A cholinergic neural organoid culture method and culture medium

Abstract

This invention discloses a method and culture medium for culturing cholinergic neural organoids. The invention constructs cholinergic neural organoids based on a matrix gel-layered nanomaterial bimetallic hydroxide complex and a culture medium. This complex effectively promotes the efficient differentiation of induced pluripotent stem cells into cholinergic neurons under three-dimensional culture conditions of organoids. The organoid construction system of this invention has a simple culture scheme and high acetylcholine transferase expression abundance, providing a powerful research model for cholinergic neuron-related central nervous system diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a culture method and culture medium for cholinergic neural organoids. Background Art

[0002] The cholinergic system plays an important role in central nervous system damage and is closely related to cognitive function, motor function, etc. Acetylcholine (ACh) is one of the important neurotransmitters in the central cholinergic system. Cholinergic nervous system disorders are an important manifestation of a variety of central nervous system degenerative diseases, such as Alzheimer's disease, myasthenia gravis, multiple sclerosis, etc. In view of the above problems, research models and treatment methods of the cholinergic nervous system are urgently needed to rebuild cholinergic function, activate the normal release of ACh, and repair damaged neural function [1-2].

[0003] The construction and application of stem cell-based nervous system functional organoids has become a hot topic in the study of central nervous system pathological mechanisms and injury repair. Organoids are a type of three-dimensional cell culture that contains some biological characteristics of representative organs. This type of in vitro culture system is a self-renewing stem cell group that can differentiate into multiple specific cell subsets representing organs, has a similar spatial configuration to the corresponding organs and reconstructs some of the functions of the corresponding organs, thereby providing a highly physiologically relevant system, which has groundbreaking value in the fields of disease mechanism research and organ regeneration.

[0004] In the past decade, research on the nervous system organoid system has achieved some landmark results, providing a new strategy for neural repair. Since the 1990s, methods for inducing 3D differentiation of neural stem cells have emerged, which is the prototype of the emergence of nervous system organoids. In 2013, Lancaster MA et al. constructed a three-dimensional organic culture system of brain organoids derived from human induced pluripotent stem cells using Matrigel as the matrix material [3]. Since then, a large amount of research work has been devoted to the development of organoids in specific brain regions. In 2016, Jo et al. developed human midbrain organoids, which developed mature dopamine neurons and neuromelanin-like granules consistent with the in vivo structure [4]; in 2021, Shim JW's research team constructed hindbrain organoids rich in serotonin neurons [5]; in 2022, the research team of Nanjing Medical University used a combination of suspension culture and adherent culture to construct basal forebrain cholinergic organoids, simulating the development of the basal forebrain in vitro [6]; however, the organoid construction scheme is complex, requiring alternating adherent and suspension culture, and the introduction of animal-derived components, which is not conducive to reproducing the development process of human organ tissues.

[0005] The introduction of new tissue engineering technologies and new biomaterials has received widespread attention in the field of constructing functional central nervous system organoids. On the one hand, tissue engineering is an important foundation of regenerative medicine. It usually uses the principles of chemistry, biology and engineering to promote the growth, development and regeneration of damaged tissues or organs. This involves cells, artificial scaffolds and biological signal factors, which can promote the regeneration of new tissues or organs. Studies have shown that 3D culture of nerve cells can be achieved through specially designed culture vessels or bionic scaffolds, among which bionic scaffolds are a modeling method that is conducive to controlling cell-to-cell interactions. On the other hand, functional biomaterials can create an adjustable central nervous system microenvironment during the in vitro development of organoids and guide the growth pattern of organoids. In 2014, a study used laminin and polyethylene glycol (PEG) scaffolds to construct a neural tube model with controlled release of retinoic acid, inducing neuroepithelial tissue with a dorsal-ventral structure [7]. In 2017, Lancaster et al. used polylactic acid-glycolic acid (PLGA) microfilaments as a scaffold material for the growth of induced pluripotent stem cells (iPSCs), physically regulating cell polarization, and cooperating with matrix gel and soluble chemical components in the culture medium to construct elongated embryoid bodies, enhancing neural ectoderm formation and subsequent cortical development [8]. In 2021, Ali Khademhosseini's research team at Harvard Medical School designed a photo-crosslinkable bio-ink and a thermosensitive suspension gel to simulate the mechanical modulus of soft tissue to form a heterogeneous neural population with neurospheres and glia [9]. However, the above-mentioned research on material-guided organoid growth is still limited to the simulation of a single tissue or organ, and there is currently no research on highly functional organoids for the central nervous system.

