Method for three-dimensional culture and induced differentiation of neural organoids and use thereof

Abstract

The present invention relates to a method for three-dimensional culture and induced differentiation of neural organoids and use thereof. The method for three-dimensional culture and induced differentiation of neural organoids comprises: adding a signal pathway inhibitor into an induced pluripotent stem cell three-dimensional culture, and culturing cells to obtain the neural organoids. The signal pathway inhibitor comprises a TGF-β signal pathway inhibitor, a WNT signal pathway inhibitor, and a BMP signal pathway inhibitor. Compared with a traditional method, the neural organoids obtained by the method of the present invention feature a large size, small individual difference, and short culture time. The method is simple and rapid, and has high stability and low cost, and only a common incubator is needed. The method has wide application prospects.

Description

A method for inducing differentiation of neural organoids through three-dimensional culture and its application Technical Field

[0001] This invention relates to the field of cell-induced differentiation, and more particularly to a method for inducing differentiation through three-dimensional culture of neural organoids and its application. Background Technology

[0002] Currently, there is no fundamental cure for neurological diseases. Taking Asia as an example, according to a 2023 report in *Neurology*, the annual mortality rate from neurological diseases increased by 60.7% from 2009 to 2019, peaking between the ages of 65 and 74, and affecting more men than women. One reason for this phenomenon is the lack of effective disease models. Furthermore, neurological diseases are closely related to environmental factors; exposure to environmental pollutants can lead to stress in the nervous system, resulting in inflammation, peroxidation, mitochondrial dysfunction, and impaired cellular function. The brain's complex structure and the difficulty in directly obtaining samples have hindered research progress at the cellular level, leading to slow development of in vitro human disease models.

[0003] In 2013, Yoshiki Sasai's and Juergen A. Knoblich's laboratories pioneered a method for culturing neural organoids. Neural organoids mimic early neural development, with neural stem cells emerging first and then differentiating into nerve cells, exhibiting a certain wreath-like tissue structure. At the same time, neural organoids can also mimic the phenotype of microcephaly.

[0004] Knoblich's method is a non-guided differentiation method. After 10 days of induced differentiation, pluripotent stem cell microspheres are embedded in matrix gel for further differentiation and cultured in microculturers, resulting in neural organoids containing multiple cell types. Sasai's method is a guided differentiation method, producing neural organoids primarily composed of cortical cell types, with relatively homogeneous individual organoids. Sasai's guided differentiation method involves treating pluripotent stem cell microspheres for 18 days with a combination of the ROCK inhibitor Y-27632 (for the first six days), the TGF-β inhibitor SB431542, and the WNT signaling pathway inhibitor IWR1-e, inducing cell differentiation into cortical neural progenitor cells. These cells are then transferred to a horizontal shaker for culture. Sasai's guided differentiation method requires a lengthy 18-day induction period for neural stem cells, followed by transfer to low-absorption culture dishes and culture in a hyperoxia incubator (40% O2, 5% CO2), resulting in demanding culture conditions.

[0005] Although the induction of neural organoids has progressed rapidly in recent years with a variety of protocols, the two protocols mentioned above are the earliest, with extensive replication and sufficient scientific data. While non-guided differentiation protocols encompass a rich variety of cell types, the size and proportion of cell types vary significantly among individual organoids. Guided differentiation methods, on the other hand, primarily yield cortical neural cell types, resulting in relatively homogeneous organoid size and smaller differences in cell type proportions, making them more suitable for downstream toxicology and pathology applications.

[0006] In summary, how to efficiently and rapidly induce induced pluripotent stem cells to differentiate into neural organoids and apply them to toxicological testing has become an urgent problem to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for three-dimensional culture-induced differentiation of neural organoids and its application. By adding signaling pathway inhibitors to the culture medium for induced pluripotent stem cells, the induced pluripotent stem cells differentiate into neural organoids. The obtained neural organoids are large in size and exhibit minimal individual variation. The culture method is simple, rapid, stable, and low in cost, and has broad application prospects.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for inducing differentiation through three-dimensional culture of neural organoids, the method comprising: adding signaling pathway inhibitors to a three-dimensional culture of induced pluripotent stem cells, culturing the cells, and obtaining the neural organoids; wherein the signaling pathway inhibitors include TGF-β signaling pathway inhibitors, WNT signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0010] The three-dimensional culture involves culturing cells in a 96-well culture plate with an ultra-low adsorption surface treatment, where the cells can spontaneously aggregate into cell spheres.

