Construction method of epilepsy brain organ disease model

Abstract

The invention relates to the technical field of biology, in particular to a construction method of an epilepsy brain organ disease model, which comprises the following steps: A, culturing human induced pluripotent stem cells until the cell density is 70-90%, and carrying out passage; b, the human induced pluripotent stem cells are dissociated through digestive juice to obtain cell suspension, after the cell suspension is centrifuged and supernatant is sucked and discarded, the cell suspension is inoculated into an AggreWell TM 80024 pore plate, EB balls are formed in an embryoid body forming culture medium and a Y27632 factor, and the embryoid body forming culture medium is prepared by adding a CHIR-99021 inhibitor and an EGF factor into an mTeSRTM culture medium; c, placing the EB ball in an embryoid body forming culture medium, and culturing until the diameter of the EB ball is 400-600 [mu] m; d, culturing in an EB2 culture medium until the diameter is 500-800 microns; e, culturing in an EB3 culture medium for three days; and F, carrying out shake cultivation in an EB4 culture medium. The method can be used for identifying brain organs and researching pathogenesis of nervous system diseases such as epilepsy and drug screening.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for constructing an epileptic brain organoid disease model. Background Art

[0002] Epilepsy is a brain disease characterized by unbalanced brain electrical activity and is related to the imbalance of neuronal excitation and inhibition transmission. Due to the lack of a suitable research model that has both a three-dimensional structure and the same human genetic information, the progress of research on the pathogenesis of various epilepsies has been hindered. The traditionally used 2D cell models and animal models have many limitations. For example, 2D cell models do not have a three-dimensional and complex structure, while the genetic information and brain structure of animal models are somewhat different from those of humans. These limitations restrict their ability to study the underlying mechanisms of the brain and epilepsy occurrence. With the rise and development of the brain organoid technology, constructing epileptic brain organoids can provide great convenience for the study of disease pathogenesis and the screening of effective therapeutic drugs. However, there are still few cultivation methods for successfully and effectively constructing a brain organoid disease model that can be used to study the pathogenesis of epilepsy, and there is an urgent need to provide a more convenient and effective method for constructing an epileptic brain organoid disease model. Summary of the Invention

[0003] To solve the above technical problems, an embodiment of the present invention provides a method for constructing an epileptic brain organoid disease model.

[0004] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0005] The present invention provides a method for constructing an epileptic brain organoid disease model, comprising the following steps:

[0006] A. Culturing human induced pluripotent stem cells until the cell density reaches 70% - 90%, and then performing passage;

[0007] B. Dissociating human induced pluripotent stem cells with a digestive solution to obtain a cell suspension, centrifuging the cell suspension and discarding the supernatant, and then inoculating it into an AggreWell TM 80024-well plate to form EB spheres in a medium for embryoid body formation and Y27632 factor. The medium for embryoid body formation: CHIR-99021 inhibitor and EGF factor are added to mTeSRTM medium;

[0008] C. Culturing the EB spheres in the medium for embryoid body formation until the diameter of the EB spheres reaches 400 - 600 μm;

[0009] D. Culturing in EB2 medium until the diameter reaches 500 - 800 μm;

[0010] E. Culturing in EB3 medium for three days;

[0011] F. Incubate in a shaker in EB4 medium.

[0012] In some embodiments, in step A, the culture conditions are as follows: culture in mTeSRTM medium containing 6 μM Y27632 factor. After 18 hours of inoculation, discard the mTeSRTM medium containing Y27632 factor, and add DMEM / F12 medium to the culture dish. Change the medium daily until the cell density reaches 70% - 90%.

[0013] In some embodiments, in step A, the embryoid body formation medium: Add 40 μL of CHIR-99021 and 35 μL of EGF to 20 mL of mTeSRTM medium.

[0014] In some embodiments, in step D, the EB2 medium: Add 0.5 mL of STEMdiff TM Cerebral Organoid Supplement B to 49.5 mL of STEMdiff TM Cerebral Organoid Basal Medium 1.

[0015] In some embodiments, in step E, the EB3 medium: Add 0.25 mL of STEMdiff TM Cerebral Organoid Supplement C and 0.5 mL of STEMdiff TM Cerebral Organoid Supplement D to 24.25 mL of STEMdiff TM Cerebral Organoid Basal Medium 2.