[0006] Nanomaterials are widely used in tissue engineering due to their excellent properties such as small size effect. The incorporation of nanomaterials into biological scaffold materials can significantly improve their mechanical properties. In addition, nanomaterials can also be used as delivery carriers for small molecule drugs or bioactive factors. A large number of studies have also shown that nanomaterials can regulate the fate of stem cells and affect cell proliferation and differentiation. In 2019, our research group applied nano-graphene oxide to mouse embryonic stem cell culture and achieved the self-renewal of embryonic stem cells

[10] . In 2021, our research group's preliminary research work developed Mg / Fe-LDH nanomaterials that can replace leukemia inhibitory factor (LIF factor), achieving LIF-free embryonic stem cell culture

[11] .

[0007] By utilizing the unique biological and mechanical properties of nanomaterials, functional cholinergic neural organoids are constructed to simulate the developmental pattern of the cholinergic nervous system in vitro and the pathological changes of the cholinergic nervous system in nerve injury / degeneration. Cholinergic organoids have important research value in the study of the mechanism of central nervous system-related diseases and clinical transformation in the field of injury repair.

[0008] [1] Li,

[0009] [2] Horio, T., Ozawa, A., Kamiie, J. & Sakaue, M. Immunohistochemical analysis for acetylcholinesterase and choline acetyltransferase in mouse cerebral cortex after traumatic brain injury. Journal of Veterinary Medical Science 82, 827-835, doi:10.1292 / jvms.19-0551(2020).

[0010] [3] Lancaster, MA et al. Cerebral organoids model human brain development and microcephaly. Nature 501,373-+, doi:10.1038 / nature12517(2013).

[0011] [4]Jo, J.et al. Midbrain-like Organoids from Human Pluripotent StemCells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. CellStem Cell 19, 248-257, doi:10.1016 / j.stem.2016.07.005(2016).

[0012] [5] Valiulahi, P. et al. Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs. Stem Cell Reports 16, 1938-1952, doi:10.1016 / j.stemcr.2021.06.006(2021).

[0013] [6] Liu Yan, Wang Da, Tang Xiaoyan, Zhang Xinyue & Xu Min. Basal forebrain cholinergic organoids prepared by inducing differentiation of hPSCs and preparation method thereof. CN114058587A.

[0014] [7]Meinhardt,A.et al.3D Reconstitution of the Patterned Neural Tubefrom Embryonic Stem Cells.Stem Cell Reports 3,987-999,doi:10.1016 / j.stemcr.2014.09.020(2014).

[0015] [8] Lancaster, MA et al. Guided self-organization and cortical plateformation in human brain organoids. Nature Biotechnology 35,659-+, doi:10.1038 / nbt.3906(2017).

[0016] [9] Li, Y.-CE et al. Toward a neurospheroid niche model: optimizingembedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs. Biofabrication 13, doi:10.1088 / 1758-5090 / abc1be(2021).

[0017]

[10] Jing,G.et al.Suspended graphene oxide nanosheets maintain the self-renewal of mouse embryonic stem cells via down-regulating the expression of Vinculin. Biomaterials 171,1-11,doi:10.1016 / j.biomaterials.2018.04.017(2018).

[0018]

[11] He, Summary of the invention

[0019] In view of the above background technology, the present invention provides a culture method and culture medium for cholinergic nerve organoids, which utilize the pluripotency of stem cells and combine with nanomaterials to construct functional cholinergic nerve organoids in vitro, induce the directed differentiation of pluripotent stem cells, and simulate the developmental pattern of the cholinergic nervous system and the pathological changes of the cholinergic nervous system in central nervous system diseases in vitro.

