is not possessed by other existing models.

[0224] Comparative Example 1

[0225] ​Referring to prior art CN15404218B, two small molecules are additionally added as an induction method of adding SMADi to induce cerebral organoids containing microglia. The specific process is as follows: the EB formation stage is generally the first 6 days of the culture process. Cells seeded into a low-adsorption 96-well plate will aggregate to form EB balls after 24 hours, and this is recorded as the first day. In the first three days of EB culture, "Cerebral organoids EB formation medium 1" (first culture medium) is used; in the 3-6 days of EB culture, the culture medium is changed to "Cerebral organoids EB formation medium 2" (second culture medium). After that, the EB balls are cultured to the 6th day, and then the culture medium is changed to the sixth culture medium. After the EB balls are formed, they are cultured to the 8th day, and the culture medium is changed to neural induction medium (NIM) (third culture medium). After the EB balls are cultured through the neural induction culture stage, the culture medium is changed to neural differentiation medium (NDM) (fourth culture medium) at the 12th day. After the EB balls are cultured through the neural differentiation culture stage, the culture medium is changed to neural maturation medium (NMM) (fifth culture medium) at the 30th day. Finally, an EB of a 96-well plate is obtained at the 60th day after the six culture media are changed after the EB is formed, and a microglia organoid containing IBA1 and CD11b positive microglia is obtained.

[0226] Compared with the method, the present application introduces a large number of lineage-purified macrophage progenitor cells at the initial stage of neural induction (day 0) through optimization of experimental methods, and about 300 EBs containing IBA1, CD11b, TMEM119 and CD45 four specific markers of microglia cells are obtained in about 45-55 days. Therefore, the present application uses fewer culture media types and obtains higher organoid yield.

[0227] Meanwhile, the iPSC differentiation of Comparative Example 1 is not controlled, the cell types in the middle stage cannot be controlled, and is in a free differentiation process, therefore, it cannot be determined that the microglia progenitor cells are generated from which specific stage. The brain organoids of Comparative Example 1 are stimulated by the same interleukin 6 as in Example 1, and the results show that no difference is detected for CD235 and SOX2, that is, the microglia cells cannot respond to the interleukin 6 stimulation. However, in Example 1 of the present application, the cell types at each stage are determined, and the ratio change process of the PMP and NPC two progenitor cells is also clear, so that the generation stage of the microglia progenitor cells can be accurately determined, and sampling research at each stage is facilitated.

[0228] Comparative Example 2

[0229] According to the culture scheme of the prior art CN113924362B, the mature organoids and macrophages are fused again on the 26th day of induction, that is, the scheme of adding free microglia progenitor cells to the mature organoids, and the specific steps are as follows: brain organoids and primitive macrophages (iMac) are generated from the same human iPSC. Microglia cells are observed in human fetal brain at 4.5 weeks. The iMac is co-cultured with the relatively young brain organoids (26 days) which simulate the early pregnancy stage fetal brain.

[0230] The results show that the infiltration scheme cannot simulate the influence of microglia cells and their progenitor cells on this process of the differentiation of neural progenitor cells into neurons, and misses the early stage of real human brain development. Because in Comparative Example 2, the organoid balling and the introduction of microglia cells are performed in sequence, and there is no overlap in time and space, therefore, the influence of microglia cells on this process cannot be achieved during the organoid balling process. Meanwhile, the immunofluorescence detection results of the example show that the microglia cells clearly have an influence on the ratio change of the two early cell types after being stimulated by the outside world, Figure 6 CD43 is a specific marker of PMP cells, and SOX2 is a specific marker of NPC cells, and both show significant differences. However, the brain organoids of Comparative Example 2 are stimulated by the same interleukin 6 as in Example 1, and the results show that no difference is detected for CD43 and SOX2 because the microglia cells do not participate in the early balling process.

[0231] Meanwhile, the proportion of microglia cells in the finally obtained brain organoids is detected by flow cytometry, and the detection steps are the same as in Example 1. The detected results show that it is less than 5%, which does not meet the proportion of microglia cells in the real human brain.