[0016] In some embodiments, in step F, the EB4 medium: Add 2 mL of STEMdiff TM Cerebral Organoid Supplement E to 98 mL of STEMdiff TM Cerebral Organoid Basal Medium 2.

[0017] The present invention has the following beneficial effects compared with the prior art:

[0018] The self-renewal ability and multi-directional differentiation ability of human induced pluripotent stem cells (iPSCs) enable them to spontaneously aggregate to form 3D aggregates, which are EB spheres. In a certain neural induction molecule and basal medium, EB spheres can expand the neuroepithelial layer and further develop neurally. The expanded neuroepithelial buds merge and then present a neural rosette-like structure. The brain organoid forms a dense center, and as the brain organoid matures, this dense center becomes denser and deeper and has the distribution of cortical cells. The mature brain organoid can reach a diameter of 3-5 mm and can be subjected to immunofluorescence staining by paraffin embedding and sectioning to verify the expression of proteins such as the mature neuron markers β-Tubulin, MAP2, and TUJ1. The mature brain organoid can be used to explore the pathogenesis of nervous system diseases and screen for effective drugs.

[0019] Even if the iPSCs used to construct the brain organoids are in a poor state or have certain differentiation, the brain organoids can still show the proper morphological characteristics and diameter size at the embryoid body formation stage, neural induction and differentiation stage, expansion stage, and mature stage and can develop smoothly at each stage. The EB can be seen with the naked eye on the 5th day (diameter 500-800 μm), with a smooth and translucent edge, indicating the formation of the neuroepithelium and a good state of the embryoid body, and it will not break during subsequent culture. The organoids on the 7th day further develop and expand to generate a neuroepithelial structure, showing a budding shape on the surface of the EB. The organoids on the 10th day show a budding morphology, indicating the expansion of the neuroepithelium. On the 13th day, the expanded neuroepithelial buds merge and form a dense center, and a neural rosette-like structure can be observed. On the 20th day, the diameter of the organoid exceeds 750 μm, and the neuroepithelial layer further expands. On the 30th day, the diameter of the organoid exceeds 1 mm and shows a tissue structure with a dense center. On the 40th day, the diameter of the organoid can reach 3-5 mm, the central part is denser, and it has the distribution of cortical cells. The brain organoids have been effectively and stably constructed and matured, and can be used for brain organoid identification and research on exploring the pathogenesis of nervous system diseases such as epilepsy and drug screening.

[0020] Currently, the mainstream methods for culturing brain organoids have relatively high requirements for the state and differentiation degree of initial iPSCs. Only when the cell state is good and the differentiation is extremely low can a certain rate of embryoid body formation be ensured. When using this culture method to construct brain organoids, even if the initial iPSCs are in a poor state or there are many differentiated cells, embryoid bodies can still be formed, and the fragmentation rate during subsequent culture is relatively low, effectively improving the success rate and stability of brain organoid construction. By resuspending iPSCs with a mixed solution of EGF, CHIR-99021, ROCKi inhibitor Y27632, and mTeSRTM medium at the initial stage of constructing epileptic brain organoids, injecting the resuspended cell suspension into an AggreWellTM 800 24-well plate, under the action of centrifugation, the cells will aggregate in the small chambers within the wells. After standing for 2 days, 300 embryoid bodies with smooth edges and good morphology can be formed. After transferring them to a low-attachment 6-well plate, continue to culture them statically with the above-mentioned mixed medium supplemented with several factors. The volume of the embryoid bodies will increase rapidly and the edges will become more translucent, indicating the start of neural induction. When changing the medium on the 5th day, change to STEMdiff TM Cerebral Organoid Supplement B and STEMdiff TM Cerebral Organoid Basal Medium 1 mixed medium can make the subsequent neural induction and construction more stable. If STEMdiff TM Cerebral Organoid SupplementA and STEMdiff TM Cerebral Organoid Basal Medium 1 mixed medium is used when constructing epileptic brain organoids, even if the state of iPSCs is good and the differentiation ratio is extremely low, and the epileptic brain organoids are constructed according to the conventional process, the number of formed embryoid bodies is small, the edges are not smooth or the state is poor, which seriously affects the subsequent development of continuous induction and often leads to the failure of constructing the epileptic brain organoid disease model. Therefore, using the method of the present invention can effectively improve the stability and sustainability of constructing the epileptic brain organoid disease model, laying a solid foundation for studying the pathogenesis of epilepsy and exploring effective therapeutic drugs. Description of the Drawings