[0020] To achieve the above purpose, the technical solution adopted by the present invention is:

[0021] In one aspect, the present invention provides a culture medium for cholinergic neural organoids, comprising:

[0022] Medium 1: Contains Bone Morphogenetic Protein (BMP) signaling pathway inhibitor and Wnt signaling pathway activator;

[0023] Medium 2: Contains Bone Morphogenetic Protein (BMP) signaling pathway inhibitor, Wnt signaling pathway activator and neural induction small molecule retinoic acid RA;

[0024] Medium 3: Contains the neuroinducible small molecule retinoic acid RA and SHH signaling pathway agonist;

[0025] Culture medium 4: contains nerve growth factor βNGF and glial cell line-derived neurotrophic factor GDNF.

[0026] As a preferred embodiment, the culture medium comprises:

[0027] Culture medium 1: contains DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement (N2 Supplement), B27 supplement (B27 Supplement), GlutaMAX, Bone Morphogenetic Protein (BMP) signaling pathway inhibitor and WNT signaling pathway activator;

[0028] Culture medium 2: contains DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement (N2 Supplement), B27 supplement (B27 Supplement), GlutaMAX, Bone Morphogenetic Protein (BMP) signaling pathway inhibitor, WNT signaling pathway activator and neural induction small molecule retinoic acid RA;

[0029] Culture medium 3: DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, neural induction small molecule retinoic acid RA and SHH signaling pathway agonist;

[0030] Culture medium 4: DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement (N2Supplement), B27 supplement (B27 Supplement), GlutaMAX, nerve growth factor βNGF and glial cell line-derived neurotrophic factor GDNF.

[0031] As a preferred embodiment, the Bone Morphogenetic Protein (BMP) signaling pathway inhibitor is LDN-193189;

[0032] Preferably, the Wnt signaling pathway activator is CHIR99021;

[0033] Preferably, the SHH signaling pathway agonist is Purmorphamine.

[0034] As a preferred embodiment, in the culture medium 1-4, the volume ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:1;

[0035] Preferably, in the culture medium 1-4, the volume fraction of penicillin-streptomycin dual antibody in the culture medium is 1% to 5%;

[0036] Preferably, in the culture medium 1 or culture medium 2, the concentration of LDN-193189 is 1 to 5 μM;

[0037] Preferably, in the culture medium 1 or culture medium 2, the concentration of CHIR99021 is 1 to 5 μM;

[0038] Preferably, in the culture medium 2 or culture medium 3, the concentration of the neuro-inducing small molecule retinoic acid RA is 0.1 to 1 μM;

[0039] Preferably, in the culture medium 3, the concentration of Purmorphamine is 0.5-1 μM;

[0040] Preferably, in the culture medium 4, the concentration of βNGF is 5 to 10 ng / mL;

[0041] Preferably, in the culture medium 4, the concentration of GDNF is 5-10 ng / mL.

[0042] In another aspect, the present invention provides use of the culture medium in preparing a biological implant, specifically, use of the culture medium in culturing cholinergic neural organoids.

[0043] In another aspect, the present invention provides a method for culturing cholinergic nerve organoids, wherein the method is to culture cholinergic nerve organoids based on the above-mentioned culture medium, comprising the following steps:

[0044] (1) culturing induced pluripotent stem cells (iPSCs) in culture medium 1-2 in sequence to induce differentiation;

[0045] (2) encapsulating the embryoid bodies obtained after culturing in step (1) in a matrix gel-layered nanomaterial complex to form cell spheres;

[0046] (3) continuing to culture the cell spheres obtained in step (2) in culture medium 3-4 to induce differentiation, thereby completing the cholinergic neural organoid culture;

[0047] In step (1), the matrix glue-layered nanomaterial composite is obtained by blending layered nanomaterial and matrix glue, the layered nanomaterial is magnesium / iron layered double hydroxide (Mg / Fe-LDH) or magnesium / aluminum layered double hydroxide (Mg / Al-LDH), and the matrix glue is preferably Matrigel matrix glue.

