Mesenchymal stem cell assisted brain organ culture method and application thereof

Abstract

The invention discloses a brain organ culture method assisted by mesenchymal stem cells. The brain organ culture method comprises the step of adding mesenchymal stem cells in an initial stage or an induction stage. It is found for the first time that the mesenchymal stem cells can be fully fused with embryonic stem cells or induced pluripotent stem cells or embryoid bodies formed by the embryonic stem cells or the induced pluripotent stem cells and survive, so that development of brain organs is promoted, the growth speed of the brain organs is increased, extracellular matrix reconstruction of the brain organs is accelerated, pluripotency of the stem cells is adjusted, and the survival rate of the brain organs is increased. The nerve maturation in the brain organs is promoted. As a model, the obtained brain organ can be used for researching the development mechanism of the brain, the pathogenesis of diseases related to the brain and drug screening.

Description

Technical Field

[0001] The present invention belongs to the field of organoid culture, and specifically relates to a mesenchymal stem cell-assisted brain organoid culture method and application thereof. Background Art

[0002] Organoids are "organ-like" miniature organs produced by stem cells through 3D culture in vitro, with self-renewal and assembly capabilities, and structures and functions highly similar to the source tissues or organs. Brain organoids refer to the self-assembly of a specific number of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into 3D tissues similar to the human brain in a 3D microenvironment. At present, a variety of brain organoid models have been successfully studied, such as regional brain organoids, whole brain organoids, and fused brain organoids. In general, two different types of methods can be used to generate brain organoids: unguided differentiation and guided differentiation. Unguided differentiation refers to the spontaneous formation of whole brain organoids with minimal external factors, which completely relies on spontaneous morphogenesis and the intrinsic differentiation ability of hPSC aggregates (embryoid bodies). Guided differentiation requires the addition of external patterning factors to induce embryonic stem cells or pluripotent stem cells to differentiate into specific cell types to form brain organoids with regional characteristics. According to the relevant literature on brain organoids at home and abroad, the method of culturing brain organoids in vitro can be summarized into these four steps: 1. Obtain a specific number of embryonic stem cells or induced pluripotent stem cells, and place them in a low-adhesion 96-well plate to quickly aggregate to form embryoid bodies; 2. Add an appropriate amount of stimulating factors, for example, LANCASTERMA et al. added the ROCK inhibitor Y-27632, which is a small molecule compound that helps to improve the survival rate of embryonic stem cells; 3. A suitable extracellular matrix is ​​needed as a support to promote the formation of the neuroepithelium. The most widely used extracellular matrix is ​​an extract from mouse sarcoma cells, and its main components include collagen, proteoglycans and a variety of different growth factors; 4. Rotating bioreactors are used to culture brain organoids. Since brain organoids lack the vascular system in the body, they will become larger and larger over time, and there will be a shortage of nutrients and oxygen inside, which will hinder the maturation and differentiation of neurons. The use of this dynamic culture system can promote the diffusion of nutrients and oxygen to a certain extent and reduce the death of cells inside brain organoids.

[0003] Mesenchymal stem cells (MSC) are a type of multipotent stem cells in the mesoderm, with strong proliferation ability and multidirectional differentiation potential, and have the ability to differentiate into a variety of cells under appropriate in vivo or in vitro environments. Clinical research on MSC has been carried out in many countries. The principle of MSC treatment is based on the potential of MSC to differentiate and form new replacement cells in diseased tissues. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to accelerate the growth rate of brain organoids and promote the neural maturation in brain organoids.

[0005] In order to solve the above technical problems, the present invention first provides a brain organoid culture method.

[0006] The brain organoid culture method provided by the present invention comprises the following steps: a1) culturing embryonic stem cells or induced pluripotent stem cells in an embryoid body formation medium to obtain embryoid bodies; a2) performing neural induction culture on the embryoid bodies in an induction medium, and then performing expansion culture and maturation culture to form brain organoids; The step a1) or step a2) comprises the step of adding mesenchymal stem cells during the culture process.

[0007] When mesenchymal stem cells are added in the step a1), the step a1) comprises the following steps: culturing embryonic stem cells or induced pluripotent stem cells and mesenchymal stem cells in an embryoid body formation medium.

[0008] Furthermore, the embryonic stem cells or the induced pluripotent stem cells and the mesenchymal stem cells are cultured in a 96-well cell culture plate containing an embryoid body formation medium; The embryonic stem cells or induced pluripotent stem cells may be added in an amount of 8,000-12,000 cells / well, specifically 10,000 cells / well; the mesenchymal stem cells may be added in an amount of 2,500-20,000 cells / well, specifically 2,500 cells / well, 5,000 cells / well, 10,000 cells / well, and 20,000 cells / well.

