Three-dimensional brain organ culture method based on electric field stimulation

Abstract

The invention discloses a three-dimensional brain organ culture method based on electric field stimulation, and belongs to the technical field of biomedicine. According to the method, firstly, embryonic stem cells or induced pluripotent stem cells are cultured and differentiated into three-dimensional brain organs, then an electric field device is used for conducting stimulation intervention in the development process of the brain organs, the structural change of the three-dimensional brain organs is detected, and it is proved that non-contact electric field stimulation promotes the maturity of the brain organs to be improved; in addition, the effect of non-contact electric field stimulation on the culture of the human MECP2 knockout Rater's syndrome brain organ disease model is researched, a reliable and effective in-vitro research model is provided for studying the nervous system development process, and a new way is provided for studying the treatment of diseases such as the Rater's syndrome related to the nervous development.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for culturing three-dimensional brain organoids based on electric field stimulation. Background Art

[0002] Brain organoids can reproduce the structural and functional characteristics of brain development in vitro and can serve as excellent in vitro models for simulating neural development. Rett Syndrome is a rare neurodevelopmental disorder that usually begins to severely deteriorate the health of patients at 6 months of age, presenting with severe intellectual disability, epilepsy, and ataxia. The main cause of Rett Syndrome is the mutation of the methyl CpG binding protein 2 gene (MECP2), which leads to impaired neuronal maturation and delayed synapse formation. Patients show a reduction in brain volume, which is closely related to a decrease in the size of neuronal cell bodies, a reduction in the complexity of neuronal processes, and a decrease in synaptic density. Currently, there are certain deficiencies in rodent models lacking MECP2: Female Mecp2 - / - homozygous mice only survive for a few weeks after birth, and the symptoms of female Mecp2 + / - heterozygous mice do not progress. The human-derived brain organoid model lacking MECP2 can reproduce the phenotypes of neurogenesis defects and reduced maturity in vitro (Molecular Psychiatry. 2018; 23: 1051 - 1065; Cell Reports. 2021; 109124), and can serve as a good model for simulating Rett Syndrome in vitro.

[0003] Currently, animal experiments have demonstrated that the pathological defects of Rett Syndrome can be rescued by implementing deep brain stimulation to restore long-term potentiation in the hippocampus and hippocampal neurogenesis (Nature. 2015; 526: 430 - 434). However, there has been no report on the research of optimizing the maturity of three-dimensional brain organoids based on non-contact electric field stimulation. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for culturing three-dimensional brain organoids based on electric field stimulation.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a method for culturing three-dimensional brain organoids based on electric field stimulation, comprising the following steps:

[0007] (1) Construct three-dimensional brain organoids: Culture embryonic stem cells or induced pluripotent stem cells and differentiate them into three-dimensional brain organoids;

[0008] (2) Electric field optimization of brain organoid development: Use an electric field device to perform stimulation intervention during the development of brain organoids, and detect the structural changes of three-dimensional brain organoids.

[0009] Based on the above technical solution, further, the embryonic stem cells are commercial human embryonic stem cells or are isolated from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0010] Based on the above technical solution, further, the embryonic stem cells in step (1) are H9 human embryonic stem cells.

[0011] Based on the above technical solution, further, the specific process of constructing three-dimensional brain organoids in step (1) is as follows: Digest embryonic stem cells or induced pluripotent stem cells into single cells, resuspend them with a neural induction medium, plate them in an ultra-low attachment U-bottom 96-well plate at a density of 5000 - 9000 cells / well to form embryoid bodies, culture for 8 - 12 days, transfer them to a low-attachment 6-well plate, discard the neural induction medium, add a neural differentiation medium, and place them in a shaker for rotary culture. Culture until the 16th - 20th day, discard the neural differentiation medium, and replace it with a maturation medium for continued rotary culture.

[0012] Based on the above technical solution, further, the rotation speed of the shaker in step (1) is set to 50 - 100 rpm.

[0013] Based on the above technical solution, further, the neural induction medium in step (1) is: DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, 2 μM XAV939.

[0014] Based on the above technical solution, further, the neural differentiation medium in step (1) is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 50 μM β-Mercaptoethanol.

