A method for culturing a 3d neuro-immune organoid containing microglia

Abstract

The application belongs to the field of stem cell biology and relates to a culture method of a 3D nerve immune organoid containing microglia cells, which comprises the following steps: S1, inducing induced pluripotent stem cells in ectoderm direction and mesoderm direction respectively to obtain nerve-like embryoid bodies and yolk sac-like embryoid bodies; S2, culturing the nerve-like embryoid bodies to make them differentiate into nerves to obtain nerve ring structures, re-digesting the nerve ring structures to obtain nerve progenitor cell single cells, and culturing the yolk sac-like embryoid bodies to make them generate macrophage progenitor cells; and S3, fusing the nerve progenitor cell single cells and the macrophage progenitor cells and continuing to culture to form the 3D nerve immune organoid containing microglia cells. The brain-like organoid of the application can realize the following functions: exploring the control of microglia cells on the proportion of progenitor cells in the development stage, the change of phagocytosis, the control on the number of mature neurons, the influence on cell proliferation and apoptosis, and simultaneously accepting external stimulation and making corresponding functional stress changes.

Description

Technical Field

[0001] This invention belongs to the field of stem cell biology and relates to a method for culturing 3D neuroimmune organoids containing microglia. Background Technology

[0002] Organoids are three-dimensional micro-organ models constructed using the self-renewal and directed differentiation capabilities of stem cells. Their core value lies in their ability to reproduce the spatiotemporal dynamics of human organ development and the complex intercellular interaction networks in vitro. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have become ideal tools for constructing organoid models due to their unique self-renewal capabilities and strong differentiation potential. By inhibiting bone morphogenetic protein (BMP) and transforming growth factor-β / NODAL signaling pathways to varying degrees (i.e., the "dual SMAD signaling pathway inhibition" strategy), hPSCs can be guided to differentiate into various neural cell types, such as neural stem cells and cortical pyramidal neurons. Currently, 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 deficiency in cellular composition: the lack of microglia, a key regulator of the neuroimmune microenvironment. As the only resident immune cells in the central nervous system, microglia play a role far beyond being immune sentinels; they are actually the multidimensional regulatory center of the nervous system. Recent single-cell sequencing studies have revealed at least 12 functional subtypes, each playing a core role in neural development. First, they are the dynamic architects of neural development, shaping neural circuits from the embryonic stage to early postnatal life. In terms of synaptic pruning, they use a "tag-eating" mechanism to identify redundant synapses and guide the pruning process. In regulating neurogenesis, microglia maintain the stemness of neural progenitor cells in the ventricular region through TGF-β signaling and stimulate the proliferation of neighboring cells by phagocytosing sphingosine-1-phosphate (S1P) released by apoptotic cells. In the cortical laminae region, they even form "cellular bridges" and secrete matrix metalloproteinase 9 (MMP9) to degrade the extracellular matrix, providing navigation for the precise laminar localization of neurons. Second, mature microglia continuously monitor the environment to maintain functional homeostasis. The absence of microglia leads to significant distortions in organoids when mimicking core physiological or pathological processes highly dependent on neuro-immune interactions, such as neuroinflammation and synaptic remodeling. Furthermore, statistics from 2024 show that over 80% of brain disease-related drug development failures stem from the inability of preclinical models to effectively predict neuroimmune interactions. Therefore, constructing brain models incorporating functional neuroimmune units has become crucial.

[0003] Currently, there are two main technical approaches to constructing organoids containing microglia: 1. Late-stage implantation: Microglia induced in vitro (e.g., differentiated from iPSCs or primary cells) are injected into pre-matured brain organoids. The drawbacks of this method are: the dense extracellular matrix in mature organoids severely hinders the migration of implanted cells, resulting in very low implantation efficiency; implanted microglia often highly express pro-inflammatory markers (such as CD68) while expressing low homeostatic markers (such as TMEM119 / P2RY12), exhibiting a "pathological activation" state that renders them inactive; and if the implantation time is inappropriate, the microglia miss the critical period of neurogenesis and cannot effectively participate in the synaptic pruning process. 2. Mixed differentiation method: In the early stages of iPSC neurogenesis, cytokines such as M-SCF and IL-34 are added to attempt to induce some stem cells to spontaneously differentiate into microglia-like cells. The drawbacks of this method are: the final proportion of microglia is usually less than 2%, and there are large differences between batches, making it difficult to control the proportion stably; the differentiation process of microglia often lags behind neurogenesis (electron microscopy shows that their synaptic phagocytic activity is only about 30% of the normal level in vivo); and because it is non-directional differentiation, it produces a large number of peripheral macrophages, which interfere with the accurate assessment of specific functions of the central nervous system.

[0004] For example, Park, D.S. et al. disclosed fusing mature neural embryoids with iMacs on day 26 to form 3D human brain organoids containing microglia. While this method can obtain mature microglia with some function, the developmental stage of the iMac during the fusion operation does not correspond to the process in the real human brain where macrophage progenitor cells migrate into the neural tube and gradually develop into microglia. Furthermore, the ratio of neural progenitor cells to microglia-like cells is difficult to precisely control during the fusion process; although the amount of iMac fused can be adjusted, the cell count of neural embryoids is difficult to accurately estimate due to batch and individual variations. This results in a significant difference between the proportion of microglia in the final brain organoid and the 5%-10% found 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:

[0010] S1. Induced pluripotent stem cells were induced in the ectoderm direction and mesoderm direction respectively to obtain neural embryoids and yolk sac embryoids;

[0011] 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;

[0012] S3, fused with neural progenitor single cells and macrophage progenitor cells, and continued to be cultured to form 3D neuroimmune organoids containing microglia.

[0013] The unique role of microglia in neural development is determined by their strict temporal window dependence and spatial specificity. During human embryonic development, macrophage progenitors originating from the yolk sac migrate into the neural tube via the bloodstream around week 4-5, and eventually differentiate into mature microglia under the induction of the local microenvironment. This specific developmental origin means that traditional organoid models containing only neural lineages cannot fundamentally generate fully functional microglia autonomously. Therefore, developing brain organoid models containing microglia that can mimic neural development and integrate neuroimmune functions is of paramount importance.

[0014] The key point of this invention lies in its recognition of the different developmental lineages of the neuroectoderm and mesoderm, and its precise control of cell ratios. This invention introduces lineage-purified macrophage progenitor cells at the initiation stage of neural induction (day 0), thereby reconstructing the necessary developmental dialogue between the neuroectoderm (neural progenitor cells) and mesoderm (macrophage progenitor cells) at the source, accurately simulating the core event of "neuro-immune co-development" during the embryonic period. Simultaneously, by precisely setting the initial inoculation ratio of neural progenitor cells to macrophage progenitor cells, it ensures that the proportion of microglia in the final organoid reaches a stable 5%-10% of the real human brain. This optimized approach more realistically simulates the interaction between neural progenitor cells and macrophage progenitor cells during neural development, the key regulatory functions of microglia, and the complete neuroimmune microenvironment, which is more consistent with the developmental patterns of the human brain. Studies have shown that the origin of microglia is that their precursor cells already exist in developing brain tissue before the formation of the neural plate, neural tube closure, and blood-brain barrier closure. Previous methods for inducing brain organoids containing microglia involved infiltrating mature microglia into brain organoids that had already developed for some time. This missed the crucial timeframes at which microglia themselves exerted their functions, including "progenitor cell pool control" and "influencing neuronal differentiation and maturation." Furthermore, this approach yields more controllable cell types that align with the developmental patterns and cell type characteristics of the human brain.

[0015] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0016] In one preferred embodiment, before step S1, mTeSR is first used. TM The induced pluripotent stem cells were pre-cultured in Plus medium for 3-4 days.

[0017] The criteria for stopping pre-culture are: cells cover 80%-90% of the bottom area of ​​a 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.

[0018] 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.

[0019] In one preferred embodiment, step S1, which involves inducing ectoderm orientation in induced pluripotent stem cells, includes:

[0020] 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.

[0021] In one preferred embodiment, the digestive fluid used for digestion is Accutase digestive fluid.

[0022] In one preferred embodiment, the culture process uses AggreWell culture plates. TM 800 culture plate.

[0023] 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.

[0024] In one preferred embodiment, the culture medium is changed periodically during the cultivation process; the replacement medium is STEMdiff. TM Neural induction culture medium.