[0009] Furthermore, the method comprises the following steps: a1) Mix the embryonic stem cell solution or induced pluripotent stem cell solution with the mesenchymal stem cell solution and inoculate them into a 96-well cell culture plate, 100 μl per well, and then centrifuge and culture for 3 days to obtain embryoid bodies; a2) The embryoid bodies are washed with induction medium and placed in a new 96-well cell culture plate, and the volume is supplemented to 300 μl with induction medium. After 3 days of induction culture in the induction medium, cell spheres are obtained, and the cell spheres are embedded in matrix gel and transferred to a 6-well cell culture plate containing expansion medium. After 3 days of expansion culture, the expansion medium in the 6-well cell culture plate is replaced with maturation medium, and the cells are cultured in the maturation medium for at least 30 days to obtain brain organoids.

[0010] In the step a1), the embryonic stem cell solution is composed of embryonic stem cells and embryoid body formation medium. The induced pluripotent stem cell solution is composed of induced pluripotent stem cells and embryoid body formation medium. The mesenchymal stem cell solution is composed of mesenchymal stem cells and embryoid body formation medium.

[0011] The embryonic stem cells and the induced pluripotent stem cells also include a step of digesting with a cell dissociation reagent GCDR before mixing; the digestion conditions can be 37° C., 5% CO2 digestion for 10 minutes.

[0012] The mesenchymal stem cells may be digested with ACCUTASE enzyme before mixing; the digestion time may be 5-10 min.

[0013] In the step a1), the centrifugation condition may be 100×g for 3 min.

[0014] In the step a1), the culture conditions may be 37° C. and 5% CO 2 .

[0015] In the step a1), the cell culture plate should not be disturbed during the culture for at least 24 hours.

[0016] In the step a1), 100 μl of pluripotent stem cell culture medium is added every day on the first and second days of the culture.

[0017] In the step a2), the conditions of the induction culture and the expansion culture can both be 37° C. and 5% CO 2 .

[0018] In the step a2), the conditions of the maturation culture may be 37° C., 5% CO 2 , 65 rpm, and the culture medium may be replaced every 3 days.

[0019] When mesenchymal stem cells are added in the step a2), the step a2) comprises the following steps: subjecting the embryoid bodies and mesenchymal stem cells to neural direction induction culture in an induction medium.

[0020] Furthermore, the embryonic stem cells or the induced pluripotent stem cells and the mesenchymal stem cells are cultured in a 96-well cell culture plate containing embryoid body formation medium; The embryonic stem cells or induced pluripotent stem cells may be added in an amount of 8,000-12,000 cells / well, specifically 10,000 cells / well; the mesenchymal stem cells may be added in an amount of 10,000-50,000 cells / well, specifically 10,000 cells / well or 50,000 cells / well.

[0021] Furthermore, the method comprises the following steps: a1) Inoculate the embryonic stem cell solution or induced pluripotent stem cell solution into a 96-well cell culture plate, 100 μl per well, and then centrifuge and culture for 3 days to obtain embryoid bodies; a2) The embryoid bodies are washed with induction medium and placed in a new 96-well cell culture plate, and the volume is supplemented to 300 μl with induction medium. After 1.5 days of induction culture in the induction medium, 200 μl of the induction medium in each well is discarded, and 200 μl of mesenchymal stem cell solution is added to each well. After 1.5 days of culture, cell spheres are obtained, and the cell spheres are embedded in matrix gel and transferred to a 6-well cell culture plate containing expansion medium. After 3 days of expansion culture, the expansion medium in the 6-well cell culture plate is replaced with maturation medium, and the cells are cultured in the maturation medium for at least 30 days to obtain brain organoids.

[0022] In the step a1), the embryonic stem cell solution is composed of embryonic stem cells and embryoid body formation medium. The induced pluripotent stem cell solution is composed of induced pluripotent stem cells and embryoid body formation medium.

[0023] The embryonic stem cells and the induced pluripotent stem cells also include a step of digesting with a cell dissociation reagent GCDR before mixing; the digestion conditions can be 37° C., 5% CO2 digestion for 10 minutes.

[0024] In the step a1), the centrifugation condition may be 100×g for 3 min.

[0025] In the step a1), the culture conditions may be 37° C. and 5% CO 2 .

[0026] In the step a1), the cell culture plate should not be disturbed during the culture for at least 24 hours.

[0027] In the step a1), 100 μl of pluripotent stem cell culture medium is added every day on the first and second days of the culture.

[0028] In the step a2), the mesenchymal stem cell solution consists of mesenchymal stem cells and an induction medium.

[0029] The mesenchymal stem cells further include a step of digesting with ACCUTASE enzyme before adding the induction medium; the digestion time may be 5-10 min.

[0030] In the step a2), the conditions of the induction culture and the expansion culture can both be 37° C. and 5% CO 2 .

[0031] In the step a2), the conditions of the maturation culture may be 37° C., 5% CO 2 , 65 rpm, and the culture medium may be replaced every 3 days.

[0032] In the above method, the embryoid body formation medium is composed of ROCK inhibitor Y27632 and pluripotent stem cell medium. The final concentration of the ROCK inhibitor Y27632 in the embryoid body formation medium is 10 μM.