[0048] As a preferred embodiment, in step (1), the culture time in the culture medium 1 is 2 days;

[0049] Preferably, in step (1), the culture time in the culture medium 2 is 7 days;

[0050] Preferably, in step (3), the culture time in the culture medium 3 is 7 days;

[0051] Preferably, in step (3), the culture time in the culture medium 4 is 3 to 25 days.

[0052] As a preferred embodiment, in step (2), the cell sphere formation is performed by constant temperature incubation at 25 to 37° C.; the constant temperature incubation time is 20 to 30 min.

[0053] In certain specific embodiments, the method for obtaining induced pluripotent stem cells is to culture induced pluripotent stem cells (iPSCs) derived from wild-type fibroblasts in mTeSR1 medium, then separate them into single cells using Versene digestion solution, and then resuspend them in mTeSR13 medium.

[0054] In certain specific embodiments, the specific operation of step (1) is: culturing the induced pluripotent stem cells in culture medium 1, and after 2 days, adding the neural inducing small molecule retinoic acid RA to the culture medium 1 and continuing to culture for 7 days.

[0055] In certain specific embodiments, the specific operation of step (2) is: encapsulating the embryoid bodies obtained in step (1) in the matrix gel-layered nanomaterial complex, and incubating at a constant temperature of 25 to 37° C. for 20 to 30 minutes to solidify and form cell spheres.

[0056] In certain specific embodiments, the specific operation of step (3) is: resuspending the cell sphere obtained in step (2) in culture medium 3, culturing for 7 days, and then transferring to culture medium 4 for culturing for 3 to 25 days.

[0057] As a preferred embodiment, the concentration of the layered nanomaterial in the matrix gel-layered nanomaterial complex is 1-100 μg / mL, preferably 10 μg / mL.

[0058] In certain specific embodiments, the method for preparing a matrix glue-layered nanomaterial composite using a magnesium / iron layered double hydroxide comprises the following steps:

[0059] S1. In an inert atmosphere, add a mixed solution of magnesium nitrate and ferric nitrate to a NaOH solution and stir in a water bath at 25 to 60°C for 30 to 60 minutes;

[0060] S2. washing the product of step S1 and subjecting it to a hydrothermal reaction at 80 to 120° C. to obtain a magnesium / iron layered double hydroxide;

[0061] S3. Physically blending the magnesium / iron layered double hydroxide obtained in step S2 with the matrix glue;

[0062] Preferably, in step S1, the molar ratio of magnesium ions to iron ions in the mixed solution of magnesium nitrate and iron nitrate is 3 to 5:1;

[0063] Preferably, the concentration ratio of magnesium ions in the magnesium nitrate to the NaOH solution is 0.5 to 0.8:1;

[0064] Preferably, the mixed solution of magnesium nitrate and ferric nitrate is added to the NaOH solution, and the final molar concentration ratio of magnesium nitrate, ferric nitrate and sodium hydroxide is 5-5.2:1.0-1.3:1;

[0065] Preferably, step S3 is to perform physical blending at a low temperature of 0 to 10°C.

[0066] In certain specific embodiments, the method for preparing a matrix glue-layered nanomaterial composite using a magnesium / aluminum layered double hydroxide comprises the following steps:

[0067] 1) In an inert atmosphere, add the mixed solution of magnesium nitrate and aluminum nitrate into the NaOH solution and stir in a water bath at 25-60°C for 30-60 minutes;

[0068] 2) washing the product of step 1) and subjecting it to a hydrothermal reaction at 80-120° C. to obtain a magnesium / iron layered double hydroxide;

[0069] 3) Physically blending the magnesium / aluminum layered double hydroxide obtained in step 2) with matrix glue.

[0070] Preferably, in step 1), the molar ratio of magnesium ions to iron ions in the mixed solution of magnesium nitrate and aluminum nitrate is 3 to 5:1;

[0071] Preferably, the concentration ratio of magnesium ions in the magnesium nitrate to the NaOH solution is 0.5 to 0.8:1;

[0072] Preferably, the mixed solution of magnesium nitrate and aluminum nitrate is added to the NaOH solution, and the final molar concentration ratio of magnesium nitrate, iron nitrate and sodium hydroxide is 5-5.2:1.0-1.3:1;

[0073] Preferably, step 3) is to perform physical blending at a low temperature of 0 to 10°C.

