A cerebral organoid containing microglia cells, and a preparation method and application thereof

Abstract

The application provides a microglial cell-containing brain organoid and a preparation method and application thereof, and belongs to the technical field of brain organoids.The present application promotes the development of microglial cells in the brain organoid, accelerates the development of neurons and neuron synapses, and can promote the development of forebrain, pre-cortical plate, hippocampus, choroid plexus and hindbrain in the brain organoid, and the brain organoid obtained can spontaneously differentiate into microglial cells, which has great significance in brain development research, nervous system disease research or drug screening and the like.

Description

Technical Field

[0001] This invention relates to the field of brain organoid technology, and more particularly to a brain organoid containing microglia, its preparation method, and its application. Background Technology

[0002] Human brain organoids are organ-like tissues formed spontaneously in vitro by the induced differentiation of human pluripotent stem cells (hPSCs). They are composed of various cell types and closely replicate the physiological and pathological characteristics of human brain tissue in terms of structure and function. Furthermore, human brain organoids differentiated from patient-derived induced pluripotent stem cells (iPSCs) carry the patient's genetic information and can serve as an excellent model for studying the mechanisms of human-specific brain diseases. They also hold great promise in biomedical and preclinical drug testing.

[0003] Currently, compared to other methods for creating brain organoids, the Lancaster method (Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly[J]. Nature, 2013, 501(7467):373-379) is the most efficient and stable method for creating brain organoids. However, this type of brain has a major drawback: it lacks microglia (MG) cells derived from the mesoderm. Microglia are essential cells for neuronal maturation and synapse formation, playing a crucial role in brain development and maturation. There are currently many methods for creating microglia-containing brain organoids, roughly divided into three types: 1. Co-culturing brain organoids with human immortalized microglia; 2. Co-culturing microglia and brain organoids derived from stem cells of the same origin; 3. Implanting human brain organoids into rat brains and fusing them with microglia in the rat brain. However, it remains unproven whether these exogenous microglia can perfectly integrate with brain organoids and exert the same effects as the microglia in the human brain.

[0004] Studies have shown that exogenously implanted microglia function differently from microglia spontaneously formed during brain organoid culture under inflammatory stimulation. Therefore, compared to the implantation of exogenous microglia, the spontaneous differentiation of microglia into brain organoids is more desirable. However, this method currently has the following problems: ① the number of microglia in the brain-like organoid is unstable; ② MG brain-like organoids are not easy to survive. Therefore, how to efficiently prepare 3D brain organoids containing microglia has become an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the present invention provides a method for efficiently preparing 3D brain organoids containing microglia.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing brain organoids, comprising the following steps:

[0008] (1) Culture human induced pluripotent stem cells, passage them every 5-7 days, and select undifferentiated human induced pluripotent stem cells;

[0009] (2) Separate the cells selected in step (1), and mix the separated cells with EB seed culture medium to prepare a cell suspension;

[0010] (3) Culture the cell suspension, and add EB formation medium at 47-49h and 95-97h respectively. After 4-5 days, select embryos with clear edges and a diameter between 300-600μm.

[0011] (4) The embryonic bodies described in step (3) are mixed with the neuroblastocyst induction medium and cultured for 46-50 hours. Embryonic bodies with transparent and irregular edges are selected for further differentiation.

[0012] (5) Add the matrix gel to the embryo body selected in step (4) to wrap the embryo body. After the matrix gel solidifies, culture it with brain organoid amplification culture medium. After 70-75 hours, select brain organoids with folded outer edges.

[0013] (6) Discard the amplification culture medium of brain organoids, add brain organoid maturation culture medium for culture, change the culture medium every 2-3 days, culture for 14-16 days to obtain mature brain organoids.

[0014] The EB formation medium, neuroblast induction medium, brain organoid amplification medium, and brain organoid maturation medium contain nicotinamide mononucleotide.

[0015] Preferably, the initial culture density of the human induced pluripotent stem cells is 0.8–1.2 × 10⁻⁶. 6 per mL.