[0011] Three-dimensional cell culture systems are relatively complex, requiring different culture equipment and techniques. Cellular metabolic functions in three-dimensional culture also differ from those in two-dimensional culture. Three-dimensional culture increases the intercellular connections and is generally considered to more closely resemble physiological conditions; therefore, under certain conditions, three-dimensional culture can lead to the formation of specific physiological structures.

[0012] This invention incorporates multiple signaling pathway inhibitors during the induction phase, accelerating the aggregation and growth of neural organoids. The volume of neural organoids increases, and individual differences among neural organoids are minimal, forming the primary structure of neural organoids. Overall, the operation is simple and easy to implement, the system is stable, and the cost is low. It only requires a common incubator and can be directly applied to toxicology experiments, showing strong application prospects.

[0013] Preferably, the TGF-β signaling pathway inhibitor includes any one of SB431542, SB505124, A83-01, Disitertide, Galunisertib, A77-01, or AZ12799734.

[0014] Preferably, the WNT signaling pathway inhibitor includes any one of IWR1, IWP4, FH535, NLS-StAx-h, KYA1797K, TAK715, Klotho-derived peptide 6, or WNTinib.

[0015] Preferably, the BMP signaling pathway inhibitor includes any one of LDN-193189, Dorsomorphin, DMH-1, DMH2, K02288, M4K2163 dihydrochloride, or ML347.

[0016] Preferably, the signaling pathway inhibitor further includes a Rock signaling pathway inhibitor.

[0017] Preferably, the Rock signaling pathway inhibitor includes Y-27632.

[0018] Preferably, the method for three-dimensional culture-induced differentiation of the neural organoids includes: culturing induced pluripotent stem cells in a culture medium containing 3-7 μM (e.g., 3 μM, 4 μM, 5 μM, 6 μM, or 7 μM) TGF-β signaling pathway inhibitors, 1-5 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM) WNT signaling pathway inhibitors and 80-120 nM (e.g., 80 nM, 90 nM, 100 nM, 110 nM, or 120 nM) BMP signaling pathway inhibitors for 9-11 days (e.g., 9 days, 9.5 days, 10 days, 10.5 days, or 11 days) to obtain the neural organoids.

[0019] The culture medium described in this invention can be any culture medium suitable for embryonic stem cells and pluripotent inducible cells, without any special limitations.

[0020] The method provided by this invention can shorten the time for inducing cortical neural organoids, can be operated in a single culture plate without changing the culture plate, and can be cultured in a common incubator (containing 5% CO2). It has the advantages of simple operation, stable system and low cost.

[0021] Preferably, the method for inducing differentiation through three-dimensional culture of neural organoids includes the following steps:

[0022] (1) Culture induced pluripotent stem cells in a medium containing 15-25 μM (e.g., 15 μM, 18 μM, 20 μM, 22 μM or 25 μM) Rock signaling pathway inhibitors, 3-7 μM (e.g., 3 μM, 4 μM, 5 μM, 6 μM or 7 μM) TGF-β signaling pathway inhibitors, 1-5 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM or 5 μM) WNT signaling pathway inhibitors and 80-120 nM (e.g., 80 nM, 90 nM, 100 nM, 110 nM or 120 nM) BMP signaling pathway inhibitors for 5-7 days (e.g., 5 days, 5.5 days, 6 days, 6.5 days or 7 days).

[0023] (2) The cells obtained in step (1) are cultured in a culture medium containing 3-7 μM (e.g., 3 μM, 4 μM, 5 μM, 6 μM or 7 μM) TGF-β signaling pathway inhibitor, 1-5 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM or 5 μM) WNT signaling pathway inhibitor and 80-120 nM (e.g., 80 nM, 90 nM, 100 nM, 110 nM or 120 nM) BMP signaling pathway inhibitor for 2-6 days (e.g., 2 days, 3 days, 4 days, 5 days or 6 days) to obtain the neural organoids.

[0024] Preferably, the neural organoids express molecular markers of neural stem cells and molecular markers of nerve cells.