[0074] In another aspect, the present invention provides cholinergic nerve organoids obtained by the above culture method.

[0075] The above technical solution has the following advantages or beneficial effects:

[0076] The present invention provides a cholinergic nerve organoid culture method and culture medium, and uses the culture medium and a composite of Mg / Fe-LDH layered nanomaterials or Mg / Al-LDH layered nanomaterials and matrigel to construct cholinergic nerve organoids, wherein the composite of layered nanomaterials and matrigel can effectively improve the formation efficiency of induced pluripotent stem cells in the three-dimensional culture conditions of organoids, and promote the efficient differentiation of cholinergic neurons. The culture scheme of the organoid construction system of the present invention is simple, the expression abundance of acetylcholine transferase is high, and mature electrical signals can be generated, providing a powerful research model for central nervous system diseases related to cholinergic neurons. This construction scheme utilizes the characteristics of layered nanomaterials in regulating the fate of stem cells, shortening the time of cholinergic neuron occurrence. Neuron occurrence was detected on the 14th day of construction, and a large number of cholinergic neurons matured on the 35th day. At the same time, highly active neural electrical signals were detected in the organoids constructed by the present invention in the 6th week. In addition, the present invention also solves the complex situation of planar-suspension alternating culture in existing construction schemes, and can directly induce pluripotent stem cells into cholinergic neural organoids in suspension, which is easy to operate. The present invention provides an effective research method for central nervous system-related diseases, and achieves breakthroughs in the field of in vitro models of the central nervous system and neural regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The characterization results of the layered nanomaterials in Example 1 of the present invention are as follows: Figure 1 (A) is the transmission electron microscopy results of Mg / Al-LDH and Mg / Fe-LDH. Figure 1 (B) is the elemental analysis of Mg / Al-LDH and Mg / Fe-LDH. Figure 1 (C) is the Fourier transform infrared spectrum results of Mg / Al-LDH and Mg / Fe-LDH. Figure 1 (D) is the X-ray diffraction pattern of Mg / Al-LDH and Mg / Fe-LDH.

[0078] Figure 2 This is a flow chart of constructing cholinergic neural organoids in Example 3 of the present invention, wherein: Figure 2 (A) Scheme for constructing cholinergic neural organoids. Figure 2 (B) Bright field morphology of cholinergic neural organoids.

[0079] Figure 3 This is a distribution diagram of the cholinergic nerve organoid nerve fibers (Tuj1) constructed in Example 3 of the present invention.

[0080] Figure 4 This is the expression of acetylcholine transferase (ChAT) in the cholinergic neural organoid constructed in Example 3 of the present invention.

[0081] Figure 5 This is the electrical signal recording of the cholinergic neural organoid constructed in Example 3 of the present invention. DETAILED DESCRIPTION

[0082] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0083] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0084] Example 1: Preparation and characterization of layered double hydroxides

[0085] Preparation and Characterization of Magnesium / Iron Layered Double Hydroxide (Mg / Fe-LDH)

[0086] (1) heating double distilled water (ddH2O) to boiling, boiling for 40 min and then naturally drying to obtain ddH2O free of CO2 for later use;

[0087] (2) Dissolve 0.544 g of sodium hydroxide (NaOH) in 80 mL of the above-mentioned ddH2O for de-CO2 removal and transfer to a three-necked flask;

[0088] (3) Place a three-necked flask in a water bath at 60°C, then fix it on a constant speed stirrer and introduce nitrogen; weigh magnesium nitrate and ferric nitrate in a molar ratio of 4:1 in a 50 mL centrifuge tube, add 20 mL of ddH2O and stir to dissolve, turn on the stirrer and set the speed to 500 rpm to obtain a salt solution with a magnesium nitrate concentration of 76.9 mg / mL and a ferric nitrate concentration of 30.3 mg / mL;

[0089] (4) The obtained nitrate solution was added dropwise to the above NaOH solution and stirred vigorously for 30 min under water bath conditions; the obtained suspension was centrifuged at 4500 rpm for 5 min, washed three times with ddH2O and then resuspended, placed in a hydrothermal kettle, and reacted at 100°C for 16 h. The product was collected and centrifuged at 8500 rpm for 15 min, washed three times and then dried naturally. The product was Mg / Fe-LDH nanomaterial.