[0016] Preferably, the generation ratio is 1:4 to 1:6.

[0017] Preferably, the cell density of the cell suspension is 85,000 to 95,000 cells / mL.

[0018] Preferably, each embryo is individually coated with matrix gel, and the volume of matrix gel corresponding to each embryo is 20-25 μL.

[0019] Preferably, embryos coated with matrix gel are cultured in 6-well low-mount plates, with 6 to 8 embryos placed in each well.

[0020] Preferably, the concentration of nicotinamide mononucleotide in EB formation medium, neuroblastoma induction medium, brain organoid amplification medium, and brain organoid maturation medium is 180–220 μM.

[0021] Preferably, the cultivation conditions described in steps (1) to (6) are: 4-6% CO2, 35-40℃.

[0022] The present invention also provides brain organoids obtained by the preparation method, wherein the brain organoids comprise microglia.

[0023] This invention provides the application of the aforementioned brain organoids in brain development research, nervous system disease research, or drug screening.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention cultured human induced pluripotent stem cells and added 180-220 μM NMN to EB formation medium, neuroblastoma induction medium, brain organoid expansion medium, and brain organoid maturation medium, respectively. This promoted the development of microglia in brain organoids, accelerated the development of neurons and neuronal synapses, and also promoted the development of the forebrain, anterior cortical plate, hippocampus, choroid plexus, and hindbrain in brain organoids. Brain organoids that can spontaneously differentiate into microglia were obtained, which is of great significance in brain development research, nervous system disease research, or drug screening. Attached Figure Description

[0025] Figure 1 Morphological images of human induced pluripotent stem cells at different culture stages.

[0026] Figure 2 This is a flowchart of a brain organoid culture method containing microglia.

[0027] Figure 3 The diagram shows the expression of neuronal and axonal and dendritic markers in brain organoids of Example 1 and Comparative Example 1.

[0028] Figure 4 This is a diagram showing the expression of microglia markers in brain organoids of Example 1 and Comparative Example 1.

[0029] Figure 5 This is a diagram showing the expression of biomarkers in different brain regions in brain organoids of Example 1 and Comparative Example 1. Detailed Implementation

[0030] This invention provides a method for preparing brain organoids, comprising the following steps:

[0031] (1) Culture human induced pluripotent stem cells, passage them every 5-7 days, and select undifferentiated human induced pluripotent stem cells with enlarged cell colonies.

[0032] (2) Separate the cells selected in step (1), and mix the separated cells with EB seed culture medium to prepare a cell suspension;

[0033] (3) The cell suspension was cultured, and EB formation medium was added at 47-49h and 95-97h respectively. After 4-5 days, embryos with clear edges and a diameter between 300-600μm were selected for further differentiation.

[0034] (4) Mix the embryonic bodies from step (3) with the neuroblastocyst induction medium and culture them for 46-50 hours. Select the embryonic bodies with transparent and irregular edges for the next differentiation.

[0035] (5) Add matrix gel to the embryo body to wrap the embryo body. After the matrix gel solidifies, culture it with brain organoid amplification medium. After 70-75 hours, select brain organoids with folded outer edges for further differentiation.

[0036] (6) Discard the brain organoid expansion culture medium, add brain organoid maturation culture medium for culture, change the culture medium every 2-3 days, and culture for 14-16 days to obtain mature brain organoids.

[0037] The EB formation medium, neuroblast induction medium, brain organoid amplification medium, and brain organoid maturation medium contain nicotinamide mononucleotide.

[0038] This invention involves culturing human induced pluripotent stem cells, changing the culture medium daily, passaged every 5-7 days, removing differentiated cells before each passage, and selecting undifferentiated stem cells with larger cell colonies, denser and brighter centers for further culture.