[0021] Figure 1 In Figure A, the iPSCs with better cell state and less self-differentiation are compared with the iPSCs with poorer cell state and more self-differentiation in Figure B;

[0022] Figure 2 In Figure A, the EB balls on the 5th day constructed with the brain organoid construction medium are compared with the EB balls on the 5th day constructed in the embodiment of the present application in Figure B;

[0023] Figure 3Figure A shows a cerebral organoid on the 15th day constructed with a cerebral organoid construction medium, and Figure B shows a cerebral organoid on the 15th day constructed with the cerebral organoid construction method of the present application.

[0024] Figure 4 Figure A shows a cerebral organoid on the 35th day constructed with a cerebral organoid construction medium, and Figure B shows a cerebral organoid on the 35th day constructed with the cerebral organoid construction method of the present application. Detailed implementation manners

[0025] Next, the technical solutions in some embodiments of the present disclosure will be clearly and completely described in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present disclosure.

[0026] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in this application can be obtained through market purchases or can be prepared by existing methods.

[0027] The iPSCs medium (mTeSRTM) is purchased from STEMCELL (85850), and 400 ml of Basal medium mTeSRTM1 and 100 ml of 5×supplement mTeSRTM1 are mixed for use.

[0028] The cerebral organoid construction medium (STEMdiffTM Cerebral Organoid Kit) is purchased from STEMCELL (08570).

[0029] The ROCKi inhibitor (Y27632 factor) is purchased from SELLECK (S6390).

[0030] Matrigel matrix glue is purchased from Corning (354277).

[0031] CHIR-99021 is purchased from STEMCELL (72052).

[0032] EGF is purchased from STEMCELL (78006).

[0033] The DMEM / F12 medium is purchased from Gibco (C11330500BT).

[0034] The human pluripotent stem cell digestive solution is purchased from CELLAPY (CA1008500).

[0035] ACCUTASE is purchased from STEMCELL (07920).

[0036] Experimental materials:

[0037] (1) Cells

[0038] Human induced pluripotent stem cells (hiPSCs) derived from epilepsy patients were obtained from the Key Laboratory of Major Obstetric Diseases of Guangdong Province, the Third Affiliated Hospital of Guangzhou Medical University.

[0039] (2) Materials

[0040] hiPSCs medium: 80% Basal medium mTeSRTM1 + 20% 5×supplement mTeSRTM1.

[0041] Embryoid body formation medium: 0.2% CHIR-99021 + 0.175% EGF + 99.625% hiPSCs medium.

[0042] EB2 medium: 1% STEMdiff TM Cerebral Organoid Supplement B + 99% STEMdiff TM Cerebral Organoid Basal Medium 1.

[0043] EB3 medium: 1% STEMdiff TM Cerebral Organoid Supplement C + 2% STEMdiff TM Cerebral Organoid Supplement D + 97% STEMdiff TM Cerebral Organoid BasalMedium 2.

[0044] EB4 medium: 2% STEMdiff TM Cerebral Organoid Supplement E + 98% STEMdiff TM Cerebral Organoid Basal Medium 2.

[0045] Matrigel matrix glue: When used for iPSCs culture, it is dissolved on ice and diluted with DMEM / F12 at a ratio of 1:1000. When used for embedding cerebral organoids, it can be directly used after being dissolved on ice.

[0046] Examples

[0047] Experimental methods:

[0048] 1. Culture and Passage of iPS Cells

[0049] 1.1 Spread Matrigel matrix glue diluted 1:1000 with DMEM / F12 on a 3.5 mm cell culture dish in advance and place it in a 37 °C cell culture incubator for 30 minutes.

[0050] 1.2 Pre - turn on a 37 °C water bath, take a 15 mL centrifuge tube, and add 5 mL of pre - warmed DEME / F12 medium.