[0025] In one preferred embodiment, step S1, which involves inducing the mesodermal orientation of induced pluripotent stem cells, includes:

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In one preferred embodiment, step S2, the step of culturing neural embryoids to achieve neural differentiation, includes:

[0030] Digest the neural embryoids to obtain a rosette structure; further expand and culture the rosette structure for 15-21 days to obtain neural progenitor cells.

[0031] In one preferred embodiment, the reagent used to digest the neural embryoid is a neural rosette selection reagent.

[0032] In one preferred embodiment, the neural rosette selection reagent is STEMdiff. TM Neural rosette selection reagent.

[0033] In one preferred embodiment, the rosette structure is further expanded using STEMdiff medium. TM Neural induction culture medium.

[0034] In one preferred embodiment, step S2, the step of culturing the yolk sac embryoid to induce neural differentiation, includes:

[0035] Macrophage progenitor cells were obtained by adding macrophage progenitor cell culture medium to yolk sac embryos and culturing for 5-21 days.

[0036] In one preferred embodiment, the macrophage progenitor cell culture medium includes a basal culture medium and additives; the additives include interleukin-3 and macrophage colony-stimulating factor; the basal culture medium is X-VIVO 15 medium.

[0037] 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.

[0038] In one preferred embodiment, step S3, the fusion step includes:

[0039] 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.

[0040] In one preferred embodiment, the fusion medium comprises macrophage progenitor cell culture medium and STEMdiff™ neural progenitor cell culture medium in a volume ratio of 1-2:1-2.

[0041] In one preferred embodiment, neural progenitor cells and macrophage progenitor cells are fused at a ratio of 5-9:3.

[0042] In one preferred embodiment, neural progenitor cells and macrophage progenitor cells are fused in a 7:3 ratio.

[0043] In one preferred embodiment, in step S3, after culturing for another 25-30 days, the brain-like organoid is embedded, and then induced to grow using a mature culture medium to form a 3D neuroimmune organoid containing microglia.

[0044] In one preferred embodiment, the culture medium used for continued culturing is a fusion medium.

[0045] In one preferred embodiment, a matrix adhesive is used for embedding.

[0046] In one preferred embodiment, the maturation culture medium comprises a basal culture medium and additives; the additives include interleukin-34 and granulocyte-macrophage colony-stimulating factor; the basal culture medium is BrainPhys TM Neuronal Medium.

[0047] In one preferred embodiment, the maturation culture medium contains 100-200 ng / ml interleukin-34 and 10-20 ng / ml granulocyte-macrophage colony-stimulating factor.

[0048] The following is an explanation of the principles of this invention:

[0049] During embryonic central nervous system (CNS) development, myeloid progenitor cells originating from the yolk sac migrate to the developing neural tube and interact with neural progenitor cells (NPCs). These cells proliferate together and eventually differentiate into various cell types that make up the nervous system, including neurons, astrocytes, oligodendrocytes, and microglia.

[0050] The core method of this invention begins with human induced pluripotent stem cells (hiPSCs). First, hiPSCs are induced towards the mesoderm using a culture 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). This promotes the differentiation of embryoid bodies (EBs) into hematopoietic progenitor cell lines with mesoderm characteristics and the potential to differentiate into primitive myeloid cells (i.e., yolk sac embryoid bodies, YS-EBs). On day 5 of differentiation, 100 ng / ml macrophage colony-stimulating factor (M-CSF) and 25 ng / ml interleukin-3 (IL-3) were added to induce YS-EB to produce myeloid progenitor cells and further differentiate into macrophage progenitor cells. These macrophage progenitor cells mimicked their behavior in neural development, were able to work synergistically with neural progenitor cells, and ultimately differentiated into microglia.

[0051] Simultaneously, another group of hiPSCs was used to form neural embryoid bodies. This process employed a neural induction medium (NIM) containing SMAD inhibitors and the neural inducing factor N2, effectively inhibiting mesoderm / endoderm differentiation and ensuring directional induction into the neural lineage. A key innovation of this invention lies in the use of a neural progenitor cell proliferation medium (STEMdiff) at this stage. TM Neural Progenitor Medium (NPP) is used instead of traditional differentiation or maturation media. This strategy aims to maximize the stemness and multi-directional differentiation potential of neural progenitor cells, thereby more accurately mimicking the microenvironment in which neural progenitor cells interact and co-differentiate with subsequently migrating macrophage progenitor cells during in vivo development.

[0052] To achieve physiological relevance in cellular composition within organoids, induced neural progenitor cells were fused with primitive macrophage progenitors (PMPs) at an optimized ratio of 7:3. This ratio ensures that the proportion of microglia in mature brain organoids can be stably maintained at approximately 8%, which closely matches the 5%-10% microglia proportion in the real human brain. This effectively addresses the significant deficiency of insufficient and difficult-to-control microglia proportion in existing brain organoid models. Furthermore, this controllable ratio provides a solid and operational foundation for establishing research models of neuroimmunological diseases.

[0053] In the initial fusion phase, a co-culture strategy was employed for multiple organoids (5-8 organoids per well), followed by embedding with 20 μl of low-factor matrix gel. This combined approach significantly reduced organoid size heterogeneity and structural dissociation risk, effectively controlled batch-to-batch variation, and improved model reproducibility. Fusion culture utilized MO-1 medium (a 1:1 mixture of NPC and PMP media), which simultaneously maintains the stemness and state of both progenitor cells, accurately simulating early developmental events of myeloid progenitor cell migration into the neural tube in vivo. On day 41, the medium was replaced with BrainPhysics containing 100 ng / ml interleukin-34 (IL-34) and 10 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF). TM Neuronal Medium (its basic components include N2, B27 supplements, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ascorbic acid). This culture medium effectively promotes neuronal maturation and, through the synergistic effect of IL-34 and GM-CSF, regulates key biological behaviors of microglia within organoids, including their proliferation, migration, and homeostasis.

[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] Using the 3D human brain organoids cultured to day 55 of this invention, immunofluorescence analysis revealed that the neurons of this invention exhibit more complex dendritic structures and dendritic spine maturity. The early fusion system showed a state of fusion between NPC and PMP cells, containing various neural lineage cells: including microglia (marked by transmembrane protein 119, TMEM119, CD11b, and CD45 positivity), neurons (marked by β3 tubulin Beta 3 Class III and TUJ1 positivity), astrocytes, and neural progenitor cells (marked by nestin Nestin positivity). This demonstrates that the model successfully constructed a complex 3D human brain organoid containing functional microglia. Attached Figure Description

[0056] Figure 1 This is a flowchart illustrating the brain organoid construction process of the present invention.

[0057] Figure 2 Immunofluorescence staining image of progenitor cells in a brain organoid section on day 55 (40×).

[0058] Figure 3 Immunofluorescence staining image of microglia in brain organoid sections on day 55 (40×).

[0059] Figure 4 Immunofluorescence staining image of neurons in a brain organoid section taken on day 55 (40×).

[0060] Figure 5 Immunofluorescence staining image (40×) showing the colocalization of microglia and presynaptic membrane in a brain organoid section on day 55.

[0061] Figure 6 A bar graph showing the changes in CD43, SOX2, SYNAPSIN I, SYNAPSIN I / CD11b, and Ki67 in brain organoids after inflammatory stimulation.

[0062] Figure 7 Flow cytometry plots showing the proportion of microglia in brain organoids induced by two iPSC strains. Detailed Implementation

[0063] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0064] 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.

[0065] The reagents and culture media used in the following examples:

[0066] I. NPCs' Inducement

[0067] Phase 1: hiPS cell pretreatment and pre-induction status assessment (preparation phase) Figure 1 (0 days ago)

[0068] 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.

[0069] 2. Pre-induction status check: NPC induction can be initiated by observing the following conditions using an inverted microscope (10× objective lens):

[0070] 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).

[0071] 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).

[0072] Phase 2: AggreWell TM 800 plate pretreatment and EB formation (days 0-1)

[0073] 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.

[0074] 2. HiPS cell washing: Take out the six-well plate containing hiPS cells to be induced, and slowly aspirate the old culture medium along the well wall with a 1 mL pipette (avoiding the cell clone clusters with the pipette tip); add 1 mL of DPBS to each well, gently tilt the plate to cover the cell surface with the buffer, let stand for 1 min, and then aspirate. Repeat the washing 3 times.