[0025] Preferably, the molecular markers of the neural stem cells include any one or a combination of at least two of SOX2, PAX6, or NESTIN.

[0026] Preferably, the molecular markers of the nerve cells include any one or a combination of at least two of MAP2, TUJ1, CTIP2, SATB2, or NEUN.

[0027] Secondly, the present invention provides a neural organoid model, which is prepared by the three-dimensional culture-induced differentiation method for neural organoids described in the first aspect.

[0028] Thirdly, the present invention provides a method for three-dimensional culture-induced differentiation of neural organoids as described in the first aspect and / or the application of neural organoid models as described in the second aspect in toxicological detection.

[0029] The toxicological tests described in this invention can be performed directly in 96-well plates for culturing neural organoids, or transferred to other culture equipment.

[0030] Preferably, the application includes any one or a combination of at least two of the following: short-term and long-term toxicological testing of bisphenols, short-term and long-term toxicological testing of anticancer drugs, and toxicological testing of alcohol.

[0031] Fourthly, the present invention provides a method for toxicological detection, the method comprising: preparing neural organoids by means of the three-dimensional culture-induced differentiation method of neural organoids described in the first aspect, culturing the neural organoids in a neural differentiation culture medium and / or a neural maturation culture medium, and observing the size of the neural organoids and detecting the expression of molecular markers after adding the test substance.

[0032] Preferably, the substance to be tested includes any one or a combination of at least two of bisphenols, anticancer drugs, and alcohol.

[0033] Preferably, the bisphenolic substances include any one or a combination of at least two of bisphenol A, bisphenol B, bisphenol F, bisphenol P, bisphenol S, bisphenol AF, and raw materials and derivatives.

[0034] Preferably, the anticancer drug includes any one or a combination of at least two of doxorubicin, paclitaxel, cisplatin, or 5FU.

[0035] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) This invention has creatively discovered that by adding TGF-β signaling pathway inhibitors, WNT signaling pathway inhibitors and BMP signaling pathway inhibitors to the culture medium for induced pluripotent stem cells, the induced pluripotent stem cells can be induced to differentiate into neural organoids. The obtained neural organoids are large in size, have very little individual variation, and are highly stable. The obtained neural organoids can be applied to toxicological studies.

[0038] (2) The method for three-dimensional culture and induction of differentiation of neural organoids provided by the present invention can be operated in one culture plate without changing the culture plate, and can be cultured in a common incubator (containing 5% CO2). The culture method is simple, fast, stable and low cost, and has broad application prospects. Attached Figure Description

[0039] Figure 1 is a bright field image of cells from Example 1 and Comparative Example 1 after 10 days of cell growth (scale bar is 500 μm).

[0040] Figure 2 is a bright field image of cells from Example 1 and Comparative Example 1 after 16 days of cell growth (scale bar is 500 μm).

[0041] Figure 3 shows the expression of molecular markers in nerve cells after 22 days of cell growth in Example 1.

[0042] Figure 4 shows the expression of KI67 in neuronal organoids after short-term toxic treatment with bisphenol A substances.

[0043] Figure 5 is a bright field image of cell growth in neural organoids after long-term toxic treatment with bisphenols (scale bar: 500 μm).

[0044] Figure 6 shows the expression of molecular markers in nerve cells after long-term toxic treatment with bisphenol A substances in nerve organoids.

[0045] Figure 7 shows the apoptosis of cells in neural organoids after short-term doxorubicin toxicity treatment.

[0046] Figure 8 shows the expression of molecular markers in nerve cells after long-term toxic treatment with doxorubicin in organoids.

[0047] Figure 9 is a bright field image of cell growth of neural organoids after alcohol treatment (scale bar: 500 μm).

[0048] Figure 10 shows the expression of molecular markers in nerve cells after alcohol treatment of nerve organoids. Detailed Implementation

[0049] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0051] The sources or preparation methods of the reagents used in the following examples are as follows:

[0052] SB431542: Purchased from Tocris, product number Cat. No. 1614;

[0053] IWR1: Purchased from Tocris, product number Cat. No. 3532;

[0054] LDN-193189: Purchased from Stemolecule, product code 04-0074;

[0055] hiPSC medium: commercial name mTeSR Plus, purchased from Stemcell, product code 100-1130;

[0056] Neural differentiation medium (100 mL): GMEM (77 mL), KSR 5X (20 mL), P / S 100X (1 mL), NEAA 100X (1 mL), Sodium Pyruvate 100X (1 mL), beta-ME (0.1 mM);

[0057] Neural maturation medium (100 mL): DMEM / F-12 (96 mL), N-2 supplement 100X (1 mL), Lipid Concentrate 100X (1 mL), P / S 100X (1 mL), Glutamax 100X (1 mL).