[0033] The induction medium is a medium obtained by mixing DMEM / F-12, N-2 additive, GlutaMAX™ additive, MEM non-essential amino acid solution, and heparin. The concentration of the N-2 additive in the induction medium is 1% (volume fraction), the concentration of the GlutaMAX™ additive in the induction medium is 1% (volume fraction), the concentration of the MEM non-essential amino acid solution in the induction medium is 1% (volume fraction), and the concentration of the heparin in the induction medium is 1 µg / ml.

[0034] The expansion medium is a medium obtained by mixing 100 ml of DMEM / F-12, 100 ml of Neurobasal™ medium, 1 ml of N-2 supplement, 2 ml of B-27™ supplement (50X, minus vitamin A), 2 ml of GlutaMAX™ supplement, 1 ml of MEM non-essential amino acid solution, 20 µl of 2-mercaptoethanol and 50 µl of insulin.

[0035] The maturation medium is prepared by mixing 100 ml of DMEM / F-12, 100 ml of Neurobasal™ medium, 1 ml of N-2 supplement, 2 ml of B-27™ supplement (50X, serum-free), 2 ml of GlutaMAX™ supplement, 1 ml of MEM non-essential amino acid solution, 20 µl of 2-mercaptoethanol, and 50 µl of insulin.

[0036] In the above method, the 96-well cell culture plate is a 96-well U-shaped low-adsorption cell culture plate. The 6-well cell culture plate is a 6-well U-shaped low-adsorption cell culture plate.

[0037] In the above method, the embryonic stem cells may be human embryonic stem cells.

[0038] The human embryonic stem cell may be human embryonic stem cell H1.

[0039] In a specific embodiment of the present invention, the human embryonic stem cell H1 is a product of Guangzhou Yuanjing Biotechnology Co., Ltd., with a product number of YC-C096.

[0040] In the above method, the induced pluripotent stem cells can be prepared by referring to conventional methods in the technical field, such as the method in the reference “Generation and characterization of two iPSC lines from human adiposetissue-derived stem cells of healthy donors. Stem Cell Res. 2020 Oct:48:101973”.

[0041] In a specific embodiment of the present invention, the method for preparing induced pluripotent stem cells comprises the following steps: ① using ImunoSep TM ① use density gradient cell separation solution to separate mononuclear cells from human whole blood samples; ② use PBMCeasy® blood cell activation medium to culture the mononuclear cells extracted in step ①; ③ use Reproeasy hiPSC reprogramming kit to reprogram the cells cultured in step ② to obtain induced pluripotent stem cells.

[0042] In the above method, the mesenchymal stem cells may be human mesenchymal stem cells.

[0043] The human mesenchymal stem cells include, but are not limited to, human bone marrow-derived mesenchymal stem cells, human umbilical cord-derived mesenchymal stem cells, and human adipose-derived mesenchymal stem cells.

[0044] In a specific embodiment of the present invention, the human umbilical cord-derived mesenchymal stem cells are products of Anhui Shouning Biotechnology Co., Ltd., with a product number of RC02003.

[0045] In the above method, the brain organoids include progenitor cells and neurons; the neurons include deep neurons and immature neurons.

[0046] The brain organoids constructed according to the above method also fall within the protection scope of the present invention.

[0047] In order to solve the above technical problems, the present invention also provides a set of reagents for culturing brain organoids.

[0048] The complete reagent set for culturing brain organoids provided by the present invention comprises the above-mentioned embryonic stem cells and the above-mentioned mesenchymal stem cells or the above-mentioned induced pluripotent stem cells and the above-mentioned mesenchymal stem cells.

[0049] Furthermore, the set of reagents may also include the above-mentioned pluripotent stem cell culture medium, the above-mentioned embryoid body formation culture medium, the above-mentioned induction culture medium, the above-mentioned expansion culture medium, and the above-mentioned maturation culture medium.

[0050] Furthermore, the reagent set may also include ROCK inhibitor Y27632, cell dissociation reagent GCDR, matrix gel, and ACCUTASE enzyme.

[0051] In order to solve the above technical problems, the present invention finally provides any of the following applications: 1) Application of mesenchymal stem cells in promoting the development of brain organoids; 2) Application of mesenchymal stem cells in the preparation of products that promote the development of brain organoids; 3) The application of mesenchymal stem cells in accelerating the growth rate of brain organoids; 4) The use of mesenchymal stem cells in the preparation of products that accelerate the growth rate of brain organoids; 5) Application of mesenchymal stem cells in promoting neural maturation in brain organoids; 6) Application of mesenchymal stem cells in the preparation of products that promote neural maturation in brain organoids; 7) Application of mesenchymal stem cells in accelerating extracellular matrix reconstruction of brain organoids; 8) Application of mesenchymal stem cells in the preparation of products that accelerate the remodeling of the extracellular matrix of brain organoids; 9) Application of mesenchymal stem cells in regulating the multipotency of stem cells; 10) Application of mesenchymal stem cells in the preparation of products for regulating the pluripotency of stem cells; 11) Application of the above methods in promoting the development of brain organoids; 12) Application of the above methods to accelerate the growth rate of brain organoids; 13) Application of the above methods to promote neural maturation in brain organoids; 14) Application of the above methods to accelerate extracellular matrix remodeling in brain organoids; 15) Application of the above methods in regulating the pluripotency of stem cells; 16) Application of the above-mentioned brain organoids as brain organoid models in studying the mechanisms of brain development or the pathogenesis of brain-related diseases or in drug screening for brain-related diseases; 17) Application of the above reagent kit in brain organoid culture; 18) Use of the above reagent set in the preparation of products for brain organoid culture.