[0075] 3. Cell digestion: Add 1 mL of Accutase digestion solution preheated to 37°C for 10 min to each well, return to the incubator and start timed digestion for 5 min; examine under a microscope every 2 min, and stop digestion immediately when the edges of the clonal clusters loosen and the intercellular spaces increase.

[0076] 4. Cell collection and centrifugation: Add 2 mL of DMEM-F12 medium to each well to stop digestion. Use a 1 mL pipette tip with the tip cut off (to avoid scratching the cells) to pipette clockwise along the well wall 3-5 times to ensure that the cells are completely detached and form a single-cell suspension. Transfer the suspension to a 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 min at room temperature. After centrifugation, a white cell precipitate will be visible at the bottom of the tube.

[0077] 5. Cell resuspension and counting: Discard the supernatant and add 1 mL STEMdiff to each tube. TM Resuspend the cells in the neural induction medium by gently pipetting 5-8 times; take 10 μL of the suspension and count the cells using an inverted microscope to ensure cell viability > 90%.

[0078] 6. Preparation of EB induction system: Transfer a suspension containing 3 million cells to a new 15 mL centrifuge tube, add STEMdiff... TM Add the neural induction medium to a total volume of 1 mL; add 1 μL of 10 mM Y-27632 and mix by pipetting 2-3 times (Y-27632 can inhibit apoptosis and cannot be omitted).

[0079] 7. Cell seeding and EB formation: AggreWell aspiration TM Add 1 mL of the above cell suspension to each well of the 800-well plate with anti-adhesion rinsing solution (avoid generating air bubbles; if air bubbles are present, remove them with a pipette tip); place the 24-well plate in a centrifuge equipped with an adapter and centrifuge at 100×g (approximately 450 rpm) for 3 min at room temperature; after centrifugation, place the plate directly in an incubator and incubate for 24 h (day 2). Microscopic examination will reveal dense and uniform EB cells with a diameter of 100-150 μm.

[0080] Phase 3: EB medium replacement culture (days 2-5)

[0081] 1. Change the medium on the second day: Remove the 24-well plate containing EB, and use a 1mL pipette to gently aspirate 0.5mL of the old medium along the well wall (be gentle to avoid bringing out the EB); slowly add 1.5mL of STEMdiff along the well wall. TM Return the neural induction medium to the incubator.

[0082] 2. Change the medium on days 3-5: Repeat the above medium change procedure daily (discard 0.5 mL of old medium and add 1.5 mL of STEMdiff). TM (Neural induction culture medium), during which the EB status is examined under a microscope to ensure that the EB is not disintegrated and that there are no obviously dispersed cells.

[0083] Phase 4: Preparation for EB adherent culture (Day 6)

[0084] 1. Matrigel coating: Take a sterile six-well plate, add 1 mL of Matrigel to each well (thaw at 4°C beforehand, operate on ice), and incubate at 37°C for 1 hour to allow the Matrigel to evenly cover the bottom of the well.

[0085] 2. EB Screening and Collection: Place a 40μm cell filter into a 50mL centrifuge tube, gently aspirate EB from the 24-well plate using a wide-mouth pipette tip (avoid squeezing the EB), and slowly add it to the filter; use 1mL STEMdiff... TM The filter was rinsed twice with neural induction medium to remove single cells and old medium, and EB was collected from the filter.

[0086] 3. EB Adherent Inoculation: Discard Matrigel from the six-well plate (a small amount remaining is not a problem), add 2 ml of LSTTEMdiff to each well. TM Neural induction medium; the collected EB was evenly inoculated into a six-well plate and placed in an incubator to allow the EB to adhere naturally to the well.

[0087] Phase 5: EB amplification (days 7-13)

[0088] 1. Medium change procedure: Starting from day 7, remove the six-well plate daily and use a 1mL pipette to discard all the old culture medium; add 2mL STEMdiff to each well. TM Return the neural induction medium to the incubator.

[0089] 2. Observation of induction state: Microscopic examination was performed daily. After EB adhered to the wall, it was observed that it gradually spread out and spindle-shaped or polygonal cells appeared at the edge (initially differentiating into the neural lineage). The medium was changed continuously for 6 days (until day 13).

[0090] Phase 6: Neural Rosette Screening and NPC Purification (Day 14)

[0091] 1. Matrigel coating: Coat a new six-well plate with Matrigel and incubate at 37°C for 1 hour for later use.

[0092] 2. Cell washing and digestion: Remove the six-well plate and discard the old culture medium; add 1 mL of DPBS to each well, let stand for 1 min, then discard the DPBS, repeating the washing once; add 1 mL of STEMdiff to each well. TMThe neural rosette selection reagent was placed in an incubator at 37°C for 1.5 hours for digestion.

[0093] 3. Collection of neural rosettes: Discard the digestion reagent, use a 1mL pipette tip to quickly pipette 3-4 times at the "rosette" structure region (neural progenitor cell enrichment area) visible under the microscope to detach the cells in this region; add 2mL of DMEM-F12 medium to resuspend, and transfer the suspension to a 15mL centrifuge tube.

[0094] 4. Cell centrifugation and resuspending: Centrifuge at 350×g (approximately 1200rpm) for 5 minutes at room temperature, discard the supernatant; add 2ml LSTTEMdiff to each tube. TM Neural induction medium, resuspend cells by pipetting 5 times.

[0095] 5. NPC inoculation: Discard the Matrigel from the prepared 6-well plate, add 1 mL of the above cell suspension to each well; discard the old solution, wash once with 1 mL of DPBS, and then add 2 mL of STEMdiff. TM Place the neural induction medium in an incubator.

[0096] Phase 7: NPC Expansion and Acquisition (Days 15-21)

[0097] Medium change procedure: On days 15-20, discard the old culture medium daily and add 2 mL of fresh STEMdiff™ neural progenitor cell culture medium to each well.

[0098] Obtaining and identifying NPCs (Neural Progenitor Cells): On day 21, the culture plate was removed, and microscopic examination revealed uniform spindle-shaped cells (typical NPC morphology). Immunofluorescence staining was performed; the immunofluorescence staining steps were as follows:

[0099] Cell pretreatment

[0100] Remove the 6-well cultured NPC (neural progenitor cells) and aspirate the culture medium from the wells in a clean bench. Wash the cells three times with DPBS buffer (Gibco#14190-144) for 5 minutes each time (at room temperature, shake at low speed of 50 rpm) to thoroughly remove residual culture medium and cell metabolic waste.

[0101] Cell fixation

[0102] Add pre-cooled (4°C) 4% paraformaldehyde fixative (paraformaldehyde powder Sigma-Aldrich #P6148, prepared with DPBS buffer, pH 7.2-7.4) to each well, ensuring complete coverage of the NPC cells on the coverslip; incubate at room temperature for 15-20 minutes; aspirate the fixative and wash three times with DPBS buffer for 5 minutes each time to thoroughly remove residual fixative and avoid damage to cell structure.

[0103] Cell membrane permeability

[0104] Add 0.3% Triton X-100 permeabilization buffer (Thermo Fisher #HFH10, 1% stock solution diluted with DPBS) to each well and incubate at room temperature for 10-15 minutes to allow the cell membrane to form micropores to facilitate antibody permeation; aspirate the permeabilization buffer and wash three times with DPBS buffer for 5 minutes each time to remove residual permeabilization buffer.

[0105] Closed

[0106] Aspirate the DPBS liquid from the wells and add 5% BSA blocking solution (bovine serum albumin Sigma-Aldrich #A7906, prepared with DPBS buffer) to the surface of NPC cells on the coverslip, 50-100 μL per coverlip, ensuring complete coverage of the cell surface; transfer the coverslip to a humidified chamber and incubate at room temperature for 30-60 minutes to block the binding of the secondary antibody to non-specific sites on the cell surface.

[0107] Primary Antibody Incubation

[0108] Discard the blocking solution (no washing required), and directly drop the diluted NPC-specific primary antibody solution (50-80 μL per tablet) onto the cell surface; the primary antibody should be a Nestin and Sox2 dual-label combination (Nestin(10C2) Mouse Monoclonal Antibody CST #33475, Sox2(D6D9) Rabbit Monoclonal Antibody CST#3579), each diluted with 5% BSA blocking solution at a ratio of 1:100-1:500 to ensure that the antibody solution evenly covers the cells; after sealing the humidified chamber, incubate it overnight (12-16 hours) at 4°C; the next day, remove the humidified chamber, warm it to room temperature for 30 minutes, and wash it three times with DPBS buffer for 10 minutes each time to completely remove unbound free primary antibody.