[0058] Example 1

[0059] This embodiment provides a method for three-dimensional culture-induced differentiation of neural organoids. The method includes: digesting hiPSCs into single cells, transferring them into ultra-low adsorption 96-well plates, adding 20 μM Rock signaling pathway inhibitor Y-27632 to a conventional CO2 incubator for the first 6 days, and culturing them for 10 days in a medium containing 5 μM TGF-β signaling pathway inhibitor SB431542, 3 μM WNT signaling pathway inhibitor IWR1, and 100 nM BMP signaling pathway inhibitor LDN-193189 to obtain neural organoids.

[0060] Example 2

[0061] This embodiment provides a method for three-dimensional culture-induced differentiation of neural organoids. The method includes: digesting hiPSCs into single cells, transferring them into ultra-low adsorption 96-well plates, adding 25 μM Rock signaling pathway inhibitor Y-27632 to a conventional CO2 incubator for the first 5 days, and culturing them for 11 days in a medium containing 3 μM TGF-β signaling pathway inhibitor SB431542, 5 μM WNT signaling pathway inhibitor IWR1, and 80 nM BMP signaling pathway inhibitor LDN-193189 to obtain neural organoids.

[0062] Example 3

[0063] This embodiment provides a method for three-dimensional culture-induced differentiation of neural organoids. The method includes: digesting hiPSCs into single cells, transferring them into ultra-low adsorption 96-well plates, adding 15 μM Rock signaling pathway inhibitor Y-27632 to a conventional CO2 incubator for the first 7 days, and culturing for 9 days in a medium containing 7 μM TGF-β signaling pathway inhibitor SB431542, 1 μM WNT signaling pathway inhibitor IWR1, and 120 nM BMP signaling pathway inhibitor LDN-193189 to obtain neural organoids.

[0064] Comparative Example 1

[0065] This comparative example provides a method for inducing differentiation of neural organoids, which includes: Sasai-guided differentiation method: The first step of inducing neural stem cells requires 18 days, which is relatively long: hiPSCs are digested into single cells and transferred to ultra-low adsorption 96-well plates. For the first 6 days, 20 μM Rock signaling pathway inhibitor Y-27632 is added, and the cells are cultured for 18 days in a medium containing 5 μM TGF-β signaling pathway inhibitor SB431542 and 3 μM WNT signaling pathway inhibitor IWR1. After that, the cells are transferred to low adsorption culture dishes and cultured in a hyperoxia incubator (40% O2, 5% CO2). For other details, see the citation Kadoshima T, et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. PNAS, 2013, 110(50):20284-20289.

[0066] Test Example 1

[0067] In this test case, neural organoids were prepared using the methods provided in Example 1 and Comparative Example 1. After culturing for 10 days, 12 cell clusters with good growth were randomly selected for photographic recording, and the average diameter of the obtained neural organoid cell clusters was counted. The results are shown in Figure 1 and Table 1, where the diameter unit in Table 1 is μm.

[0068] Table 1

[0069] 1234567 Example 1734.967719.503739.022748.738723.849731.826719.605 Comparative Example 1600.574576.300545.551498.231565.442581.674523.692

[0070] 89101112 Mean Standard Deviation Example 1 707.83 1739.78 4654.04 1691.86 8686.21 4716.43 727.365 Comparative Example 1 456.34 4516.48 4542.44 4531.72 4554.81 3541.10 639.592

[0071] As can be seen from Figure 1 and Table 1, the neural organoid induction differentiation method provided by the present invention can accelerate cell proliferation. In the same time period, the neural organoids prepared using the method provided by the present invention are larger in volume, which facilitates subsequent research and observation.

[0072] Test Example 2

[0073] In this test case, neural organoids were prepared using the methods provided in Example 1 and Comparative Example 1. After culturing for 16 days, 48 ​​cell clusters with good growth were randomly selected for photographic recording, and the results are shown in Figure 2.