[0090] Preparation and Characterization of Magnesium / Aluminum Layered Double Hydroxide (Mg / Al-LDH)

[0091] (1) heating ddH2O to boiling, boiling for 40 min and then naturally drying to obtain ddH2O with CO2 removed for later use;

[0092] (2) Dissolve 0.544 g of sodium hydroxide (NaOH) in 80 mL of the above-mentioned CO2-free ddH2O and transfer to a three-necked flask;

[0093] (3) placing a three-necked flask in a water bath at 60°C, then fixing it on a constant speed stirrer and introducing nitrogen; weighing magnesium nitrate and aluminum nitrate in a molar ratio of 3:1 into a 50 mL centrifuge tube, adding 20 mL of ddH2O and stirring to dissolve, turning on the stirrer and setting the speed to 500 rpm to obtain a salt solution with a magnesium nitrate concentration of 76.9 mg / mL and an aluminum nitrate concentration of 37.5 mg / mL;

[0094] (4) The obtained nitrate solution was added dropwise to the above NaOH solution, and the mixture was vigorously stirred for 30 min in a water bath; the obtained suspension was centrifuged at 4500 rpm for 5 min, washed three times with ddH2O, resuspended, placed in a hydrothermal kettle, reacted in a drying oven at 100°C overnight, and the product was collected and centrifuged at 8500 rpm for 15 min, washed three times, and naturally dried. The product is the Mg / Al-LDH nanomaterial.

[0095] The two LDH nanomaterials were observed using transmission electron microscopy. Figure 1 (A), both of them show a hexagonal lamellar nanostructure with a particle size of about 100 nm; elemental analysis was used to characterize the LDH composed of two ions, such as Figure 1 (B), both show characteristic peaks of elements composed of two metal ions; Figure 1 (C) Fourier transform infrared spectroscopy was used to detect the two ion compositions of LDH. The 3440 cm -1 The vibration absorption peaks of the OH functional group and 1380 cm -1 About (NO3 - )'s stretching vibration characteristic peak; Figure 1 (D) The LDH composed of two ions was characterized by X-ray diffractometer, and the characteristic functional group absorption peaks of LDH, diffraction peak (003) and diffraction peak (006), were detected.

[0096] Example 2: Preparation of Matrigel-Layered Nanomaterial Composites:

[0097] Pre-cool PBS buffer, pipettes and centrifuge tubes to 4°C and thaw on ice. Matrigel; the LDH layered nanomaterial prepared in Example 1 was dispersed with pre-cooled PBS buffer at a concentration range of 10 to 1000 μg / mL; then the LDH suspension was physically mixed evenly with thawed Matrigel on ice at a volume ratio of 9:1.

[0098] In this example, in the prepared Matrigel-layered nanomaterial complex, the final concentration of LDH in Matrigel is 1-100 μg / mL.

[0099] Example 3: Construction and culture of cholinergic neural organoids

[0100] like Figure 2 As shown in (A), the construction and culture process of the cholinergic neural organoid in this embodiment includes the following steps:

[0101] (1) Fibroblast-derived iPSCs were cultured in mTeSR1 medium in a 6-well plate coated with Matrigel. When the cloning rate reached about 80%, the iPSCs clones were separated into single cells using Versene, resuspended in mTeSR1, and seeded into a low-adsorption 96-well round-bottom plate at a cell number of 30,000 cells / well;

[0102] (2) The culture plate was left to stand overnight and replaced with medium 1 after the cells were integrated into a cluster. Medium 1 consisted of a 1:1 volume mixture of DMEM / F-12 medium and Neurobasal medium, with the addition of 1% double antibody, N2Supplement (100×), B27Supplement (50×), GlutaMAX (100×), 1 μM LDN-193189, and 2.5 μM CHIR99021.

[0103] (3) Step (1) was initially counted as day 0. On day 3, the medium was changed to medium 2, specifically, 0.1 μM RA was added to medium 1.