[0052] The present invention provides a method for culturing brain organoids assisted by mesenchymal stem cells, which includes the step of adding mesenchymal stem cells in the initial stage or in the induction stage. The present invention first discovered that mesenchymal stem cells can fully fuse and survive with embryonic stem cells or induced pluripotent stem cells or embryoid bodies formed thereof, thereby promoting the development of brain organoids, accelerating the growth rate of brain organoids, accelerating the reconstruction of the extracellular matrix of brain organoids, regulating the pluripotency of stem cells, and promoting neural maturation in brain organoids. The brain organoids obtained by the present invention can be used as a model to study the development mechanism of the brain, the pathogenesis of brain-related diseases, and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 , the growth process of brain organoids with different numbers of MSCs added at the initial stage of construction.

[0054] Figure 2 , the expression of various markers of brain organoids with different numbers of MSCs added at the initial stage of construction.

[0055] Figure 3 , KEGG Pathway analysis results of differentially expressed genes in brain organoids without and with MSCs added (H1 / MSC-5000 group) at the initial stage of construction.

[0056] Figure 4 , GO analysis results of differentially expressed genes in brain organoids without and with MSCs added (H1 / MSC-5000 group) at the initial stage of construction.

[0057] Figure 5 , construct the growth process of brain organoids by adding different numbers of MSCs during the induction stage.

[0058] Figure 6 , the expression of various markers of brain organoids with different numbers of MSCs added during the induction stage.

[0059] Figure 7 , KEGG Pathway analysis results of differentially expressed genes in brain organoids without and with MSCs added (H1 / MSC-10000 group) at the initial stage of construction.

[0060] Figure 8 , GO analysis results of differentially expressed genes in brain organoids without and with MSCs added (H1 / MSC-10000 group) at the initial stage of construction. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0062] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0063] The embryonic stem cells (embryonic stem cells H1) in the following examples are products of Guangzhou Yuanjing Biotechnology Co., Ltd., with a product number of YC-C096.

[0064] The induced pluripotent stem cells in the following examples were prepared by the method described in the reference "Generation and characterization of two iPSC lines from human adipose tissue-derived stem cells of healthy donors. Stem Cell Res. 2020 Oct:48:101973". The specific preparation method includes the following steps: ① Using ImunoSep TM ① Use density gradient cell separation medium (Jinzhun Biotechnology (Tianjin) Co., Ltd., catalog number LSB1077) to separate mononuclear cells from human whole blood; ② Use PBMCeasy® Blood Cell Activation Medium (Beijing Saibei Biotechnology Co., Ltd., catalog number CA3408025) to culture the mononuclear cells extracted in step ①; ③ Use ReproeasyhiPSC Reprogramming Kit (Beijing Saibei Biotechnology Co., Ltd., catalog number CA5002002) to reprogram the cells cultured in step ② to obtain induced pluripotent stem cells.

[0065] The mesenchymal stem cells (derived from umbilical cord) in the following examples are products of Anhui Shouning Biotechnology Co., Ltd., with the product number RC02003.

[0066] The pluripotent stem cell culture medium in the following examples is a product of STEMCELL Technologies, with the trade name mTeSR™1 and the product number 85850.

[0067] The ROCK inhibitor Y27632 in the following examples is a product of Selleck, with the product number S1049.

[0068] The cell dissociation reagent GCDR in the following examples is a product of STEMCELL Technologies, with the trade name of Gentle Cell Dissociation Reagent (GCDR) and the product number of 07174.

[0069] The ACCUTASE enzyme in the following examples is a product of STEMCELL Technologies, with a product number of 07920.

[0070] The induction medium in the following examples is a medium obtained by mixing DMEM / F-12 (Thermo Fisher Scientific Inc., Catalog No. 11320033), N-2 additive (Thermo Fisher Scientific Inc., Catalog No. 17502001), GlutaMAX™ additive (Thermo Fisher Scientific Inc., Catalog No. 35050061), MEM non-essential amino acid solution (Thermo Fisher Scientific Inc., Catalog No. 11140050), and heparin (Sigma, Catalog No. H3149). The concentration of N-2 additive in the induction medium is 1% (volume fraction), the concentration of GlutaMAX™ additive in the induction medium is 1% (volume fraction), the concentration of MEM non-essential amino acid solution in the induction medium is 1% (volume fraction), and the concentration of heparin in the induction medium is 1 µg / ml.

[0071] The matrix gel in the following examples is a product of Corning, with a product number of 354277.