[0109] Secondary antibody incubation

[0110] Add diluted fluorescent secondary antibody solution (50-80 μL per tablet) to the cell surface; the secondary antibody should be a fluorescein-labeled antibody that matches the species of the primary 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), diluted with 5% BSA blocking buffer at a ratio of 1:200-1:1000; incubate the humidified chamber at room temperature in the dark for 60 minutes; discard the secondary antibody solution, wash three times with DPBS buffer in the dark for 10 minutes each time to avoid fluorescein residue causing background interference.

[0111] Nuclear staining

[0112] Add 1 μg / mL DAPI staining solution (CST#4083, prepared with DPBS buffer) to the cell surface and incubate at room temperature in the dark for 5 minutes. Discard the staining solution and wash twice with DPBS buffer in the dark for 5 minutes each time to remove residual DAPI.

[0113] Cover

[0114] Take a clean glass slide and place 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 with the mounting medium on the glass slide, avoiding air bubbles; use sterile filter paper to absorb any excess mounting medium from the edges of the coverslip and allow it to air dry at room temperature for 10 minutes.

[0115] Observation imaging

[0116] The mounted slides were placed under a Zeiss LSM980 laser confocal microscope. The corresponding fluorescence channel was selected to observe and collect Nestin and Sox2-specific fluorescence signals and nuclear staining signals in NPC cells. Cell morphology and marker expression localization were recorded to obtain experimental results. The results showed that the NPC-specific markers Nestin had a positive rate ≥92%, Sox2 a positive rate ≥90%, and cell viability ≥88%, thus qualifying the cells as qualified NPCs. Subsequently, STEMdiff was used... TM The neural progenitor cell culture medium was used for the culture and expansion of NPCs.

[0117] II. PMP Induction

[0118] Phase 1: hiPSC pretreatment and status determination (1 day before induction)

[0119] 1. hiPSC culture: Take hiPSCs in the logarithmic growth phase and use mTeSR TM Regular culture in Plus medium, with fresh medium changed daily to ensure cells are in the logarithmic growth phase and avoid excessive confluence (consolidation rate controlled at 70%-80%).

[0120] 2. Status verification: Observation with an inverted microscope confirms that hiPSCs form a dense clonal cluster with no obvious differentiated cells (differentiated cells are judged by: extended cell bodies, increased protrusions, and significant differences in morphology from the clonal cluster), and that cell viability is ≥95%, before proceeding to the subsequent YS-EB induction.

[0121] Phase 2: Induction of YS-EB formation (days 0-4)

[0122] 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. Ensure that the solution evenly covers the bottom of the well. After incubation, discard the anti-adhesion rinsing solution and set aside for later use.

[0123] 2. Preparation of YS-EB induction medium: Take an appropriate amount of mTeSR TM Add BMP4 (50 ng / mL), SCF (20 ng / mL), and VEGF (50 ng / mL) to the Plus medium according to the final concentration. Gently invert the centrifuge tube 3-5 times to mix well. Filter with a 0.22 μm filter membrane to sterilize, avoiding repeated blowing to prevent air bubbles.

[0124] 3. hiPSC digestion and inoculation:

[0125] 1) Remove the pretreated hiPSCs, discard the old culture medium, and wash once with 1 mL DPBS; add 1 mL Accutase digestion solution to each well, digest at 37°C for 5 min, and examine under a microscope until the edges of the cloning clusters are loose.

[0126] 2) Add 2 mL of DMEM-F12 medium to stop digestion, gently pipette the mixture into a single-cell suspension, transfer it to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min at room temperature, and discard the supernatant.

[0127] 3) Resuspend the cells in the YS-EB induction medium prepared above and adjust the cell concentration to 1×10⁻⁶. 6 Cells / mL, per well to pretreated AggreWell TM Add 1 mL of cell suspension (containing 1 × 10⁸ cells) to each of 800 wells. 6 (hiPSC).

[0128] 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.

[0129] 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.

[0130] 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.

[0131] Phase 3: YS-EB transfer and hPMP (Primitive Macrophage Precursor) culture (days 5-13)

[0132] 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.

[0133] 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.

[0134] Days 5-9: Every 2 days, discard 5 mL of old culture medium and add 5 mL of fresh PMP culture medium;

[0135] 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.

[0136] Phase 4: Collection and yield detection of hPMP (Primitive Macrophage Precursor) (Days 14-21)

[0137] 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).

[0138] 2. hPMP production detection: Cell counting was performed using a cell counting chamber, combined with immunofluorescence detection of hPMP-specific markers (CD235). + CD43 + The positive rate, cumulative yield calculation, and immunofluorescence staining detection steps are as follows:

[0139] Cell pretreatment

[0140] Remove the culture flask containing PMP, aspirate the PMP cell suspension in the laminar flow hood, and seed it into 6-well plates pre-coated with 0.1% Matrigel (Corning #354234) (seeding density approximately 5 × 10⁻⁶). 4 Cells per well); after culturing in a 37°C, 5% CO2 cell culture incubator for 24 hours, the culture medium in the wells is aspirated; the cells are washed three times with DPBS buffer (Gibco#14190-144) for 5 minutes each time (at room temperature, shaker at low speed of 50 rpm) to thoroughly remove residual culture medium and cell metabolic waste.

[0141] Cell fixation

[0142] Add pre-cooled (4°C) 4% paraformaldehyde fixative (paraformaldehyde powder Sigma-Aldrich #P6148, prepared with DPBS buffer, pH 7.2-7.4) to each well, ensuring complete coverage of PMP cells in the well; incubate at room temperature for 15-20 minutes; aspirate the fixative and wash three times with DPBS buffer for 5 minutes each time to thoroughly remove residual fixative and avoid damage to cell structure.

[0143] Cell membrane permeability

[0144] Add 0.3% Triton X-100 permeabilization buffer (Thermo Fisher #HFH10, 1% stock solution diluted with DPBS buffer) to each well and incubate at room temperature for 10-15 minutes to allow the cell membrane to form micropores to facilitate antibody permeation; aspirate the permeabilization buffer and wash three times with DPBS buffer for 5 minutes each time to remove residual permeabilization buffer.

[0145] Closed

[0146] Aspirate all DPBS from the wells and add 5% BSA blocking solution (bovine serum albumin Sigma-Aldrich #A7906, prepared with DPBS buffer) to the surface of PMP cells, 50-100 μL per well, ensuring complete coverage of the cell surface; place the 6-well plate in a humidified chamber and incubate at room temperature for 30-60 minutes to block the binding of the secondary antibody to non-specific sites on the cell surface.

[0147] Primary Antibody Incubation

[0148] Discard the blocking solution (no washing required), and directly add diluted PMP-specific primary antibody solution (50-80 μL per well) to the cell surface. The primary antibody should be a CD235 and CD43 dual-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 evenly covers the cells. After sealing the humidified chamber, incubate it overnight (12-16 hours) at 4°C. The next day, remove the humidified chamber, warm it to room temperature for 30 minutes, and wash it three times with DPBS buffer for 10 minutes each time to completely remove unbound free primary antibody.

[0149] Secondary antibody incubation

[0150] Add diluted fluorescent secondary antibody solution (50-80 μL per well) to the cell surface; the secondary antibody should be a fluorescein-labeled antibody that matches the species of the primary antibody (Anti-rabbit IgG(H+L), F(ab')2 Fragment(Alexa Fluor®488Conjugate)CST#4412, Anti-mouse IgG(H+L), F(ab')2 Fragment(Alexa Fluor®488Conjugate)CST#4408), diluted with 5% BSA blocking buffer at a ratio of 1:200-1:1000; place the humidified chamber at room temperature and incubate in the dark for 60 minutes; discard the secondary antibody solution and wash three times with DPBS buffer in the dark, 10 minutes each time (shaking slowly on a shaker) to avoid fluorescein residue causing background interference.

[0151] Nuclear staining

[0152] Add 1 μg / mL DAPI staining solution (CST#4083, prepared with DPBS buffer) to the cell surface and incubate at room temperature in the dark for 5 minutes. Discard the staining solution and wash twice with DPBS buffer in the dark for 5 minutes each time to remove residual DAPI.