[0074] As can be seen from Figure 2, the volume of the three organoids marked with red dashed lines in Comparative Example 1 is significantly smaller than that of the other organoids, while the individual differences of the neural organoids induced in Example 1 are even smaller. The neural organoids prepared by the method provided in this invention have relatively uniform volume among individual organoids.

[0075] Test Example 3

[0076] In this test case, neural organoids were prepared using the method provided in Example 1. After culturing for 22 days, the organoids were fixed with 4% PFA (paraformaldehyde fixative), dehydrated with 30% sucrose, and embedded in OCT for frozen sectioning. Immunofluorescence staining was performed on the sections using primary antibodies SOX2 (mouse anti) and MAP2 (rabbit anti), and secondary antibodies 488 donkey anti-mouse and 568 donkey anti-rabbit. After mounting, images were taken using a confocal microscope. The results are shown in Figure 3. The neural organoids prepared using the method of this invention can express the molecular markers of neural stem cells (SOX2) and nerve cells (MAP2), indicating that the organoids contain neural stem cells and nerve cells and possess some tissue structure, thus forming neural organoids.

[0077] Test Example 4

[0078] In this test case, neural organoids were prepared using the method provided in Example 1. The organoids were then cultured in neural differentiation medium for 4 days, followed by 2 days of culture in neural differentiation medium containing 20 μM bisphenol A or 20 μM bisphenol B. After fixation with 4% PFA, frozen sections were prepared and cell proliferation was detected by KI67 immunofluorescence. The results are shown in Figure 4.

[0079] In this test case, neural organoids were prepared using the method provided in Example 1. The culture medium was replaced with neural differentiation medium and cultured for 4 days, followed by 12 days of culture using neural differentiation medium containing 20 μM bisphenol A or 20 μM bisphenol B. The results are shown in Figure 5. The expression of the neural cell markers SOX2 / MAP2 was then detected, and the results are shown in Figure 6.

[0080] As shown in Figures 4-6, bisphenol A substances affect the early development of neural organoids, reduce cell division, and decrease the volume of neural organoids.

[0081] Test Example 5

[0082] In this test case, neural organoids were prepared using the method provided in Example 1. The cells were then cultured in neural differentiation medium for 4 weeks and neural maturation medium containing 0.4 μM doxorubicin for 2 days. TUNEL assay was performed, with cells not treated with doxorubicin serving as the control group. The results are shown in Figure 7.

[0083] In this test case, neural organoids were prepared using the method provided in Example 1. The organoids were then cultured in neural differentiation medium for 4 weeks and in neural maturation medium containing 0.4 μM doxorubicin for 10 days. The expression of neural stem cells and the neural cell molecular markers SOX2 / MAP2 was detected. Cells not treated with doxorubicin were used as the control group. The results are shown in Figure 8.

[0084] Figures 7 and 8 show that short-term doxorubicin treatment increases apoptosis, while long-term doxorubicin treatment alters the internal structure of neural organoids.

[0085] Test Example 6

[0086] In this test case, neural organoids were prepared using the method provided in Example 1. They were cultured for 4 days in untreated 6 cm culture dishes using neural differentiation medium, followed by 14 days in neural differentiation medium containing 100 mM ethanol. The size of the neural organoids was observed and recorded. Cells not treated with ethanol were used as a control group. The results are shown in Figure 9. Frozen sections of the neural organoids were then prepared, and the expression of the neural cell markers SOX2 / MAP2 was detected. Cells not treated with ethanol were used as a control group. The results are shown in Figure 10.

[0087] As shown in Figures 9 and 10, alcohol treatment reduced the volume of neural organoids and decreased the number of SOX2-positive neural stem cells.

[0088] In summary, this invention provides a method for three-dimensional culture-induced differentiation of neural organoids. By adding a compound inhibitor to the culture medium for induced pluripotent stem cells, the induced pluripotent stem cells differentiate into neural organoids. The obtained neural organoids are large in size and have very little individual variation. The culture method is simple, rapid, stable, and low in cost, and has broad application prospects.