[0072] The expansion medium in the following examples is 100 ml DMEM / F-12 (Thermo Fisher Scientific Inc., Catalog No. 11320033), 100 ml Neurobasal™ Medium (Thermo Fisher Scientific Inc., Catalog No. 21103049), 1 ml N-2 Supplement (Thermo Fisher Scientific Inc., Catalog No. 17502001), 2 ml B-27™ Supplement (50X, without vitamin A) (Thermo Fisher Scientific Inc., Catalog No. 12587010), 2 ml GlutaMAX™ Supplement (Thermo Fisher Scientific Inc., Catalog No. 35050061), 1 ml MEM Non-Essential Amino Acid Solution (Thermo Fisher Scientific Inc., Catalog No. 11140050), 20 µl 2-Mercaptoethanol (Thermo Fisher Scientific Inc., Catalog No. 21985023), and 50 µl The culture medium was obtained by mixing with insulin (Sigma, catalog number I9278-5ML).

[0073] The maturation medium used in the following examples was prepared by mixing 100 ml DMEM / F-12 (Thermo Fisher Scientific Inc., Catalog No. 11320033), 100 ml Neurobasal™ Medium (Thermo Fisher Scientific Inc., Catalog No. 21103049), 1 ml N-2 Supplement (Thermo Fisher Scientific Inc., Catalog No. 17502001), 2 ml B-27™ Supplement (50X, Serum-free) (Thermo Fisher Scientific Inc., Catalog No. 17504044), 2 ml GlutaMAX™ Supplement (Thermo Fisher Scientific Inc., Catalog No. 35050061), 1 ml MEM Non-essential Amino Acids Solution (Thermo Fisher Scientific Inc., Catalog No. 11140050), 20 µl 2-mercaptoethanol (Thermo Fisher Scientific Inc., Catalog No. 21985023), and 50 µl The culture medium was obtained by mixing with insulin (Sigma, catalog number I9278-5ML).

[0074] Example 1: Method for culturing brain organoids with the aid of mesenchymal stem cells in the initial stage of construction 1. Add ROCK inhibitor Y27632 to the pluripotent stem cell culture medium to a final concentration of 10 μM to obtain embryoid body (EB) formation medium.

[0075] 2. Wash the embryonic stem cells twice with DPBS, add 1 ml of cell dissociation reagent GCDR, and place in a 37°C, 5% CO2 incubator for 10 minutes.

[0076] 3. Discard GCDR, add EB formation medium to resuspend cells into single cells, transfer to a 1.5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, count, and dilute with EB formation medium to obtain an embryonic stem cell solution with a concentration of 200,000 cells / ml.

[0077] 4. Wash the mesenchymal stem cells twice with DPBS, add 1 ml ACCUTASE enzyme to digest for 5-10 min, then add EB formation medium to resuspend the cells into single cells, transfer to a 1.5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and count. Dilute with EB formation medium to obtain mesenchymal stem cell solutions with concentrations of 50,000 / ml, 100,000 / ml, 200,000 / ml, and 400,000 / ml, respectively.

[0078] 5. Mix the embryonic stem cell solution with a concentration of 200,000 cells / ml with the mesenchymal stem cell solution with different concentrations in equal volumes, and then inoculate in a 96-well U-shaped low-adsorption cell culture plate, 100 μl per well, and then centrifuge at 100×g for 3 min, place in a 37°C, 5% CO2 incubator, and do not disturb the culture plate for at least 24 hours. The experiment is recorded as Day 0, and the 1st day, 2nd day, 3rd day, ..., 70th day thereafter are recorded as Day 1, Day 2, Day 3, ..., Day 70, respectively. At the same time, the group without mesenchymal stem cells was used as the control group (H1 / MSC-0).

[0079] 6. On day 1 and day 2, add 100 μl of pluripotent stem cell culture medium to each well.

[0080] 7. On the third day, the embryoid bodies formed in the 96-well U-shaped low-adsorption cell culture plate were aspirated into a 6-well low-adsorption cell culture plate using a 200 μl wide-mouth pipette tip, washed twice with induction medium, and placed back into a new 96-well U-shaped low-adsorption cell culture plate, and the volume was supplemented to 300 μl with induction medium.

[0081] 8. On day 4.5, discard 200 μl of induction medium in each well and add 200 μl of induction medium to each well.

[0082] 9. On day 6, the cell spheres induced in the 96-well U-shaped low-adhesion cell culture plate were embedded in 15 μl of matrix gel and transferred to a 6-well low-adhesion cell culture plate containing expansion medium and cultured in the expansion medium for 3 days.

[0083] 10. On day 9, replace the expansion medium in the 6-well low-adhesion cell culture plate with the maturation medium, and then culture in a shaking incubator at 37°C, 5% CO2, and 65 rpm. Change the medium every 3 days.

[0084] 11. Immunofluorescence detection and RNAseq detection were performed on day 70. The specific steps of immunofluorescence detection are as follows: 1) Fixation, embedding, and sectioning of organoids: Take brain organoids, wash them twice with PBS buffer, and then fix them with 4% paraformaldehyde for one day. Change to 75% ethanol overnight. The next day, change to 90%, 100%, 100%, xylene, xylene, and wax immersion for 1 hour each, embed, section, and bake.