[0039] The human induced pluripotent stem cells of this invention are preferably male neonatal foreskin cells infused with transcription factors (OCT4 / SOX2 / KLF4 / MYC), purchased from the Chinese Academy of Sciences Cell Bank. This step uses serum-free mTeSR1 medium for culture. Preferably, the human induced pluripotent stem cells are seeded in six-well plates at a density of 0.8–1.2 × 10⁻⁶. 6 The concentration of cells / mL is more preferably 0.9–1.1 × 10⁻⁶. 6 Cells / mL, more preferably 1×10⁻⁶ 6Cells / mL. The culture medium is changed daily during the culture process, and passages are performed every 5–7 days, more preferably every 6 days, with a passage ratio of 1:4–1:6, more preferably 1:4.5–1:5.5, and even more preferably 1:5. The culture is preferably carried out in an incubator at a temperature of 35–40°C, more preferably 36–38°C, and even more preferably 37°C; the CO2 concentration is 4–6%, more preferably 4.5–5.5%, and even more preferably 5%. Before each passage, differentiated cells are removed, and undifferentiated stem cells with larger cell colonies, denser centers, and brighter centers are selected for the next stage of culture.

[0040] In this invention, undifferentiated stem cells with enlarged cell colonies and dense, bright centers were selected. The cells were washed 1-2 times with 1×PBS, digested with a mild separation solution, and after the cell edges thickened and curled, the mild separation solution was removed. The cells were then washed 1-2 times with DMEM / F12 medium, gently resuspended in EB seed medium, centrifuged, and the medium was removed. Seed medium was added to resuspend the cells, resulting in a cell suspension. The cells were then counted using a hemocytometer. The mild separation solution is preferably 0.5 mM EDTA; the digestion time is 5–8 min, more preferably 6–7 min, and even more preferably 6.5 min; the volume of the first added EB seed culture medium is 2.5–3.5 mL, more preferably 3 mL; the centrifugation time is 4–6 min, more preferably 4.5–5.5 min, and even more preferably 5 min; the centrifugation speed is 700–900 rpm, more preferably 750–850 rpm, and even more preferably 800 rpm; the volume of the second added EB seed culture medium is 1.5–2.5 mL, more preferably 2 mL; the cell density in the cell suspension is 85,000–95,000 cells / mL, more preferably 87,000–92,000 cells / mL, and even more preferably 90,000 cells / mL.

[0041] In this invention, the cell suspension is added to the wells of each 96-well low-adhesion plate, and the 96-well low-adhesion plate is placed in an incubator for 24 hours without interference. EB formation medium is gently added at 47–49 hours and 95–97 hours of culture, respectively. After 4–5 days, embryos with clear edges and a diameter between 300–600 μm are selected for further differentiation. The volume of cell suspension added to the 96-well low-adhesion plate is 80–120 μL / well, more preferably 90–110 μL / well, and more preferably 100 μL / well; the timing of adding EB formation medium is at 47–49 hours and 95–97 hours of culture, more preferably 48 hours and 96 hours of culture; the diameter of the embryos for further differentiation is 300–600 μm, more preferably 400–500 μm, and more preferably 450 μm.

[0042] In this invention, neuroblastoma induction medium is added to a 24-well low-adhesion plate. Using a 200 μL wide-bore pipette tip, embryos from a 96-well plate are carefully transferred to the 24-well low-adhesion plate (one embryo per well). The 24-well plate is shaken 3-4 times and incubated at 37°C. The embryos are observed daily for the appearance of transparent, irregular edges. After continuous incubation for 46-50 hours, preferably 47-49 hours, and more preferably 48 hours, embryos with transparent, irregular edges are selected for further differentiation. The volume of neuroblastoma induction medium added to the 24-well low-adhesion plate is 0.8-1.2 mL / well, preferably 0.9-1.1 mL / well, and more preferably 1 mL / well.