[0089] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for inducing differentiation through three-dimensional culture of neural organoids, characterized in that, The method for inducing differentiation of neural organoids through three-dimensional culture includes: adding signaling pathway inhibitors to a three-dimensional culture of induced pluripotent stem cells, culturing the cells, and obtaining the neural organoids; the signaling pathway inhibitors include TGF-β signaling pathway inhibitors, WNT signaling pathway inhibitors, and BMP signaling pathway inhibitors.

2. The method for inducing differentiation through three-dimensional culture of neural organoids according to claim 1, characterized in that, The TGF-β signaling pathway inhibitors include any one of SB431542, SB505124, A83-01, Disitertide, Galunisertib, A77-01, or AZ12799734; Preferably, the WNT signaling pathway inhibitor includes any one of IWR1, IWP4, FH535, NLS-StAx-h, KYA1797K, TAK715, Klotho-derived peptide 6, or WNTinib; Preferably, the BMP signaling pathway inhibitor includes any one of LDN-193189, Dorsomorphin, DMH-1, DMH2, K02288, M4K2163 dihydrochloride, or ML347.

3. The method for inducing differentiation through three-dimensional culture of neural organoids according to claim 1 or 2, characterized in that, The signaling pathway inhibitors also include Rock signaling pathway inhibitors; Preferably, the Rock signaling pathway inhibitor includes Y-27632; Preferably, the method for three-dimensional culture-induced differentiation of the neural organoids includes: culturing induced pluripotent stem cells in a culture medium containing 3-7 μM TGF-β signaling pathway inhibitor, 1-5 μM WNT signaling pathway inhibitor and 80-120 nM BMP signaling pathway inhibitor for 9-11 days to obtain the neural organoids; Preferably, the method for inducing differentiation through three-dimensional culture of neural organoids includes the following steps: (1) Culture induced pluripotent stem cells for 5-7 days in a culture medium containing 15-25 μM Rock signaling pathway inhibitor, 3-7 μM TGF-β signaling pathway inhibitor, 1-5 μM WNT signaling pathway inhibitor and 80-120 nM BMP signaling pathway inhibitor; (2) The cells obtained in step (1) were cultured for 2 to 6 days in a culture medium containing 3 to 7 μM TGF-β signaling pathway inhibitor, 1 to 5 μM WNT signaling pathway inhibitor and 80 to 120 nM BMP signaling pathway inhibitor to obtain the neural organoids.

4. The method for inducing differentiation through three-dimensional culture of neural organoids according to any one of claims 1 to 3, characterized in that, The neural organoids express molecular markers of neural stem cells and neural cells; Preferably, the molecular markers of the neural stem cells include any one or a combination of at least two of SOX2, PAX6, or NESTIN; Preferably, the molecular markers of the nerve cells include any one or a combination of at least two of MAP2, TUJ1, CTIP2, SATB2, or NEUN.

5. A neural organoid model, characterized in that, The neural organoid model was prepared by the method for three-dimensional culture-induced differentiation of neural organoids as described in any one of claims 1 to 4.

6. The method for three-dimensional culture-induced differentiation of neural organoids as described in any one of claims 1 to 4 and / or the application of the neural organoid model as described in claim 5 in toxicological testing.

7. The application according to claim 6, characterized in that, The applications include any one or a combination of at least two of the following: short-term and long-term toxicological testing of bisphenols, short-term and long-term toxicological testing of anticancer drugs, and toxicological testing of alcohol.

8. A method for toxicological detection, characterized in that, The toxicological detection method includes: preparing neural organoids using the three-dimensional culture-induced differentiation method for neural organoids as described in any one of claims 1 to 4; culturing the neural organoids in a neural differentiation culture medium and / or a neural maturation culture medium; and observing the size of the neural organoids and detecting the expression of molecular markers after adding the test substance.

9. The method for toxicological detection according to claim 8, characterized in that, The substances to be tested include any one or a combination of at least two of bisphenols, anticancer drugs, and alcohol.

10. The method for toxicological detection according to claim 9, characterized in that, The bisphenol compounds include any one or a combination of at least two of bisphenol A, bisphenol B, bisphenol F, bisphenol P, bisphenol S, bisphenol AF, and raw materials and derivatives; Preferably, the anticancer drug includes any one or a combination of at least two of doxorubicin, paclitaxel, cisplatin, or 5FU.

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