[0085] 2) Dewax the paraffin sections to make them hydrated: sequentially place the sections in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and wash with distilled water.

[0086] 3) Antigen repair: Sodium citrate antigen repair solution (Proteintech Group Inc., catalog number PR30001) is boiled in advance on an induction cooker, and the slices are placed in it and boiled on medium heat for 10-15 minutes, and the boiling antigen repair solution is added in the middle. Then cool naturally.

[0087] 4) Wash tissue sections three times with PBS and block with 10% goat serum (Thermo Fisher Scientific Inc., catalog number 16210064) at room temperature for 1 hour. 5) No need to wash, add 50μL of primary antibody (1:50) at 4 degrees overnight or at room temperature for 1 hour. The slides should be taken out of the refrigerator in advance after overnight at 4 degrees and rewarmed at room temperature for 1 hour. The primary antibody is prepared with 10% goat serum (Thermo Fisher Scientific Inc., catalog number 16210064). The primary antibodies include TUJ1 (BioLegend Inc., catalog number MMS-435P), TBR1 (Proteintech Group Inc., catalog number 20932-1-AP), SOX2 (Cell Signaling Technology Inc., catalog number 4900S), MAP2 (Proteintech Group Inc., catalog number 17490-1-AP), and KI67 (Cell Signaling Technology Inc., catalog number 9449S).

[0088] 6) Wash tissue sections three times with PBS, add fluorescently labeled secondary antibodies (1:500), and incubate at room temperature in the dark for 1 hour. Secondary antibodies include Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™488 (Thermo Fisher Scientific Inc., Catalog No. A-11001) and Goat anti-Rabbit IgG (H+L)Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Thermo Fisher Scientific Inc., Catalog No. A-21245).

[0089] 7) Wash tissue sections three times with PBS, add DAPI (Thermo Fisher Scientific Inc., catalog number D21490), and incubate at room temperature for 5 minutes in the dark.

[0090] 8) The tissue sections were washed twice with PBS, mounted with anti-fluorescence quenching solution (Beyotime Biotechnology, catalog number P0126), protected from light, and photographed.

[0091] The RNAseq test was commissioned to Shenzhen BGI Genomics Service Co., Ltd.

[0092] Brain organoids were observed under a microscope and photographed on Day 0, Day 1, Day 2, Day 3, Day 12, Day 22, Day 30, Day 49, and Day 70 to record the growth and development process of brain organoids.

[0093] The growth and development of brain organoids Figure 1 As shown in the figure, H1 / MSC-2500 means that 200,000 H1 cells / ml and 50,000 MSC cells / ml were mixed and inoculated into a 96-well U-shaped low-adsorption cell culture plate on day 0, with 100 μl per well, containing 10,000 H1 cells and 2,500 MSC cells; H1 / MSC-5000 means that 200,000 H1 cells / ml and 50,000 MSC cells / ml were mixed and inoculated into a 96-well U-shaped low-adsorption cell culture plate on day 0, with 100 μl per well, containing 10,000 H1 cells and 2,500 MSC cells; H1 cells at 200,000 / ml and MSC cells at 100,000 / ml were mixed and inoculated into a 96-well U-shaped low-adsorption cell culture plate, with 100 μl per well, containing 10,000 H1 cells and 5,000 MSC cells; H1 / MSC-10000 means that H1 cells at 200,000 / ml and MSC cells at 200,000 / ml were mixed and inoculated into a 96-well U-shaped low-adsorption cell culture plate, with 100 μl per well, containing 10,000 H1 cells and 10,000 MSC cells; H1 / MSC-20000 means that H1 cells at 200,000 / ml and 400,000 / ml MSC cells were mixed and inoculated into a 96-well U-shaped low-adsorption cell culture plate, with 100 μl per well, containing 10,000 H1 cells and 20,000 MSC cells. As can be seen from the figure: compared with the control group, the addition of MSCs in the initial stage can increase the volume of brain organoids, and over time, the size of brain organoids is similar to that of the control group.

[0094] The results of marker expression level detection are as follows Figure 2 As shown, the results showed that with the increase of MSC cells added, the expression levels of deep neuronal cell marker TUJ1, immature neuronal cell marker TBR1 and neuronal cell marker MAP2 increased, while the expression level of proliferative cell marker KI-67 decreased, and the expression level of progenitor cell marker SOX2 remained basically unchanged.

[0095] The results of differential gene analysis were as follows Figure 3 and Figure 4 The results of KEGG Pathway analysis of differentially expressed genes showed that the up-regulated genes were mainly enriched in extracellular matrix-related pathways, such as the ECM-receptor interaction pathway; the results of GO analysis of differentially expressed genes also showed that the up-regulated genes were mainly enriched in extracellular matrix remodeling pathways, such as the extracellular matrix organization pathway, and the up-regulated genes were also enriched in signaling pathways against viral infection, such as the type I interferon signaling pathway and the defense response to virus pathway.