[0051] 1.3 Take out iPS cells from the liquid nitrogen tank or - 80 °C refrigerator ( Figure 1 In Figures A and B in it, both are iPSCs derived from epilepsy patients. The state of Figure A is better and that of Figure B is worse. The cell counting of cells with the same culture process here is about 5x10 5 , and here we choose Figure 1 the cells in Figure B), and quickly dissolve them at 37 °C until only small ice crystals remain in the cryopreservation tube, then bring them into the biosafety cabinet.

[0052] 1.4 Transfer the liquid in the cryopreservation tube to the above - mentioned centrifuge tube containing pre - warmed DMEM / F12 medium, and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0053] 1.5 Aspirate as much supernatant of the centrifuge tube as possible, and add 2 mL of iPSCs medium to resuspend the cell pellet in the centrifuge tube. Aspirate the pre - spread Matrigel matrix glue in the culture dish, add the above cell suspension to the culture dish, and add 6 μM Y27632 factor. Shake well cross - wise and place it in a 37 °C, 5% CO 2 incubator for culture.

[0054] 1.6 After 18 hours, aspirate the original iPSCs medium containing Y27632 factor, add 1 mL of DMEM / F12 medium along the side wall of the culture dish, gently shake for washing, aspirate it, and then add 2 mL of iPSCs medium without Y27632 factor. After that, change the medium every day and observe the cell state and density.

[0055] 1.7 Observe the cell density after 2 - 3 days. When the cell density grows to 70% - 90%, passage the cells. Aspirate the original medium, add 1 mL of DMEM / F12 medium along the side wall of the culture dish, gently shake for washing twice, aspirate the DMEM / F12 medium, and then add 1 mL of iPSC cell digestive solution, and place it in a 37 °C incubator for 4 minutes.

[0056] 1.8 Prepare a 15 mL centrifuge tube, add 3 mL of DMEM / F12 medium to the tube, blow - pipette the iPSC cells away from the culture dish with the above iPSC cell digestive solution and aspirate them into the centrifuge tube, using 1

[0057] Wash the remaining iPSC cells with mLDMEM / F12 medium 2 - 3 times and transfer them into the above centrifuge tube. Centrifuge at 1000 rpm for 5 minutes at room temperature.

[0058] 1.9 Aspirate the pre - coated Matrigel matrix gel in the culture dish. Aspirate as much supernatant as possible from the centrifuge tube, and add 6 mL of iPSCs medium to resuspend the cell pellet in the centrifuge tube. Aspirate the pre - coated Matrigel matrix gel in the culture dish. Add 2 mL of the above cell suspension to the culture dish, and add 6 μM Y27632 factor to each culture dish. Shake well cross - wise and place in an incubator at 37°C and 5% CO 2 Incubate in the incubator.

[0059] 2. Brain organoid construction

[0060] 2.1 Prepare the embryoid body formation medium: Add 40 μL of CHIR - 99021 (STEMCELLTM) and 35 μL of EGF to 20 mL of mTeSRTM medium to form the embryoid body formation medium, namely EB1 medium. Preheat it for later use.

[0061] 2.2 Observe the state and density of the above iPSC cells under a microscope. When the cell density is 70%, it is suitable for constructing brain organoids.

[0062] 2.3 Aspirate the mTeSRTM medium, add 1 mL of DMEM / F12 medium along the side wall of the culture dish, gently shake and wash twice. After aspiration, add 1 mL of Accutase and place it in a 37°C incubator for 4 minutes.

[0063] 2.4 Prepare a 15 - mL centrifuge tube, add 3 mL of DMEM / F12 medium to the tube. Use the above Accutas to pipette the iPSC cells away from the culture dish and aspirate them into the centrifuge tube. Wash the remaining iPSC cells with 1 mL of DMEM / F12 medium 2 - 3 times and transfer them into the above centrifuge tube. Mix well, then pipette 10 μL of the cell suspension and 10 μL of trypan blue to mix, and pipette 10 μL to an automatic cell counting plate and place it in an automatic cell counter for cell counting.