[0043] In this invention, individual embryos are transferred one by one to a 100mm culture dish using a 200μL wide-bore pipette tip. Under ice bath conditions, 20-25μL of Matrigel is pipetted onto the embryo (EB) to coat each EB individually. The 100mm culture dish is then placed in an incubator for incubation. The incubation temperature is 35-40℃, more preferably 36-39℃, and even more preferably 37℃. The CO2 concentration during incubation is 4-6%, more preferably 4.5-5.5%, and even more preferably 5%. The incubation time is 28-32 min, more preferably 29-31 min, and even more preferably 30 min. After the Matrigel solidifies, the Matrigel-coated embryos are transferred to a 6-well low-adhesion plate, brain organoid amplification medium is added, and the 6-well low-adhesion plate is incubated for 72 hours. Brain organoids with highly folded outer edges are then selected for further differentiation. The number of embryos in the 6-well low-adhesion plate is 6-8 per well, more preferably 7 per well; the added brain organoid amplification culture medium is 2.5-3.5 ml per well, more preferably 2.7-3.2 ml per well, and even more preferably 3 ml per well. The culture temperature is 35-40°C, more preferably 36-39°C, and even more preferably 37°C; the CO2 concentration of the culture is 4-6%, more preferably 4.5-5.5%, and even more preferably 5%.

[0044] In this invention, the brain organoid amplification medium is aspirated from a 6-well plate, washed with PBS, and then added to a brain organoid maturation medium. The plate is then placed on a shaker in an incubator and cultured. The medium is changed every 2-3 days. Mature brain organoids are formed on days 14-16. The volume of the brain organoid maturation medium added in this invention is 2.5-3.5 ml / well, more preferably 2.7-3.2 ml / well, and more preferably 3 ml / well. The culture temperature is 35-40°C, more preferably 36-39°C, and more preferably 37°C; the CO2 concentration is 4-6%, more preferably 4.5-5.5%, and more preferably 5%; the shaker speed is 60-70 rpm, more preferably 62-68 rpm, and more preferably 65 rpm.

[0045] In this invention, the concentration of nicotinamide mononucleotide (NMN) in EB formation medium, neuroblastoma induction medium, brain organoid amplification medium, and brain organoid maturation medium is 180-220 μM, more preferably 190-210 μM, and more preferably 200 μM.

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] In an embodiment of the present invention, human induced pluripotent stem cells were purchased from the Chinese Academy of Sciences Cell Bank and were obtained by reprogramming male neonatal foreskin cells and introducing transcription factors (OCT4 / SOX2 / KLF4 / MYC).

[0048] The matrix gel was purchased from the Corning website, and the non-essential amino acids were purchased from the Gibico website.

[0049] Example 1

[0050] In this embodiment, the culture media for each stage of brain organoid formation are prepared as follows:

[0051] EB (embryoid body) formation medium: 225ml DMEM / F12 medium, 5ml glutamine, 5ml non-essential amino acids, 15ml fetal bovine serum for embryonic stem cells, 225ml E8 complete medium, 3.5μl 2-mercaptoethanol. Mix the above reagents and add NMN (final concentration 200μM). Filter through a 0.2μm microporous filter and store at 4℃ for 2 weeks.

[0052] EB seed culture medium: Y-27632 (final concentration 10 μM) was added to EB formation medium.

[0053] Neuroblastoma induction medium: 485 ml DMEM / F12 medium, 5 ml N2 supplement, 5 ml glutamine, 5 ml non-essential amino acid solution, 50 μg heparin (final concentration 0.1 μg / ml). After mixing the above reagents, add NMN (final concentration 200 μM), filter through a 0.2 μm microporous filter, and store at 4°C for 2 weeks.

[0054] Brain organoid amplification medium: 250ml BrainPhys™ medium, 1.25ml N2 supplement, 2.5ml Vitamin A-free NeuroCult medium. TM SM1 supplement: 2.5 ml glutamine, 2.5 ml penicillin and streptomycin, 87.5 μl 1% 2-mercaptoethanol. Mix the above reagents and add NMN (final concentration 200 μM). Filter through a 0.2 μm filter. Store at 4°C for 2 weeks.