[0153] Cover

[0154] Take a clean glass slide and place 1-2 drops of anti-fluorescence quenching mounting medium (Thermo Fisher #P36961, ProLong) in the center. TM Diamond); Carefully remove the coverslip from the 6-well plate with sterile forceps (if no coverslip is used, directly transfer the cell smear to the slide using a pipette), and slowly cover the cell side down onto the mounting medium, avoiding air bubbles; use sterile filter paper to absorb any excess mounting medium from the edges of the coverslip, and allow it to air dry at room temperature for 10 minutes.

[0155] Observation imaging

[0156] The mounted slides were placed under a Zeiss LSM980 laser confocal microscope. The corresponding fluorescence channels were selected (FITC: 488nm excitation wavelength, DAPI: 350nm excitation wavelength) to observe and collect CD235 and CD43-specific fluorescence signals and nuclear staining signals in PMP cells. Cell morphology and marker expression localization were recorded to obtain experimental results. The results showed that the initial hiPSC seeding density was 3 × 10^6 cells; within 3 months of culture, the cumulative hPMP yield reached approximately 1.2 × 10^8 cells, about 40 times the initial hiPSC quantity. Identification was performed using immunofluorescence staining, and the CD235+ / CD43+ double positivity rate was ≥85%.

[0157] III. Induction of brain organoids containing microglia

[0158] Phase 1: AggreWell TM 800-well fusion culture (days 21-24)

[0159] 1. NPC digestion: Take NPC cultured to day 21, discard the old culture medium, wash once with 1 mL DPBS; add 1 mL of 37℃ preheated Accutase digestion solution to each well, put in an incubator to digest for 5 min, and examine under a microscope until the intercellular spaces increase and the cells become loose.

[0160] 2. Preparation of MO-1 induction medium: Take PMP medium and STEMdiff medium TM The neural progenitor cell culture medium was prepared at a volume ratio of 1:1, gently mixed, and then filtered through a 0.22 μm filter membrane for sterilization.

[0161] 3. Termination and Resuspension: Add 2 mL of DMEM-F12 medium to terminate digestion, gently pipette 8-10 times with a 1 mL pipette tip to prepare a single-cell suspension; transfer to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant, and resuspend in 1 mL of MO-1 medium. Count the cells and adjust the concentration to 2.1 × 10⁻⁶ cells / mL. 6 per mL.

[0162] 4. PMP suspension preparation: Take PMP in the logarithmic growth phase, centrifuge at 800 rpm for 3 min at room temperature, discard the supernatant, resuspend in MO-1 medium, and adjust the concentration to 0.9 × 10⁻⁶. 6 per mL.

[0163] 5. Cell mixing ratio: Mix 1 mL of NPC single-cell suspension (2.1 million cells) with 1 mL of PMP suspension (900,000 cells) at a ratio of 7:3 (NPC:PMP = 7:3). Gently pipette three times to mix thoroughly, obtaining 1 mL of fused cell suspension (total cell count 3 × 10⁻⁶). 6 indivual).

[0164] 6. Culture plate pretreatment: Take AggreWell... TM Add 1 mL of anti-adhesion rinsing solution to each well of an 800 24-well plate, incubate at room temperature for 10 min, and then aspirate and reuse.

[0165] 7. Inoculation and centrifugation: Add 1 mL of fusion cell suspension (containing 3 million mixed cells) to the pretreated wells, avoiding the generation of air bubbles; place the plate in a centrifuge equipped with an adapter and centrifuge at 100×g (about 450 rpm) for 3 min at room temperature to promote cell aggregation.

[0166] 8. Fusion culture and medium change: After centrifugation, place in an incubator and culture continuously for 5 days, performing a complete medium change daily: gently aspirate the old culture medium in the well with a wide-mouth pipette tip (avoid touching the organoid rudiments), and slowly add 1 mL of fresh MO-1 culture medium along the well wall. On day 25, brain-like organoid rudiments with a diameter of 200-250 μm and dense morphology can be seen.

[0167] Phase 2: Scale-up culture in ultra-low adsorption 96-well plates (days 25-30)

[0168] 1. Brain organoid transfer: Gently aspirate the brain organoid from day 25 using a wide-mouth pipette tip, transfer it to a 15mL centrifuge tube, centrifuge at 800rpm for 3min at room temperature, and discard the supernatant.

[0169] 2. Inoculation and centrifugation: Take an ultra-low adsorption 96-well plate, add 200 μL of fresh MO-1 medium to each well, and inoculate at a density of 5-8 brain-like organs per well; place the plate in a centrifuge and centrifuge at 100 rpm for 3 min at room temperature to allow the brain-like organs to settle to the bottom of the well.

[0170] 3. Culture and medium change: Incubate in an incubator for 5 days, and change the medium every other day: discard 150 μL of old culture medium from the well and add 150 μL of fresh MO-1 culture medium to prevent the brain-like organs from drying out; on the 30th day, microscopic examination showed that the diameter of the brain-like organs increased to 300-350 μm and the boundaries were clear.

[0171] Phase 3: Matrix gel embedding and shaker culture (days 31-40)

[0172] 1. Preparation of matrix gel: Thaw the low factor matrix gel overnight at 4°C in advance, and handle on ice to avoid solidification; take 20μL of low factor matrix gel and drop it into a sterile culture dish for later use.

[0173] 2. Brain-like organoid embedding: Use a wide-mouth pipette tip to remove the brain-like organoid on day 30 and gently place it into a low factor matrix gel droplet, ensuring that each droplet embeds one brain-like organoid; place it in an incubator at 37°C and let it stand for 30 minutes until the low factor matrix gel completely solidifies (becoming a transparent gel).

[0174] 3. Shaking incubation setup: Take an ultra-low adsorption 6-well plate and add 3 mL of MO-1 medium to each well; gently transfer the solidified low factor matrix gel-brain organoid complex into the wells, place them in a constant temperature shaking incubator (60 rpm), and incubate in an incubator.

[0175] 4. Medium change procedure: Perform a half medium change every 4 days: aspirate 1.5 mL of old culture medium from the well and slowly add 1.5 mL of fresh MO-1 culture medium along the well wall. On day 40, the diameter of the brain-like organ can reach 400-450 μm, and fine nerve processes can be seen on the surface.

[0176] Phase 4: Microglia induction and maturation culture (days 41-55)

[0177] 1. Preparation of mature culture medium: Take BrainPhys TM For neuronal culture medium, add IL-34 (100 ng / mL) and GM-CSF (10 ng / mL) to the final concentration, mix gently, and then filter through a 0.22 μm filter membrane for sterilization. Prepare and use immediately.

[0178] 2. Culture medium replacement: Discard the old MO-1 culture medium in the 6-well plate, gently wash once with 2 mL DPBS, add 3 mL of mature culture medium to each well, and continue to culture on a shaker.

[0179] 3. Medium change and culture: Perform a complete medium change every 4 days: carefully aspirate the old culture medium with a wide-mouth pipette tip (avoid touching the matrix gel complex), add 3 mL of fresh mature culture medium; continue culturing until day 55 to obtain brain organoids containing microglia.

[0180] Phase 5: Extended culture (after day 55)

[0181] The brain organoids were continued on day 55 with BrainPhysics containing IL-34 (50 ng / mL) and GM-CSF (5 ng / mL). TM Neuronal culture medium (maturation medium) with complete medium replacement every 6 days and extended culture for 2-4 weeks can significantly improve the maturity of brain organoids, manifested as a denser neural network and enhanced expression of synaptic markers.

[0182] Organoids mature around 55 days, with the specific sampling time depending on experimental requirements. Sampling during the early developmental stage occurs around 35 days, while sampling during the mature stage occurs at 55 days and beyond. The final organoid morphology and size changes are shown in [see attached image]. Figure 1 .

[0183] The obtained brain organoids were then subjected to immunofluorescence staining detection. The specific steps are as follows:

[0184] I. Frozen Slices

[0185] Sample pretreatment: Take brain organoids (500-1000μm in diameter) cultured to the target stage and gently rinse 3 times with DPBS buffer (Gibco#14190-144) for 5 minutes each time (room temperature, shaker at low speed of 50 rpm) to thoroughly remove culture medium residue.

[0186] Sample fixation: Transfer brain organoids to centrifuge tubes containing 4% paraformaldehyde fixative (prepared with paraformaldehyde powder Sigma-Aldrich #P6148, pH 7.2-7.4), with the fixative volume being more than 10 times the sample volume; incubate at 4°C for 24 hours, gently inverting the centrifuge tubes once every 6 hours to ensure full penetration of the fixative; after fixation, wash three times with DPBS buffer for 10 minutes each time to remove residual fixative.