[0064] 2.5 Calculate the volume required to obtain 3×106 cells, transfer the cell suspension of this volume to another new centrifuge tube, and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0065] 2.6 Aspirate as much supernatant as possible from the centrifuge tube, and add 2 mL of embryoid body formation medium and 6 μM Y27632 factor to resuspend the cell pellet in the centrifuge tube. Add it to the AggreWellTM 800 24 - well plate, set the speed increase to 1 and the speed decrease to 1 at room temperature, centrifuge at 300 rcf for 4 minutes, and then place it in an incubator at 37°C and 5% CO 2 Incubate in the incubator.

[0066] 2.7 After standing for 48 hours, observe whether the iPSC cells aggregate into EB spheres. Blow up the EB spheres in the wells and transfer them to a low-attachment 24-well plate. Add 3 ml of EB1 medium. Observe under a microscope that the diameter of the EB is about 100 - 200 μm. After cross-shaking evenly, place it statically in an incubator at 37 °C and 5% CO 2 incubator.

[0067] 2.8 Prepare EB2 medium: Add 0.5 mL of STEMdiff TM Cerebral Organoid Supplement B to 49.5 mL of STEMdiff TM Cerebral Organoid Basal Medium 1.

[0068] 2.9 On the 5th day, observe that the diameter of the EB is greater than 300 μm (400 - 600 μm), and it shows a round and smooth edge. These EB spheres can be subjected to the second-stage operation. Transfer the EB spheres to a new low-attachment 24-well plate, slowly add 3 mL of EB2 medium. After cross-shaking evenly, place it statically in an incubator at 37 °C and 5% CO 2 incubator.

[0069] 2.10 On the 7th day, the EB spheres are visible to the naked eye (diameter 500 - 800 μm), and have a smooth and translucent edge, indicating the formation of neuroepithelium. These EB spheres can enter the third-stage culture.

[0070] 2.11 Prepare EB3 medium: Add 0.25 mL of STEMdiff TM Cerebral Organoid Supplement C and 0.5 mL of STEMdiff TM Cerebral Organoid Supplement D to 24.25 mL of STEMdiff TM Cerebral Organoid Basal Medium 2. Thaw the Matrigel matrix glue at 2 - 8 °C on ice for 1 - 2 hours.

[0071] 2.12 Add a sterile Organoid Embedding Sheet to a sterile 100 mm culture dish. Use a wide-mouth 200 μL pipette tip to aspirate 25 - 50 μL of the medium containing EB spheres from one well of the 24-well plate and transfer it to the embedding sheet. Repeat this step until 12 - 16 EB spheres are placed on the embedding sheet.

[0072] 2.13 Using a standard 200 μL pipette tip, aspirate the excess culture medium except for each EB. Add 15 μL of Matrigel to each EB, and use a new 10 μL pipette tip to position the EB at the center of the droplet. Incubate at 37 °C for 20 minutes and wait for the Matrigel to solidify.

[0073] 2.14 After the Matrigel has solidified, use sterile forceps to lift the embedding sheet containing the solidified Matrigel droplets. Aspirate the EB3 medium with a 1 mL pipette and gently wash the Matrigel droplets from the embedding sheet into a low-attachment 6-well plate. Repeat the above steps so that each well contains 10 Matrigel-embedded organoids and 3 mL of EB3 medium. Incubate at 37 °C in a 5% CO 2 incubator for 3 days.

[0074] 2.15 Prepare EB4 medium: Add 2 mL of STEMdiff TM Cerebral Organoid Supplement E to 98 mL of STEMdiff TM Cerebral Organoid Basal Medium 2.

[0075] 2.16 On the 10th day, observe under the microscope that the embedded organoids will develop and expand to form neuroepithelial structures, which appear as budding on the surface of the EB. These EBs can be cultured in the fourth stage. Aspirate the original EB3 medium, add 3 mL of EB4 medium, and place it on a horizontal shaker at 70 rpm. Incubate at 37 °C in a 5% CO2 incubator.

[0076] 2.17 Perform a complete medium change every 3 days. Tilt the 6-well plate so that the cerebral organoids lean to one side. Aspirate the original EB4 medium on the other side and add 3 mL of fresh EB4 medium. Place it on a horizontal shaker at 65 rpm. Incubate at 37 °C in a 5% CO 2 incubator.