[0232] Comparative Example 3

[0233] The medium used in Comparative Example 2 was replaced with the medium of the present application, and at day 5-6, the induction medium consisting of DMEM:F12, IX N2 supplement (Invitrogen), 10 pg / mL heparin (Sigma), IX penicillin / streptomycin, IX non-essential amino acids, IX Glutamax, 4 ng / mL WNT-3A (R&D Systems), 1 mM CHIR99021 (Cellagentech), and 1 mM SB-431542 (Cellagentech) was replaced with the neural progenitor maturation medium of the present application, and the co-culture (at day 26) was performed using the organoid growth medium supplemented with 100 ng / mL CSF-1 was replaced with the post-15-day maturation medium of the present application, and then the DAY55 organoids at the same time point were subjected to the relevant detection. The results showed that, although the medium was replaced, due to the fusion mode of the microglial precursor cells and the difference in the fusion time, the proportion of microglial cells was still much lower than 8%. At the same time, the staining results of the microglial cells showed that the co-localization proportion with the neuronal cells was about 5%, which was lower than the model prepared in the embodiment of the present application, indicating that the phagocytosis function of the microglial cells in the comparative example was poor based on the interaction with other cell types.

[0234] Comparative Example 4

[0235] According to the scheme of the prior art CN18389433A, the medium of the prior art CN18389433A was used, and at day 30, the mature brain-like organoids were co-cultured with free mature microglial cells, and finally IBA1-positive brain organoids containing microglial cells were obtained. The specific operation steps are as follows: (1) using pluripotent stem cells, CD43+ microglial precursor cells were induced and differentiated by using medium I, medium II, medium III, and medium VIII in turn; (2) using pluripotent stem cells, brain-like bodies were induced and differentiated by using medium IV, medium V, medium VI, and medium VII in turn; (3) the CD43+ microglial precursor cells and brain-like bodies obtained in steps (1) and (2) were co-cultured by using medium VIII, medium IX, and medium X in turn, and a neural immune brain-like body was obtained.

[0236] The proportion of microglial cells in the finally obtained neural immune brain-like body was detected by flow cytometry, and the detection steps were the same as in Example 1. The detected proportion of microglial cells was 2.39%, which did not conform to the proportion of microglial cells in the real human brain.

[0237] Meanwhile, compared with Comparative Examples 1-4, only the cell type identification of microglia cells is explored, and the model is not further proved to have the immune response function of the real human brain. Moreover, the experimental design of the comparative examples cannot explore the influence of microglia cells on the entire organoid sphere formation process at any stage of development, especially at the early stage. In Example 1, since the two kinds of germ layer cells are mixed before sphere formation, this process also involves microglia cells and their progenitor cells. At the same time, Example 1 realizes the response to immune changes at the system level on the in vitro human brain organoid model, and the final result is that IL-6 as a common immune factor directly acts on microglia cells and significantly affects the function of microglia cells, leading to a significant down-regulation of the proportion of progenitor cells in the immune change group, down-regulation of microglia cell phagocytosis, and impaired cell proliferation. Compared with other comparative examples, Example 1 has a more perfect immune response system and cell-to-cell functional interaction, and is a more ideal immune human brain organoid model.

[0238] Comparative Example 5

[0239] Optimization of culture medium

[0240] The components of the NPC culture medium in MO-1 culture medium are replaced with the following components before the induction of the brain organoid containing microglia cells, i.e., within 21-40 days of Example 1:

[0241] Neurobasal medium (Thermo Fisher Scientific) and DMEM / F12 medium are mixed at a mass ratio of 1:1, and small molecules 1xN2, 1x B27-RA (Thermo Fisher Scientific), FGF2 (20 ng / ml, Peprotech) are added.

[0242] Other than Example 1. The 3D human brain organoids cultured by the above method to the 55th day were identified by immunofluorescence, and the positive proportion of their specific marker NESTIN was about 60%, and the proportion of their broad-spectrum proliferation marker Ki67 was about 40%, which were both significantly lower than the results of Example 1. At the same time, the brain organoid yield was about 15 or so per batch (per 96-well culture plate), and the growth diameter of the brain organoids at the 45th day was 80-100 μm.

[0243] Comparative Example 6

[0244] Optimization of culture medium

[0245] In the post-induction phase of brain organoids containing microglia, i.e., after day 41 of Example 1, the maturation medium was replaced with a mixture of Neurobasal medium and DMEM / F12 medium at a mass ratio of 1:1, and small molecules were added: 1×N2 (Thermo Fisher Scientific), BDNF (20 ng / ml, Peprotech), GDNF (20 ng / ml, Peprotech), dibutyryl-cyclic AMP (1 mM, Sigma), ascorbic acid (200 nM, Sigma), IL-34 (100 ng / ml, Peprotech), and Granulocyte macrophage colony-stimulating factor (GM-CSF, 10 ng / ml, Peprotech).