[0096] After replacing the embryonic stem cells in the above method with induced pluripotent stem cells, the growth and development process, marker expression levels and differential gene analysis results of brain organoids showed no significant differences from those of embryonic stem cells.

[0097] The above results show that adding MSC-assisted culture methods in the initial stage can accelerate the growth rate of brain organoids, accelerate the reconstruction of the extracellular matrix of brain organoids, and promote neural maturation in brain organoids.

[0098] Example 2: Method for culturing brain organoids with the aid of MSCs during the induction phase 1. Add ROCK inhibitor Y27632 to the pluripotent stem cell culture medium to a final concentration of 10 μM to obtain embryoid body (EB) formation medium.

[0099] 2. Wash the embryonic stem cells twice with DPBS, add 1 ml of cell dissociation reagent GCDR, and place in a 37°C, 5% CO2 incubator for 10 minutes.

[0100] 3. Discard GCDR, add EB formation medium to resuspend cells into single cells, transfer to a 1.5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, count, and dilute with EB formation medium to obtain an embryonic stem cell solution with a concentration of 100,000 cells / ml.

[0101] 4. Inoculate the embryonic stem cell solution with a concentration of 100,000 cells / ml into a 96-well U-shaped low-adsorption cell culture plate, 100 μl per well, then centrifuge at 100×g for 3 minutes and place in a 37°C, 5% CO2 incubator. Do not disturb the culture plate for at least 24 hours.

[0102] 5. On day 1 and day 2, add 100 μl of pluripotent stem cell culture medium to each well.

[0103] 6. On the third day, the embryoid bodies formed in the 96-well U-shaped low-adsorption cell culture plate were aspirated into a 6-well low-adsorption cell culture plate using a 200 μl wide-mouth pipette tip, washed twice with induction medium, and placed back into a new 96-well U-shaped low-adsorption cell culture plate, and the volume was supplemented to 300 μl with induction medium.

[0104] 7. On day 4.5, wash the mesenchymal stem cells twice with DPBS, add ACCUTASE enzyme to digest for 5-10 min, then add induction medium to resuspend the cells into single cells, transfer to a 1.5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and count. Dilute with induction medium to obtain mesenchymal stem cell solutions with concentrations of 50,000 cells / ml and 250,000 cells / ml, respectively.

[0105] 8. Discard 200 μl of induction medium in each well, and add 200 μl of mesenchymal stem cell solution of different concentrations to each well. The experiment is recorded as Day 0. The 1st day, 2nd day, 3rd day, ..., 65th day thereafter are recorded as Day 1, Day 2, Day 3, ..., Day 65, respectively. Meanwhile, the group without mesenchymal stem cells was used as the control group (H1 / MSC-0).

[0106] 9. On day 6, the cell spheres induced in the 96-well U-shaped low-adhesion cell culture plate were embedded in 15 μl of matrix gel and transferred to a 6-well low-adhesion cell culture plate containing expansion medium and cultured in the expansion medium for 3 days.

[0107] 10. On day 9, replace the expansion medium in the 6-well low-adhesion cell culture plate with the maturation medium, and then culture in a shaking incubator at 37°C, 5% CO2, and 65 rpm. Change the medium every 3 days.

[0108] 11. Immunofluorescence detection and RNAseq detection were performed on the 65th day.

[0109] Observe and take photos under a microscope on Day 0, Day 1, Day 2, Day 7, Day 17, Day 25, Day 34, and Day 65 to record the growth and development process of brain organoids.

[0110] The growth and development of brain organoids Figure 5 As shown in the figure, H1 / MSC-10000 means that 10,000 MSC cells were added to every 10,000 H1 cells to form embryoid bodies, and H1 / MSC-50000 means that 50,000 MSC cells were added to every 10,000 H1 cells to form embryoid bodies. It can be seen from the figure that the addition of MSCs to embryoid bodies in the early induction stage can increase the volume of brain organoids compared with the control group, and over time, the size of brain organoids is similar to that of the control group.

[0111] The results of marker expression level detection are as follows Figure 6 As shown, the results showed that with the increase of MSC cells added, the expression levels of deep neuronal cell marker TUJ1, immature neuronal cell marker TBR1 and neuronal cell marker MAP2 increased, while the expression level of proliferative cell marker KI-67 decreased, and the expression level of progenitor cell marker SOX2 remained basically unchanged.

[0112] The results of differential gene analysis were as follows Figure 7 and Figure 8The results of KEGG Pathway analysis of differentially expressed genes showed that the up-regulated genes were mainly enriched in stem cell development regulation pathways, such as Signaling pathways regulating pluripotency of stem cells pathway; the results of GO analysis of differentially expressed genes showed that the up-regulated genes were mainly enriched in neural development pathways, such as neuron differentiation pathway and central nervous system morphogenesis pathway.

[0113] After replacing the embryonic stem cells in the above method with induced pluripotent stem cells, the growth and development process, marker expression levels and differential gene analysis results of brain organoids showed no significant differences from those of embryonic stem cells.