[0187] Trypan blue staining: Prepare trypan blue staining solution by diluting 0.4% trypan blue solution (Sigma-Aldrich #T8154) 30 times; transfer brain organoids to 6-well cell culture plates using a wide-mouth pipette tip, and add an appropriate amount of trypan blue staining solution (ensuring complete coverage of the sample); place the culture plate on a horizontal shaker and stain at room temperature for 10 minutes at 80 rpm; discard the trypan blue staining solution, and wash 3 times with DPBS buffer for 5 minutes each time.

[0188] Preparation of embedding agent: Mix OCT embedding agent (Sakura#4583) and 20% sucrose solution (prepared with sucrose Sigma-Aldrich#S9378) at a volume ratio of 2:1, and mix thoroughly to prepare a brain organoid embedding agent.

[0189] Sample embedding: Take a sterile embedding mold, add 200-300 μL of the prepared embedding medium to the bottom of the mold, carefully transfer the brain organoid to the center of the embedding mold with a wide-mouth pipette tip, and then add more embedding medium to completely cover the sample; quickly place the embedding mold in liquid nitrogen for 5-10 minutes until the embedding medium is completely solidified.

[0190] Cryosectioning: Remove the solidified embedded block from the mold and install it on the sample stage of the cryostat (Leica CM1950). Set the section thickness to 15-20 μm and perform continuous sectioning. Gently adhere the cut brain organoid sections to poly-L-lysine coated slides (Fisher Scientific #12-550-15) and allow them to air dry at room temperature for later use.

[0191] II. Immunofluorescence staining

[0192] Baking: Place the slides with the brain organoid sections attached on a slide baking machine and bake at 60°C for 1 hour to enhance the adhesion between the sections and the slides.

[0193] Looping for localization: Use a hydrophobic pen (Vector Laboratories#H-4000) to draw circles around the brain organoid slice on the slide to clearly define the staining area and avoid reagent loss.

[0194] Secondary fixation: Add 1 mL of 4% paraformaldehyde fixative (prepared by Sigma-Aldrich #P6148) to the circled area and let it stand at room temperature for 20 minutes; discard the fixative and wash 3 times with DPBS buffer (Gibco #14190-144) for 5 minutes each time.

[0195] Cell membrane permeability: Add 1 mL of 1% Triton X-100 permeability buffer (Thermo Fisher #HFH10) to the circled area and incubate at room temperature for 15 minutes to allow micropores to form in the cell membrane to facilitate antibody permeation; aspirate the permeability buffer and wash three times with DPBS buffer for 5 minutes each time.

[0196] Blocking: Aspirate all DPBS liquid and add 5% BSA blocking solution (Bovine Serum Albumin Sigma-Aldrich #A7906, prepared with DPBS) to the surface of the slide, 100-150 μL per slide, ensuring complete coverage of the slide; place the slide in a humidified chamber and block at room temperature for 1 hour to block non-specific binding sites.

[0197] Primary antibody incubation: Dilute the brain organoid-specific primary antibody to 1:200 using a 1% BSA + 0.3% Triton X-100 mixture (BSA#A7906, Triton X-100#HFH10). (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) XP® Rabbit Monoclonal Antibody CST#5297, beta3-Tubulin (D71G9) Rabbit Monoclonal Antibody) (CST#5568); Discard the blocking solution, do not wash, and directly drop 200 μL of diluted primary antibody solution onto the surface of the slide, ensuring even coverage of the slide; place the humidified chamber in a 4°C refrigerator and incubate overnight (12-16 hours); the next day, remove the humidified chamber, allow it to warm to room temperature for 30 minutes, and then wash three times with DPBS buffer for 10 minutes each time to completely remove unbound free primary antibody.

[0198] Secondary antibody and nuclear staining: Dilute the fluorescent secondary antibody and DAPI nuclear staining solution to 1:400 with 1% BSA + 0.3% Triton X-100 mixture (recommended secondary antibodies: Anti-rabbit IgG (Alexa Fluor® 488 Conjugate) CST#4412, Anti-mouse IgG (Alexa Fluor® 594 Conjugate) CST#8890; DAPI CST#4083); add 200 μL of the diluted secondary antibody and DAPI mixture to the slide surface, and incubate in a humidified chamber at room temperature in the dark for 1 hour; discard the mixture, and wash 3 times with DPBS buffer in the dark, 10 minutes each time.

[0199] Mounting: Take a clean coverslip and place 1-2 drops of anti-fluorescence quenching mounting medium (Thermo Fisher #P36961) in the center; carefully pick up the coverslip with tweezers and slowly cover the slide with the cell side down, avoiding air bubbles; seal the edges of the coverslip with nail polish (Sigma-Aldrich #Z665850); store at 4°C protected from light.

[0200] Imaging observation: Using a Zeiss LSM980 laser confocal microscope, the corresponding fluorescence channels (FITC: 488nm, Cy3 / 594: 550nm, DAPI: 350nm) were selected for observation. Fluorescence signal images of the sections were acquired and the results were recorded. The experimental results are as follows: Figures 2-5 As shown.

[0201] Figure 2 To induce brain-like structures on day 35 (day 15 post-fusion), which is the initial stage of neurogenesis, immunofluorescence was used to identify the distribution of progenitor cells in the human brain organoid containing microglia. NESTIN is a specific marker for neural progenitor cells (NPCs), and CD235 is a specific marker for macrophage progenitor cells (PMPs). It was observed that both types of progenitor cells were evenly distributed within the organoid, indicating that this model can simulate the developmental process of neural and macrophage lineages and allows for continuous observation of the entire process of neural development, which is not available in other models.

[0202] Figure 3 , Figure 4 , Figure 5 To induce brain-like behavior on day 55 (day 35 after fusion). Figure 3 The results were obtained using immunofluorescence to identify microglia in human brain organoids. TMEM119 is a microglia-specific marker, and CD11b is a macrophage lineage marker. At this stage, the brain is in the mid-to-late stages of neural development, when most macrophage lineage cells have begun or have already differentiated into microglia. Therefore, TMEM119, as a specific marker of mature microglia, can be observed to be evenly distributed throughout the organoid. CD11b, as a macrophage lineage marker, can label all cells differentiated from PMPs. By comparing the ratios of TMEM119 and CD11b, it can be observed that some mature microglia appeared on day 35 after fusion, while some macrophage lineage cells were still in the developmental stage. This indicates that the model has the ability to induce mature microglia and can simulate the entire developmental process from macrophage progenitors to mature microglia.

[0203] Figure 4 This is the result of immunofluorescence identification of neurons in human brain organoids containing microglia. TUJ1 is a specific marker for mature neurons; SOX2 is a marker for neural progenitor cells. Similar to obtaining mature microglia, mature neurons were also labeled in this 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. The overall distribution conforms to the distribution characteristics of peripheral neurons and internal neural progenitor cells in the cerebral cortex.

[0204] Figure 5This is an immunofluorescence assay of the co-localization of microglia and presynaptic membranes in human brain organoids containing microglia. The core mechanism by which microglia participate in shaping neural circuits is synaptic pruning, identifying redundant synapses, and guiding the pruning process. SYNAPSIN I was used to localize the presynaptic membrane of neurons, and the co-localization of the presynaptic membrane with CD11b microglia was observed. The figure shows a large number of areas co-stained with green and red (orange areas), indicating significant co-localization between the presynaptic membrane and CD11b microglia. This confirms that microglia play an important role in synaptic pruning in organoids, demonstrating that the human brain organoids containing microglia induced by this method contain functional, mature microglia.

[0205] Based on the above immunofluorescence results, it can be seen that the human brain organoids containing microglia induced by the method of the present invention can not only specifically reflect the development and differentiation process of neural lineage and macrophage lineage cells, but also control the proportion. Furthermore, it can yield morphologically and functionally mature and normal microglia and neurons, which can serve as a model of a normal human brain for subsequent experiments.

[0206] Overall, this model showed a relatively uniform mixed distribution pattern of NPC and PMP cells in the early stage (approximately 35 days), see [see details]. Figure 2 It is evident that the two cell types exhibit a spatial distribution pattern similar to that of the human brain, with neurons located in the superficial cortex and progenitor cells located internally. Figure 4 Co-localization of the microglia-specific marker CD11b and the presynaptic membrane-specific marker SYNAPSIN I revealed that this model not only possesses cell types approximating the developmental stages of the human brain, but also that the spatial distribution and function of each cell type are similar to those of the real human brain. (See...) Figure 5 .