[0055] Brain organoid maturation culture medium: 250ml BrainPhys TM Culture medium, 1.25 ml N2 additive, 2.5 ml glutamine, 62.5 μl insulin, 2.5 ml penicillin and streptomycin, 87.5 μl 1% 2-mercaptoethanol. Mix the above reagents and add NMN (final concentration 200 μM). Filter through a 0.2 μm filter and store at 4°C for 2 weeks.

[0056] (1) Human induced pluripotent stem cells (at a density of 1×10⁻⁶) were cultured using the serum-free mTeSR1 culture system from STEM CELL. 6 The cells were seeded in six-well plates and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed daily, and the cells were passaged every 6 days at a ratio of 1:5. Differentiated cells were removed before each passage. Undifferentiated human pluripotent stem cells with larger, denser, and brighter central colonies were selected for brain organoid differentiation.

[0057] (2) Selected cells were washed once with 1×PBS, digested with mild separation buffer (0.5mM EDTA) for 6 min, and after the cell edges thickened and curled, the mild separation buffer was removed. The cells were washed once with DMEM / F12 medium, and the cells were gently resuspended with 3 ml of EB seed medium. The cells were centrifuged for 5 min at 800 rpm, the medium was removed, and the cells were resuspended with 2 ml of EB seed medium. The cells were counted using a hemocytometer to achieve a cell density of 90,000 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well low-adhesion plate. The 96-well low-adhesion plate was placed in an incubator and left undisturbed for 24 hours. 100 μL of LEB was gently added to form culture medium at 48 and 96 h of culture. After 5 days, embryonic bodies with clear edges and a diameter between 300 and 600 μm were selected for further differentiation.

[0058] (3) Add 1 mL of neuroblastoma induction medium to a 24-well low-adhesion plate. Carefully transfer the embryos from the 96-well plate into the 24-well low-adhesion plate using a 200 μL pipette tip with a wide diameter. Place one embryo per well. Shake the 24-well low-adhesion plate three times and incubate it in a 37°C incubator. Observe the embryos daily to see if they develop transparent irregular edges. After 48 hours of continuous culture, select the embryos with transparent irregular edges for the next differentiation step.

[0059] (4) Using a 200μL pipette tip, transfer each individual embryo to a 100mm culture dish. Under ice bath conditions, use a pipette to add 20μL of Matrigel onto the embryo to coat each embryo. Place the 100mm culture dish in a 37℃ incubator for 30 minutes. After the Matrigel solidifies, carefully transfer the Matrigel-coated embryos to a 6-well plate with 7 embryos per well. Add 3ml of brain organoid amplification medium per well. Place the 6-well plate in an incubator for 72 hours (37℃, 5% CO2). Select brain organoids with highly folded outer edges for further differentiation.

[0060] (5) Aspirate the brain organoid amplification medium from the 6-well plate, wash with PBS, add 3 ml of brain organoid maturation medium per well, place on a shaker in an incubator (37℃, 5% CO2) at a shaking speed of 65 rpm, change the medium every three days, and culture until day 15 when mature brain organoids are formed.

[0061] Example 2

[0062] In this embodiment, the NMN concentration in the EB formation medium, neuroblast induction medium, brain organoid amplification medium, and brain organoid maturation medium is 180 μM, and the other components of the medium are the same as in Example 1.

[0063] (1) Human induced pluripotent stem cells (at a density of 0.8 × 10⁻⁶) were cultured using the serum-free mTeSR1 culture system from STEM CELL. 6 The cells were seeded in six-well plates and cultured in a 4% CO2 incubator at 35°C. The culture medium was changed daily, and the cells were passaged every 5 days at a ratio of 1:4. Differentiated cells were removed before each passage. Undifferentiated human pluripotent stem cells with larger, denser, and brighter central colonies were selected for brain organoid differentiation.