[0104] (4) On the 10th day, the embryoids were taken out and encapsulated in the matrix gel-layered nanomaterial complex prepared in Example 2 (v(LDH):v(Matrigel)=1:9), and placed in a 37°C incubator for 30 min until the droplets solidified; the encapsulated embryoids were resuspended in culture medium 3 and inoculated in a 6-well plate with ultra-low adsorption. The composition of culture medium 3 was DMEM / F-12 culture medium and Neurobasal culture medium mixed in a volume ratio of 1:1, 1% double antibody, N2 Supplement (100×), B27 Supplement (50×), GlutaMAX (100×), 0.1M RA and 0.5μM Purmorphamine were added, and the well plate was placed on a shaker in the incubator and maintained at 65rpm;

[0105] (5) On the 17th day of construction, the medium was replaced with medium 4, which was composed of DMEM / F-12 medium and Neurobasal medium mixed in a 1:1 volume ratio, and 1% double antibody, N2 Supplement (100×), B27 Supplement (50×), GlutaMAX (100×), 10 ng / mL β-NGF and 10 ng / mL GDNF were added to promote the maturation of organoids.

[0106] The initial step of step (1) was counted as day 0, and the cholinergic neural organoid samples constructed on days 14, 21, and 35 were collected for bright field image collection and immunofluorescence staining. The morphology of the organoids was as follows: Figure 2 (B) As shown. The specific operation of immunofluorescence staining is as follows: the organoids are fixed with 4% PFA overnight, dehydrated in gradients, and then embedded in OCT and frozen overnight. The embedded samples are cut into 14μm slices using a freezing microtome. The organoid sample sections are blocked with a blocking solution containing 5% donkey serum at room temperature for 1 hour, and then incubated with the primary antibody (Tuj1 and ChAT) at 4°C overnight. Wash three times with PBS, dilute the secondary antibody with blocking solution, and incubate at room temperature for 1 hour. Wash three times with PBS and dry the sections. Figure 3 The labeled Tuj1 is a neuronal synaptic marker. The results showed that a large number of nerve fibers were already distributed in the cholinergic neural organoids on the 14th day. Figure 4 The labeled ChAT is acetylcholine transferase. The results showed that a large number of mature cholinergic neurons could be detected in the organoids on the 35th day of construction; in addition, LDH composed of magnesium iron and magnesium aluminum at a concentration of 10 μg / mL combined with matrigel had a good promoting effect on the occurrence of cholinergic neurons in organoids.

[0107] The initial step (1) was marked as day 0. At week 5, a six-well MEA plate was taken, each well containing 64 microelectrodes. It was pre-coated with 10μg / ml Mg / Al-LDH and 10μg / ml Mg / Fe-LDH combined with matrix gel in the incubator for 30 minutes. A 5-week-old cholinergic neural organoid was placed in the coated MEA well, covering the 64 microelectrodes. The cholinergic organoid was fixed in the incubator for 10 minutes, and then fresh neural culture medium was added and cultured in the incubator for 1 week. The Maestro pro MEA system and AxIS Software spontaneous nerve configuration were used to record electrical signals for 10-15 minutes. The distribution of microelectrodes in the six-well plate and the heat map of neuronal activity per second are shown in Figure 2. Figure 5 (A) shows the electrical signal of a single neuron on a single electrode within 20 milliseconds. Figure 5 (B) shown.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of a culture medium in culturing cholinergic neural organoids, characterized in that: The culture medium consists of: Culture medium 1: composed of DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, Bone Morphogenetic Protein signaling pathway inhibitor and WNT signaling pathway activator; Culture medium 2: DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, Bone Morphogenetic Protein signaling pathway inhibitor, WNT signaling pathway activator and neural induction small molecule retinoic acid RA; Medium 3: composed of DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, neural induction small molecule retinoic acid RA and SHH signaling pathway agonist; Medium 4: composed of DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, nerve growth factor βNGF and glial cell line-derived neurotrophic factor GDNF; The Bone Morphogenetic Protein signaling pathway inhibitor is LDN-193189; the WNT signaling pathway activator is CHIR99021; the SHH signaling pathway agonist is Purmorphamine; In the culture medium 3, the concentration of the neuro-inducing small molecule retinoic acid RA is 0.1-1 μM; the concentration of Purmorphamine is 0.5-1 μM; In the culture medium 4, the concentration of nerve growth factor βNGF is 5-10 ng / mL; the concentration of glial cell-derived neurotrophic factor GDNF is 5-10 ng / mL.