[0207] The study also verified whether the obtained brain organoids could simulate the responses of the real human brain to external stimuli during development. Microglia, as the most abundant immune cells in the brain, are also the only cell type in the brain with IL-6 membrane receptors. Brain organoids were stimulated with IL-6 at a concentration of 10 ng / mL for 10 days, followed by sampling. Specifically, brain organoids cultured to day 55 were selected, and IL-6 at a concentration of 10 ng / mL was added to the maturation culture medium. The culture was continued for 10 days, with complete medium changes performed daily. Brain organoids were collected on day 10 (day 65). Immunofluorescence detection was performed on frozen sections of the sampled brain organoids, following the same procedure as above. ImageJ software was used to analyze and detect changes in model function and cell types. The results are as follows... Figure 6As shown, SYNAPSIN I / CD11b indicates co-localization, suggesting that microglia have phagocytosed into the synapses of some neurons, indicating that microglia are performing phagocytic function. The SYNAPSIN I / CD11b ratio indicates the proportion of staining with co-localization. The results show that the brain organoid obtained in Example 1 can respond to IL-6 stimulation and can influence the main functions of microglia during development under stimulation. In the first part of the results, CD43 is a PMP cell-specific marker, and SOX2 is an NPC cell-specific marker, which are progenitor cell indicators in the early stages of embryoid formation. This shows that the brain organoid system constructed in this invention can explore cell-cell interactions and functions in the early stages of fusion of two progenitor cells, which is not available in other models. Secondly, SYNAPSIN I labels the presynaptic membrane, and CD11b is a microglia-specific marker. The co-staining of these two markers indicates that microglia are performing phagocytosis. This result shows that microglia can receive external stimuli and change their phagocytic function accordingly. This stress response function is also unique to the brain organoid obtained in this invention. Finally, Ki67, as a broad-spectrum proliferation-related marker, indicates that the entire system of this invention is affected by external stimuli when microglia are involved, thus altering the proliferation state of the entire system. This result also demonstrates that this invention is not simply a cell type fusion, but a neural developmental system containing microglia with complex interactive functions.

[0208] In summary, the brain-like organoids of the present invention can achieve the following functions: exploring the control of the proportion of progenitor cells by microglia during the developmental stage, changes in phagocytic function, control of the number of mature neurons, influence on cell proliferation and apoptosis, and simultaneously accepting external stimuli and making corresponding functional stress changes.

[0209] Example 2

[0210] Two iPS cells from different sources (ACS1011, purchased from the ATCC cell bank; and T-iPSC, constructed according to existing technology) were used to conduct experiments and two brain-like organoids were obtained according to the method in Example 1.

[0211] The obtained brain organoids were analyzed by flow cytometry, and the specific steps are as follows:

[0212] Single-cell suspension preparation: Brain organoids cultured to the target stage were aseptically transferred to 15 mL centrifuge tubes; papain-Accutase mixed digestion solution (papain Worthington#LS003119, Accutase Thermo Fisher#A1110501, volume ratio 1:1) was added, with the digestion solution volume being more than 5 times the volume of the organoids; the tubes were placed in a 37℃, 5% CO2 incubator for 45 minutes for digestion. During digestion, the centrifuge tubes were removed every 10 minutes, and the organoids were gently ground with a sterile disposable blade (avoiding excessive force to avoid damaging the cells). The digestion status was observed under an optical microscope until most of the tissue was dispersed into single cells.

[0213] Filtration and purification: Prepare a 0.45μm sterile cell sieve (Corning#352340) in advance and place it on a new 15mL centrifuge tube; slowly filter the digested cell suspension through the cell sieve, rinse the sieve twice with 1mL PBS buffer (Gibco#14190-144), collect the filtered single-cell suspension, and remove undigested tissue fragments.

[0214] Cell counting and concentration adjustment: Mix 20 μL of single-cell suspension with 20 μL of trypan blue staining solution (Sigma-Aldrich #T8154, 0.4% concentration) and incubate at room temperature for 5 minutes. Count viable cells (trypan blue-rejected cells) using a hemocytometer or automated cell counter (ThermoFisher #C10227). Adjust the cell concentration to 1 × 10⁻⁶ cells using PBS buffer based on the counting results. 6 -5×10 6 cells / mL.

[0215] Sample grouping and loading: Take sterile flow cytometry tubes (BD#352052) and label them as experimental group, isotype control group, and blank control group (PBS only). Set up 3 replicates for each group. Add 100 μL of single-cell suspension with adjusted concentration to each flow cytometry tube. Add only 100 μL of PBS buffer to the blank control group.

[0216] 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 cytometer of the experimental group and the isotype control group, and incubate at room temperature in the dark for 15 minutes to block non-specific binding sites on the cell surface.

[0217] Fluorescent antibody incubation: Add fluorescently labeled specific antibodies to the flow cytometry tubes of the experimental group according to the recommended ratio in the antibody instructions (recommended antibody combinations and parameters are as follows); add an equal amount of the corresponding species' isotype control antibody to the isotype control group; no antibody is added to the blank control group. The antibody names and types are as follows:

[0218] IBA1-PE (IBA1(E4O4W)Rabbit Monoclonal Antibody, PE ConjugateBioLegend#690504, 2μL / tube);

[0219] TMEM119-FITC (TMEM119(E4B9S) Mouse Monoclonal Antibody, FITC ConjugateCST#98778-FITC, 3μL / tube);

[0220] CD45-APC (CD45(D3F8Q)Rabbit Monoclonal Antibody, APC Conjugate CST#70257-APC, 2μL / tube);

[0221] CD11b-PerCP-Cy5.5 (CD11b(M1 / 70) Mouse Monoclonal Antibody, PerCP-Cy5.5Conjugate BioLegend#101228, 2μL / tube).

[0222] Gently mix using a vortex mixer (500 rpm for 5 seconds), and incubate at 4°C in the dark for 30 minutes to avoid quenching of the fluorescent antibody and non-specific binding.

[0223] Washing to remove free antibodies: After incubation, add 2 mL of PBS buffer to each flow cytometer, centrifuge at 1000 rpm for 5 minutes (room temperature), and carefully discard the supernatant; repeat washing twice to completely remove unbound free antibodies.

[0224] Flow cytometry detection and data analysis: Open the flow cytometer data acquisition software, establish the detection protocol, first run the blank control group, adjust the threshold to exclude debris and noise signals, set the gate with FSC (forward scattered light) vs SSC (side scattered light), and select the target cell population.

[0225] Samples from the control group and the experimental group were loaded sequentially. 1×10^4-1×10^5 target cell events were collected from each tube, and fluorescence signal intensity data were recorded.

[0226] During the testing process, a blank control was run every 10 samples to calibrate the instrument drift.

[0227] Import the raw data into flow cytometry software (such as FlowJo, BDFACSDiva) and perform data preprocessing.

[0228] Statistical methods (such as independent samples t-test) were used to analyze differences between groups.

[0229] Generate flow cytometry scatter plots, histograms, and statistical analysis reports to obtain the results of brain organoid flow cytometry detection, such as... Figure 7 As shown.

[0230] The results showed the flow cytometry findings of brain organoids containing microglia on day 70. Two iPSC lines from healthy adult Chinese men were used in the experiment. Microglia were sorted using IBA1, and the proportions of IBA1+ microglia obtained were 10.60% and 9.72%, respectively. This result is consistent with the proportion of microglia in the real human brain, with very small batch-to-batch variation and extremely high reproducibility—a characteristic not found in other existing models.

[0231] Comparative Example 1

[0232] Referring to existing technology CN15404218B, a method using SMADi to induce brain organoids containing microglia is employed, adding two additional small molecules as a protocol. The specific process is as follows: The EB formation phase generally lasts for the first 6 days of culture. Cells seeded into low-adsorption 96-well plates aggregate to form EB spheres after 24 hours, which is recorded as day 1. For the first three days of EB culture, "Cerebral organoids EB formation medium 1" (first medium) is used; from day 3 to day 6, the medium is changed to "Cerebral organoids EB formation medium 2" (second medium). Afterwards, the EB spheres are cultured until day 6 for the neural induction phase. After EB sphere formation, the medium needs to be changed to the sixth medium on day 6. After EB spheres form, the culture medium needs to be changed to neural induction medium (NIM) (third medium) on day 8. After the neural induction culture stage, the culture medium needs to be changed to neural differentiation medium (NDM) (fourth medium) on day 12. After the neural differentiation culture stage, the culture medium needs to be changed to neural maturation medium (NMM) (fifth medium) on day 30. Finally, after changing the six different culture media after EB formation, a 96-well plate of EB is obtained on day 60, yielding microglial organoids containing IBA1 and CD11b positive cells.