[0064] (2) Selected cells were washed once with 1×PBS, and digested for 5 min with mild separation buffer (0.5 mM EDTA). After the cell edges thickened and curled, the mild separation buffer was removed, and the cells were washed once with DMEM / F12 medium. The cells were gently resuspended with 2.5 ml of EB seed medium and centrifuged for 4 min at 700 rpm. The medium was removed, and the cells were resuspended with 1.5 ml of EB seed medium. The cells were counted using a hemocytometer to achieve a cell density of 85,000 cells / mL. 80 μL of the cell suspension was added to each well of a 96-well low-adhesion plate. The 96-well low-adhesion plate was placed in an incubator and left undisturbed for 24 h. 80 μL of EB was gently added to form culture medium at 47 and 95 h of culture. After 5 days, embryonic bodies with clear edges and a diameter between 400 and 500 μm were selected for further differentiation.

[0065] (3) Add 0.8 mL of neuroblastoma induction medium to a 24-well low-adhesion plate. Carefully transfer the embryos from the 96-well plate to the 24-well low-adhesion plate using a 200 μL pipette tip with a wide diameter. Place one embryo per well. Shake the 24-well low-adhesion plate three times and incubate it in a 37°C incubator. Observe the embryos daily to see if they develop transparent irregular edges. After 46 hours of continuous culture, select the embryos with transparent irregular edges for the next differentiation step.

[0066] (4) Using a 200μL pipette tip, transfer each individual embryo to a 100mm culture dish. Under ice bath conditions, use a pipette to add 20μL of Matrigel onto the embryo to coat each embryo. Place the 100mm culture dish in a 35℃ incubator for 28 minutes. After the Matrigel solidifies, carefully transfer the Matrigel-coated embryos to a 6-well plate with 6 embryos per well. Add 2.5ml of brain organoid amplification medium per well. Place the 6-well plate in an incubator for 72 hours (35℃, 4% CO2). Select brain organoids with highly folded outer edges for further differentiation.

[0067] (5) Aspirate the brain organoid amplification medium from the 6-well plate, wash with PBS, add 2.5 ml of brain organoid maturation medium per well, place on a shaker in an incubator (35℃, 4% CO2) at a shaking speed of 60 rpm, change the medium every three days, and culture until day 14 when mature brain organoids are formed.

[0068] Example 3

[0069] In this embodiment, the NMN concentration in the EB formation medium, neuroblast induction medium, brain organoid amplification medium, and brain organoid maturation medium is 220 μM, and the other components of the medium are the same as in Example 1.

[0070] (1) Human induced pluripotent stem cells (at a density of 1.2 × 10⁻⁶) were cultured using the serum-free mTeSR1 culture system from STEM CELL. 6 The cells were seeded in six-well plates and cultured in a 6% CO2 incubator at 40°C. The culture medium was changed daily, and the cells were passaged every 5 days at a ratio of 1:6. Differentiated cells were removed before each passage. Undifferentiated human pluripotent stem cells with larger, denser, and brighter central colonies were selected for brain organoid differentiation.

[0071] (2) Selected cells were washed once with 1×PBS, digested with mild separation buffer (0.5mM EDTA) for 8 min, and after the cell edges thickened and curled, the mild separation buffer was removed. The cells were washed once with DMEM / F12 medium, and the cells were gently resuspended with 3.5 ml of EB seed medium. The cells were centrifuged for 6 min at 900 rpm, the medium was removed, and the cells were resuspended with 2.5 ml of EB seed medium. The cells were counted using a hemocytometer to achieve a cell density of 95,000 cells / mL. 120 μL of the cell suspension was added to each well of a 96-well low-adhesion plate. The 96-well low-adhesion plate was placed in an incubator and left undisturbed for 24 h. 120 μL of LEB was gently added at 49 and 97 h of culture to form culture medium. After 5 days, embryonic bodies with clear edges and a diameter between 300 and 500 μm were selected for further differentiation.

[0072] (3) Add 1.2 mL of neuroblastoma induction medium to a 24-well low-adhesion plate. Carefully transfer the embryos from the 96-well plate into the 24-well low-adhesion plate using a 200 μL pipette tip with a wide diameter. Place one embryo per well. Shake the 24-well low-adhesion plate 4 times and incubate it in a 37°C incubator. Observe the embryos daily to see if they develop transparent irregular edges. After 50 hours of continuous culture, select the embryos with transparent irregular edges for the next differentiation step.