[0114] The above results show that adding MSC-assisted culture methods during the induction stage can accelerate the growth rate of brain organoids, regulate the pluripotency of stem cells, and promote neural maturation in brain organoids.

[0115] Comparative Example 1 According to the method in Example 1, brain organoids were constructed by fixing the number of MSC cells and adjusting the number of H1 cells, wherein the number of MSC cells was 5000 / well, and the number of H1 cells was 2500 / well, 5000 / well, 10000 / well, and 20000 / well, respectively.

[0116] The results showed that when the number of H1 cells was 2500 / well and 5000 / well, the formation of embryoid bodies was limited, and multiple embryoid bodies of different sizes were formed. When the number of H1 cells was 20000 / well, the embryoid bodies were too large and the overall data were not ideal.

[0117] Comparative Example 2 According to the method in Example 1, brain organoids were constructed by fixing the number of H1 cells and adjusting the number of MSC cells, wherein the number of H1 cells was 5000 / well, and the number of MSC cells was 2500 / well, 5000 / well, 10000 / well, and 20000 / well, respectively.

[0118] The results showed that when there were too few MSC cells (2500 and 5000 MSC cells / well, respectively), there was no obvious difference between adding MSC and not adding MSC, and the formed embryoid bodies were small and uneven. However, when there were too many MSC cells (10000 and 20000 MSC cells / well, respectively), the formation of embryoid bodies was limited.

[0119] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for culturing brain organoids, the method comprising the following steps: a1) culturing embryonic stem cells or induced pluripotent stem cells in an embryoid body formation medium to obtain embryoid bodies; a2) performing neural induction culture on the embryoid bodies in an induction medium, and then performing expansion culture and maturation culture to form brain organoids; The step a1) or step a2) comprises the step of adding mesenchymal stem cells during the culture process.

2. The method according to claim 1, characterized in that: The step a1) comprises the following steps: culturing embryonic stem cells or induced pluripotent stem cells and mesenchymal stem cells in an embryoid body formation medium.

3. The method according to claim 2, characterized in that: The embryonic stem cells or induced pluripotent stem cells are added in an amount of 8000-12000 cells / well; The amount of mesenchymal stem cells added is 2500-20000 per well.

4. The method according to claim 1, characterized in that: The step a2) comprises the following steps: subjecting the embryoid bodies and mesenchymal stem cells to neural induction culture in an induction medium.

5. The method according to claim 4, characterized in that: The embryonic stem cells or induced pluripotent stem cells are added in an amount of 8000-12000 cells / well; The amount of mesenchymal stem cells added is 10,000-50,000 per well.

6. The method according to any one of claims 1 to 5, characterized in that: In the step a1), the culturing time is 3 days.

7. The method according to any one of claims 1 to 6, characterized in that: In the step a2), the induction culture time is 3 days; Or, the expansion culture time is 3 days; Alternatively, the maturation culture time is at least 30 days.

8. A brain organoid constructed according to any one of the methods described in claims 1-7.

9. A set of reagents for culturing brain organoids, the set of reagents comprising embryonic stem cells and mesenchymal stem cells or induced pluripotent stem cells and mesenchymal stem cells.

10. Any of the following applications: 1) Application of mesenchymal stem cells in promoting the development of brain organoids; 2) Application of mesenchymal stem cells in the preparation of products that promote the development of brain organoids; 3) The application of mesenchymal stem cells in accelerating the growth rate of brain organoids; 4) The use of mesenchymal stem cells in the preparation of products that accelerate the growth rate of brain organoids; 5) Application of mesenchymal stem cells in promoting neural maturation in brain organoids; 6) Application of mesenchymal stem cells in the preparation of products that promote neural maturation in brain organoids; 7) Application of mesenchymal stem cells in accelerating extracellular matrix reconstruction of brain organoids; 8) Application of mesenchymal stem cells in the preparation of products that accelerate the remodeling of the extracellular matrix of brain organoids; 9) Application of mesenchymal stem cells in regulating the multipotency of stem cells; 10) Application of mesenchymal stem cells in the preparation of products for regulating the pluripotency of stem cells; 11) Use of the method according to any one of claims 1 to 8 in promoting the development of brain organoids; 12) Use of the method according to any one of claims 1 to 8 for accelerating the growth rate of brain organoids; 13) Use of the method according to any one of claims 1 to 8 in promoting neural maturation in brain organoids; 14) Use of the method according to any one of claims 1 to 8 for accelerating the remodeling of the extracellular matrix of brain organoids; 15) Use of the method according to any one of claims 1 to 8 for regulating the pluripotency of stem cells; 16) Use of the brain organoid according to claim 8 as a brain organoid model in studying the mechanism of brain development or the pathogenesis of brain-related diseases or drug screening for brain-related diseases; 17) Use of the reagent set of claim 9 in culturing brain organoids; 18) Use of the reagent set according to claim 9 in the preparation of products for culturing brain organoids.

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