[0077] In Figure 1 Figure A shows that the edges of the iPSC clones derived from epilepsy patients are clear and smooth, are flat and dense circles, and grow in a colony-like manner. The stem cells inside the colony are small and round, with uniform morphology, indicating that the iPSC state is good and there are almost no differentiated cells.

[0078] Figure B shows that the edges of the iPSC clones derived from epilepsy patients are not smooth, and there are a large area of differentiated cells around them, with non-uniform morphology and poor cell state.

[0079] In Figure 2In Figure A, the EB sphere on the 5th day constructed using the brain organoid construction medium is relatively small in volume, with an uneven edge, the edge tissue is not translucent, and the whole is a dense sphere.

[0080] In Figure B, the diameter of the EB sphere on the 5th day constructed using the brain organoid construction method of the present application is about 300 microns, and the outer edge is smooth, and the edge tissue becomes translucent, indicating the induction and formation of the primitive neuroectoderm.

[0081] In Figure 3 Figure A shows the brain organoid on the 15th day constructed using the brain organoid construction medium. It can be seen that the volume of the brain organoid is relatively small and the neuroblast buds are hardly visible, that is, there is almost no neuroinductive expansion and development.

[0082] Figure B shows the brain organoid on the 15th day constructed using the brain organoid construction method of the present application. It can be seen that the buds grow and expand, the budding range is large, arranged radially, and the rosette-like structure also further increases.

[0083] In Figure 4 Figure A shows the brain organoid on the 35th day constructed using the brain organoid construction medium. Compared with the 15th day, only the volume has increased, and there is still no neuroexpansion development.

[0084] Figure B shows the brain organoid on the 35th day, with a diameter of about 3 - 5 mm. The central part of the organoid is dense and deep, and has the distribution of cortical cells and a gyrus-like structure.

[0085] In the description of this specification, specific features, structures, materials, or characteristics may be combined in a suitable manner in any one or more embodiments or examples.

[0086] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A method for constructing an epileptic brain organoid disease model, characterized in that: The following steps are involved: A. Cultivate human induced pluripotent stem cells to a cell density of 70% to 90% and perform passaging; B. Human induced pluripotent stem cells were dissociated by digestion solution to obtain cell suspension, which was centrifuged and the supernatant was discarded before inoculation in AggreWell TM In an 80024-well plate, EB spheres were formed in an embryoid body formation medium containing an embryoid body formation medium and a Y27632 factor, wherein the embryoid body formation medium is mTeSRTM medium with CHIR-99021 inhibitor and EGF factor added thereto; C. Place the EB spheres in embryoid body formation medium and culture until the diameter of the EB spheres is 400-600 μm; D. Cultivate in EB2 medium until the diameter is 500-800 μm; E, cultured in EB3 medium for three days; F. Culture in EB4 medium on a shaking platform.

2. The construction method according to claim 1, characterized in that: In step A, the culture conditions are mTeSRTM medium containing 6 μM Y27632 factor. After 18 hours of inoculation, the mTeSRTM medium containing Y27632 factor is discarded, and DMEM / F12 medium is added to the culture dish. The medium is changed every day until the cell density reaches 70% to 90%.

3. The construction method according to claim 1, characterized in that: In step A, the embryoid body culture medium: 40 μL CHIR-99021 and 35 μL EGF were added to 20 mL mTeSR™ medium.

4. The construction method according to claim 1, characterized in that: In step D, the EB2 medium: 0.5 mL of STEMdiff TM Add Cerebral Organoid Supplement B to 49.5 mL of STEMdiff TM CerebralOrganoid Basal Medium 1 in.

5. The construction method according to claim 1, characterized in that: In step E, the EB3 medium: 0.25 mL STEMdiff TM Cerebral Organoid Supplement C and 0.5mL STEMdiff TM Cerebral OrganoidSupplement D Added 24.25mL STEMdiff TM Cerebral Organoid Basal Medium 2.

6. The construction method according to claim 1, characterized in that: In step F, the EB4 medium: 2 mL of STEMdiff TM Add Cerebral Organoid Supplement E to 98 mL of STEMdiff TM CerebralOrganoid Basal Medium 2 Medium.

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