[0073] (4) Using a 200μL wide-bore pipette tip, each individual embryo was transferred to a 100mm culture dish. Under ice bath conditions, 25μL of Matrigel was pipetted onto the embryo to coat each embryo. The 100mm culture dish was placed in a 40℃ incubator for 32 minutes. After the Matrigel solidified, the Matrigel-coated embryos were carefully transferred to 6-well low-profile plates, with 6 embryos per well. 3.5ml of brain organoid amplification medium was added to each well. The 6-well low-profile plates were placed in an incubator for 72 hours (40℃, 6% CO2). Brain organoids with highly folded outer edges were selected for further differentiation.

[0074] (5) Aspirate the brain organoid amplification medium from the 6-well plate, wash with PBS, add 3.5 ml of brain organoid maturation medium per well, place on a shaker in an incubator (40℃, 6% CO2) at a shaking speed of 70 rpm, change the medium every three days, and culture until day 16 when mature brain organoids are formed.

[0075] Comparative Example 1

[0076] Unlike Example 1, the EB formation medium, neuroblast induction medium, brain organoid amplification medium, and brain organoid maturation medium do not contain nicotinamide mononucleotide (NMN), and the preparation process is the same as in Example 1.

[0077] Experimental Example 1

[0078] Total RNA from mature brain organoids prepared in Example 1 and Comparative Example 1 was extracted using an RNA extraction kit. The RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit. A 100 μL enzyme-free, sterile eight-tube PCR tube was used, and the following reaction mixture was added sequentially: 1 μL cDNA (100–200 μg / μL), 10 μL 2×SYBR Green fluorescent mix, 0.4 μL Rox, 0.3 μL upstream primer, 0.3 μL downstream primer, and 8.0 μL ddH2O. The tube was placed in a real-time quantitative PCR instrument, and the amplification conditions were set as follows: 94℃ pre-denaturation for 30 s, 94℃ denaturation for 5 s, 58–61℃ annealing for 15 s, and 72℃ extension for 10 s. This denaturation, annealing, and extension cycle was performed for 40 times. The amplification and melting curves were checked to ensure they met the requirements, and the ΔCT value was calculated. The expression of neuronal and axonal and dendritic markers in brain organoids of Comparative Example 1 and Example 1 was determined using the real-time quantitative PCR technique described above. The results are as follows: Figure 3 As shown. Figure 3The results showed that, compared with Comparative Example 1, the expression levels of neurons (TUJ1), presynaptic membrane (hSyn), postsynaptic membrane (PSD95), and microtubule-associated protein (MAP2) genes were increased in the MG-containing brain organoids of Example 1, indicating that NMN can accelerate the development of neurons and neuronal synapses.

[0079] Experiment Example 2

[0080] The expression of microglial cell markers in Comparative Example 1 and Example 1 was determined using real-time quantitative PCR. The experimental method was the same as in Example 1, and the results are as follows: Figure 4 As shown. Figure 4 The results showed that, compared with Comparative Example 1, the expression levels of microglia genes (IBA1, CX3CR1) in brain organoids of Example 1 were increased, indicating that NMN can promote the development of microglia in brain organoids.

[0081] Experimental Example 3

[0082] The expression of biomarkers in different brain regions in Comparative Example 1 and Example 1 was determined using real-time quantitative PCR. The experimental method was the same as in Example 1, and the results are as follows: Figure 5 As shown. Figure 5 The results showed that, compared with Comparative Example 1, the gene expression levels of the functional regions of the brain organoid in Example 1, namely the forebrain (FOXG1), anterior cortical plate (TBR1), hippocampus (PROX1), choroid plexus (TTR), and hindbrain (ISL1), were increased, indicating that NMN can promote the development of the forebrain, anterior cortical plate, hippocampus, choroid plexus, and hindbrain in the brain organoid.