[0246] Everything else is the same as in Example 1. Using the above method, 3D human brain organoids were cultured for 55 days, with a yield of approximately 15 organoids per batch (each 96-well culture plate). In terms of electrophysiology and microglia function, the organoids constructed in Comparative Example 6 exhibited weaker spontaneous firing ability. Simultaneously, the microglia function in synaptic phagocytosis and enhancing neuronal firing synchronization was significantly weaker than in Example 1. The growth efficiency of the brain organoids was reduced; the diameter of the brain organoids cultured for 45 days was 80-100 μm, while the diameter of the brain organoids cultured in Example 1 for the same number of days was 450-500 μm.

[0247] This demonstrates that after adjusting the type of basal culture medium in Example 1, the number of small molecules was reduced, achieving model construction with as few small molecules as possible. Simultaneously, the model after adjusting the culture medium showed improved batch-to-batch consistency and brain organoid growth rate, and also exhibited better spontaneous firing ability. Furthermore, microglia demonstrated functional advantages in several aspects, including synaptic phagocytosis and enhanced neuronal firing synchronization.

[0248] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for culturing 3D neuroimmune organoids containing microglia, characterized in that, Includes the following steps: S1. Induced pluripotent stem cells were induced in the ectoderm direction and mesoderm direction respectively to obtain neural embryoids and yolk sac embryoids; S2. Culture neural embryoids to induce neural differentiation, obtain neural rosette structures, and then digest them to obtain neural progenitor single cells; culture yolk sac embryoids to produce macrophage progenitor cells; S3, fused with neural progenitor single cells and macrophage progenitor cells, and continued to be cultured to form 3D neuroimmune organoids containing microglia.

2. The cultivation method according to claim 1, characterized in that, Step S1, the step of inducing ectoderm orientation in induced pluripotent stem cells, includes: First, induced pluripotent stem cells are digested into single cells; neural induction medium is added to the single cells for induction, and they are cultured for 7-13 days to obtain neural embryos.

3. The cultivation method according to claim 2, characterized in that, The culture plates used in the culturing process are AggreWell. TM 800 culture plates; the neural induction medium was STEMdiff containing 0.01-0.02 mM Y-27632. TM Neural induction culture medium.

4. The cultivation method according to claim 1, characterized in that, Step S1, the step of inducing induced pluripotent stem cells to undergo mesoderm orientation, includes: Induced pluripotent stem cells were digested to obtain single cells; induction culture medium was added to the single cells for 1-2 days to obtain yolk sac embryoids; the induction culture medium included basal culture medium and additives; the additives included: bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and Y-27632; the basal culture medium was mTeSR. TM Plus medium.

5. The cultivation method according to claim 1, characterized in that, Step S2, the step of culturing neural embryoids to achieve neural differentiation, includes: Digest the neural embryoid to obtain a rosette structure; further expand and culture the rosette structure for 15-21 days to obtain neural progenitor cells; The reagent used for digesting neural embryoids was the neural rosette selection reagent; the medium used for further amplification of the rosette structure was STEMdiff. TM Neural induction culture medium.

6. The cultivation method according to claim 1, characterized in that, Step S2, the steps of culturing yolk sac embryos to induce neural differentiation, include: Macrophage progenitor cells were obtained by adding macrophage progenitor cell culture medium to yolk sac embryos and culturing for 5-21 days. The macrophage progenitor cell culture medium includes a basal medium and additives; the additives include interleukin-3 and macrophage colony-stimulating factor; the basal medium is X-VIVO 15 medium.

7. The cultivation method according to claim 1, characterized in that, In step S3, the fusion steps include: Neural progenitor cells were digested into single neural progenitor cells; the single neural progenitor cells and macrophage progenitor cells were fused in fusion medium for 1-5 days.

8. The cultivation method according to claim 7, characterized in that, The fusion medium contains macrophage progenitor cell culture medium and STEMdiff™ neural progenitor cell culture medium in a volume ratio of 1-2:1-2; neural progenitor cells and macrophage progenitor cells are fused at a ratio of 5-9:

3.

9. The cultivation method according to claim 1, characterized in that, In step S3, after culturing for another 25-30 days, the brain-like organoids are embedded and then induced to form 3D neuroimmune organoids containing microglia using a mature culture medium.

10. The cultivation method according to claim 9, characterized in that, The maturation culture medium includes a basal medium and additives; the additives include interleukin-34 and granulocyte-macrophage colony-stimulating factor; the basal medium is BrainPhys TM Neuronal Medium.

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