[0233] Compared to this method, this invention, through optimization of experimental procedures, introduces a large number of lineage-purified macrophage progenitor cells at the initial stage of neural induction (day 0). Within a period of approximately 45-55 days, approximately 300 EB microglia organoids containing four microglia-specific markers—IBA1, CD11b, TMEM119, and CD45—can be collected. Therefore, this invention uses fewer types of culture media while achieving higher organoid yields.

[0234] Meanwhile, in Comparative Example 1, iPSC differentiation was uncontrolled, and the intermediate cell types were uncontrollable, resulting in a free differentiation process. Therefore, it was impossible to determine the specific stage from which the microglia progenitor cells originated. Using the same method as in Example 1, i.e., stimulating the brain organoids of Comparative Example 1 with the same interleukin-6, the results showed that since microglia did not participate in the early spheroidization process, no difference was detected in CD235 and SOX2, meaning they could not respond to interleukin-6 stimulation. However, in Example 1 of this invention, the cell types at each stage were determined, and the ratio of PMP and NPC progenitor cells was clearly defined. Therefore, the generation stage of microglia progenitor cells can be accurately determined, facilitating sampling and research at each stage.

[0235] Comparative Example 2

[0236] Following the culture protocol of existing technology CN113924362B, mature organoids and macrophages were fused on day 26 after induction, specifically by adding free microglia progenitor cells to the mature organoids. The specific steps were: generating brain organoids and primitive macrophage-like cells (iMacs) from identical human iPSCs. Microglia were observed in the human fetal brain at 4.5 weeks. The iMacs were then co-cultured with relatively young brain organoids (day 26) simulating the fetal brain in early pregnancy.

[0237] The results showed that the infiltration scheme could not simulate the influence of microglia and their progenitor cells on the differentiation of neural progenitor cells into neurons, thus missing the early stages of real human brain development. This is because in Comparative Example 2, organoid formation and the introduction of microglia occurred sequentially, without temporal or spatial overlap; therefore, the influence of microglia on this process during organoid formation could not be replicated. Meanwhile, the immunofluorescence results from the examples clearly showed that microglia, upon external stimulation, significantly altered the ratio of the two early cell types. Figure 6 CD43 is a specific marker for PMP cells, and SOX2 is a specific marker for NPC cells, both showing significant differences. However, when the brain organoids of Comparative Example 2 were stimulated with the same method as in Example 1, i.e., with the same interleukin-6, no difference was detected between CD43 and SOX2 because microglia did not participate in the early spheroidization process.

[0238] Simultaneously, the proportion of microglia in the final brain-like organoid was detected using flow cytometry, following the same detection procedure as in Example 1. The results showed that the proportion was less than 5%, which does not conform to the proportion of microglia in the real human brain.

[0239] Comparative Example 3

[0240] The culture medium used in Comparative Example 2 was partially replaced with the culture medium of the present invention. On days 5-6, the induction medium consisting of DMEM:F12, 1XN2 additive (Invitrogen), 10 μg / mL heparin (Sigma), 1X penicillin / streptomycin, 1X non-essential amino acids, 1X Glutamax, 4 ng / mL WNT-3A (R&D Systems), 1 μM CHIR99021 (Cellagentech), and 1 μM SB-431542 (Cellagentech) was replaced with the neural progenitor cell maturation medium of the present invention. The organoid growth medium supplemented with 100 ng / mL CSF-1 used during co-culture (day 26) was replaced with the maturation medium of the present invention after 15 days. The DAY55 organoids at the same time points were then subjected to relevant tests. The results showed that although the culture medium was replaced, the proportion of microglia was still far below 8% due to the fusion mode of microglia precursor cells and the difference in fusion time. Meanwhile, the staining results of microglia showed that their colocalization ratio with neurons was about 5%, which was lower than that of the model prepared in the embodiment of the present invention, suggesting that the phagocytic function of microglia based on interactions with other cell types was poor in the comparative scheme.

[0241] Comparative Example 4

[0242] Referring to the scheme of the existing technology CN18389433A, using the culture medium of the existing technology CN18389433A, mature brain-like organisms and free mature microglia were co-cultured on day 30 to finally obtain IBA1-positive brain organoids containing microglia. The specific operation steps are as follows: (1) Pluripotent stem cells were used to induce the differentiation of CD43+ microglia precursor cells in sequence using culture medium I, culture medium II, culture medium III and culture medium VIII; (2) Pluripotent stem cells were used to induce the differentiation of brain-like organisms in sequence using culture medium IV, culture medium V, culture medium VI and culture medium VII; (3) The CD43+ microglia precursor cells and brain-like organisms obtained in steps (1) and (2) were co-cultured in sequence using culture medium VIII, culture medium IX and culture medium X to obtain neuroimmune brain-like organisms.

[0243] The proportion of microglia in the final neuroimmune brain-like organism was detected by flow cytometry, following the same procedure as in Example 1. The detected microglia proportion was 2.39%, which does not conform to the proportion of microglia in the real human brain.

[0244] Meanwhile, compared to Comparative Examples 1-4, all only explored the cell type identification of microglia and did not deeply demonstrate that the model possesses the immune response function of the real human brain. Furthermore, the experimental designs of the comparative examples could not explore the influence of microglia on the entire organoid formation process at any stage of development, especially in the early stages. In Example 1, because the two germ layer cells are mixed before organoid formation, microglia and their progenitor cells are also involved in this process. Furthermore, Example 1 achieved a systemic response to immune changes in the in vitro human brain organoid model. The final result showed that IL-6, as a common immune factor, directly acts on microglia and significantly affects their function, leading to a significant decrease in the proportion of progenitor cells, a decrease in microglia phagocytic function, and impaired cell proliferation in the immune-altered group. Compared to other comparative examples, Example 1 has a more complete immune response system and intercellular functional interactions, making it a more ideal immune human brain organoid model.

[0245] Comparative Example 5

[0246] Optimization of culture medium

[0247] During the pre-induction phase of brain organoids containing microglia, specifically days 21-40 of Example 1, the components of the NPC medium in the MO-1 medium were replaced with:

[0248] Neurobasal medium (Thermo Fisher Scientific) and DMEM / F12 medium were mixed at a mass ratio of 1:1, and small molecules 1×N2, 1×B27-RA (Thermo Fisher Scientific), and FGF2 (20 ng / ml, Peprotech) were added.

[0249] Everything else is the same as in Example 1. Using the above method, 3D human brain organoids cultured to day 55 were identified by immunofluorescence. The positive rate of the specific marker NESTIN was approximately 60%, and the positive rate of the broad-spectrum proliferation marker Ki67 was approximately 40%. Both of these figures are significantly lower than the results in Example 1. Meanwhile, the yield of brain organoids was approximately 15 per batch (each 96-well culture plate), and the growth diameter of the brain organoids on day 45 was 80-100 μm.

[0250] Comparative Example 6

[0251] Optimization of culture medium

[0252] 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).

[0253] 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.

[0254] 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.

[0255] 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 brain 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 brain organoids containing microglia; 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 embryoids; The neural induction medium was STEMdiff containing 0.01-0.02 mM Y-27632. TM Neural induction culture medium; 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 culture medium; In step S3, the fusion steps include: Neural progenitor cells were digested into neural progenitor single cells; neural progenitor single cells and macrophage progenitor cells were fused in fusion medium for 1-5 days; 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; 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; In step S3, after culturing for another 25-30 days, the brain organoids are embedded, and then induced to form 3D brain organoids containing microglia using a mature culture medium. The culture medium used for further culturing is fusion medium; 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 The fusion in steps S1 and S3 both used AggreWell culture plates. TM 800 culture plate.

2. 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.

3. 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.

4. The cultivation method according to claim 1, characterized in that, Neural progenitor cells and macrophage progenitor cells were fused at a ratio of 5-9:3.

Images