[0083] As can be seen from the above embodiments, the present invention provides a brain organoid containing microglia, its preparation method, and its application. The present invention promotes the development of microglia in brain organoids and accelerates the development of neurons and neuronal synapses by adding a certain concentration of NMN to EB formation medium, neuroblastoma induction medium, brain organoid expansion medium, and brain organoid maturation medium. It also promotes the development of the forebrain, anterior cortical plate, hippocampus, choroid plexus, and hindbrain in brain organoids.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a brain organoid, characterized in that, Includes the following steps: (1) Culture human induced pluripotent stem cells, passage them every 5-7 days, and select undifferentiated human induced pluripotent stem cells; (2) Separate the cells selected in step (1), and mix the separated cells with EB seed culture medium to prepare a cell suspension; (3) The cell suspension was cultured, and EB formation medium was added at 47-49h and 95-97h respectively. After 4-5 days, embryos with clear edges and a diameter between 300-600μm were selected. (4) Mix the embryonic bodies described in step (3) with the neuroblastoma induction medium and culture them for 46-50 hours. Then select the embryonic bodies with transparent and irregular edges for the next differentiation. (5) Add the matrix gel to the embryo body selected in step (4) to wrap the embryo body. After the matrix gel solidifies, culture it with brain organoid amplification medium. After 70-75 hours, select brain organoids with folded outer edges. (6) Discard the amplification culture medium of brain organoids, add brain organoid maturation culture medium for culture, change the culture medium every 2-3 days, culture for 14-16 days to obtain mature brain organoids; EB formation medium: 225ml DMEM / F12 medium, 5ml glutamine, 5ml non-essential amino acids, 15ml fetal bovine serum for embryonic stem cells, 225ml E8 complete medium, 3.5μl 2-mercaptoethanol, 180~220μM nicotinamide mononucleotide; EB seed culture medium: EB formation medium was supplemented with Y-27632 at a final concentration of 10 μM; Neuroblast induction medium: 485ml DMEM / F12 medium, 5ml N2 supplement, 5ml glutamine, 5ml non-essential amino acid solution, 50μg heparin, 180~220μM nicotinamide mononucleotide; Brain organoid amplification medium: 250ml BrainPhys™ medium, 1.25ml N2 additive, 2.5ml NeuroCult™ SM1 supplement without vitamin A, 2.5ml glutamine, 2.5ml penicillin and streptomycin, 87.5μl 1% 2-mercaptoethanol, 180~220μM nicotinamide mononucleotide; Brain organoid maturation culture medium: 250ml BrainPhys TM Culture medium: 1.25 ml N2 additive, 2.5 ml glutamine, 62.5 μl insulin, 2.5 ml penicillin and streptomycin, 87.5 μl 1% 2-mercaptoethanol, 180~220 μM nicotinamide mononucleotide.

2. The preparation method according to claim 1, characterized in that, The initial culture density of the human induced pluripotent stem cells was 0.8~1.2×10⁻⁶. 6 per mL.

3. The preparation method according to claim 1, characterized in that, The generation ratio is 1:4 to 1:

6.

4. The preparation method according to claim 1, characterized in that, The cell density of the cell suspension is 85,000 to 95,000 cells / mL.

5. The preparation method according to claim 1, characterized in that, Each embryo is individually coated with matrix gel, and the volume of matrix gel corresponding to each embryo is 20~25μL.

6. The preparation method according to claim 1 or 5, characterized in that, Embryo bodies coated with matrix gel were cultured in 6-well low-mount plates, with 6-8 embryo bodies placed in each well.

7. The preparation method according to claim 1, characterized in that, The cultivation conditions described in steps (1) to (6) are: 4-6% CO2, 35-40℃.

8. The brain organoid prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The brain organoids contain microglia.

9. The application of the brain organoids described in claim 8 in brain development research, nervous system disease research, or drug screening.

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