2. The use according to claim 1, characterized in that: In the culture medium 1-4, the volume ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:

1.

3. The use according to claim 1, characterized in that: In the culture medium 1-4, the volume fraction of penicillin-streptomycin dual antibody in the culture medium is 1% to 5%.

4. The use according to claim 1, characterized in that: In the culture medium 1 or culture medium 2, the concentration of LDN-193189 is 1 to 5 μM.

5. The use according to claim 1, characterized in that: In the culture medium 1 or culture medium 2, the concentration of CHIR99021 is 1 to 5 μM.

6. The use according to claim 1, characterized in that: In the culture medium 2, the concentration of the nerve-inducing small molecule retinoic acid RA is 0.1-1 μM.

7. A method for culturing cholinergic neural organoids, characterized in that: The culture method comprises the following steps: (1) culturing induced pluripotent stem cells in culture medium 1-2 in sequence to induce differentiation; (2) encapsulating the embryoid bodies obtained after culturing in step (1) in a matrix gel-layered nanomaterial complex to form cell spheres; (3) continuing to culture the cell spheres obtained in step (2) in culture medium 3-4 to induce differentiation, thereby completing the cholinergic neural organoid culture; In step (2), the matrix glue-layered nanomaterial composite is obtained by blending layered nanomaterial and matrix glue, the layered nanomaterial is magnesium / iron layered double metal hydroxide or magnesium / aluminum layered double metal hydroxide, and the matrix glue is Matrigel matrix glue; in: Culture medium 1: composed of DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, Bone Morphogenetic Protein signaling pathway inhibitor and WNT signaling pathway activator; Culture medium 2: DMEM / F-12 culture medium, Neurobasal culture medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, Bone Morphogenetic Protein signaling pathway inhibitor, WNT signaling pathway activator and neural induction small molecule retinoic acid RA; Medium 3: composed of DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, neural induction small molecule retinoic acid RA and SHH signaling pathway agonist; Medium 4: composed of DMEM / F-12 medium, Neurobasal medium, penicillin-streptomycin double antibody, N2 supplement, B27 supplement, GlutaMAX, nerve growth factor βNGF and glial cell line-derived neurotrophic factor GDNF; The Bone Morphogenetic Protein signaling pathway inhibitor is LDN-193189; the WNT signaling pathway activator is CHIR99021; the SHH signaling pathway agonist is Purmorphamine; In the culture medium 3, the concentration of the neuro-inducing small molecule retinoic acid RA is 0.1-1 μM; the concentration of Purmorphamine is 0.5-1 μM; In the culture medium 4, the concentration of nerve growth factor βNGF is 5-10 ng / mL; the concentration of glial cell-derived neurotrophic factor GDNF is 5-10 ng / mL; The concentration of the layered nanomaterial in the matrix glue-layered nanomaterial complex is 1-100 μg / mL.

8. The culture method according to claim 7, characterized in that: In step (1), the culture time in the culture medium 1 is 2 days.

9. The culture method according to claim 7, characterized in that: In step (1), the culture time in the culture medium 2 is 7 days.

10. The culture method according to claim 7, characterized in that: In step (3), the culture time in the culture medium 3 is 7 days.

11. The culture method according to claim 7, characterized in that: In step (3), the culture time in the culture medium 4 is 3 to 25 days.

12. The culture method according to claim 7, characterized in that: In step (2), the cell spheres are formed by constant temperature incubation at 25 to 37° C.; the constant temperature incubation time is 20 to 30 minutes.

13. The culture method according to claim 7, characterized in that: The concentration of the layered nanomaterial in the matrix gel-layered nanomaterial complex is 10 μg / mL.

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