[0015] Based on the above technical solution, further, the maturation medium described in step (1) is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, 20 ng / ml BDNF.

[0016] Based on the above technical solution, further, the parameters of the electric field in step (2) are a direct current electric field, the field strength is 1 - 3 V, it is processed for 0.5 - 1 h every day, and processed for 2 - 20 days.

[0017] Based on the above technical solution, further, the starting time point of the stimulation intervention in step (2) is the 30th - 40th day of the development of the brain organoids.

[0018] In the second aspect, the present invention provides the application of the above-mentioned method for culturing three-dimensional brain organoids based on electric field stimulation in constructing a Rett syndrome brain organoid disease model.

[0019] Based on the above technical solution, further, human MECP2 is knocked out in the Rett syndrome brain organoid disease model.

[0020] The beneficial effects of the present invention compared with the prior art are as follows:

[0021] The present invention for the first time proves that non-contact electric field stimulation promotes the improvement of the maturity of brain organoids, provides a method for culturing three-dimensional brain organoids based on electric field stimulation, provides a reliable and effective in vitro research model for studying the process of nervous system development, and provides a new approach for studying the treatment of Rett syndrome and other diseases related to neurodevelopment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.

[0023] Figure 1 It is the time axis of the electric field intervening in the development of brain organoids;

[0024] Figure 2 It is the device and operation panel diagram of the electric field intervention. Among them, (A): the electric field stimulation device diagram conforming to a 6-well plate; (B): the MCS electrical stimulation instrument; (C) the MCS software interface diagram.

[0025] Figure 3Immunofluorescence comparison diagrams of brain organoids in the electric field stimulation group and the control group. Among them, (A): Comparison of the number of neurons (NeuN) in the electric field stimulation group and the control group; (B): Comparison of the synaptic density (SYN) in the electric field stimulation group and the control group; (C): Comparison of the proportion of apoptotic cells (TUNEL) in the electric field stimulation group and the control group.

[0026] Figure 4 In vivo development results diagram of brain organoids transplanted into the host primary motor cortex in the electric field stimulation group and the control group; among them, (A): Comparison of the number of neurons (NeuN) in the electric field stimulation group and the control group; (B): Comparison of the integration effect of grafts and host-derived neuronal myelin (MBP) in the electric field stimulation group and the control group.

[0027] Figure 5 Verification results of the Rett syndrome brain organoid model with MECP2 knockout (A) and immunofluorescence diagrams of brain organoids in the control group, MECP2 knockout group, and MECP2 knockout + electric field intervention group (B). Detailed implementation manners

[0028] The present invention will be described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0029] Example 1

[0030] Cerebral organoid construction: When the confluence of H9 human embryonic stem cells reaches 80%, digest with accutase for 10 minutes to form a single-cell suspension. After resuspending with a neural induction medium containing 50 μM Y27632 (DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, 2 μM XAV939), inoculate the cells into an ultra-low attachment U-bottom 96-well plate at a ratio of 9000 cells / well and 150 μL / well to form embryoid bodies. Replace half of the medium with the neural induction medium without Y27632 every 2 days. On the 10th day of culture, transfer them to a low-attachment 6-well plate using a 5 mL Pasteur pipette, with 6-8 embryoid bodies per well, and 3 mL of neural differentiation medium (50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 50 μM β-Mercaptoethanol), and culture with rotation on a shaker at 80 rpm, replacing the medium completely every 2 days; on the 18th day of culture, change to the maturation medium (50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, 20 ng / ml BDNF), and replace the medium completely every 3-4 days.

[0031] Example 2

[0032] Electric field stimulation intervention in the development of brain organoids: Starting from the 33rd day of brain organoid development, transfer 8 brain organoids to a new 6-well plate containing 3 mL of mature medium using a wide-bore Pasteur pipette, ensuring that the brain organoids are located at the center of the well plate. Carefully insert the electrodes (IonOptix, Milton, MA, USA) designed to match the 6-well plate into the bottom of the 6-well plate, avoiding direct contact with the brain organoids inside the well plate, and ensuring that the brain organoids are located between the two electrode plates. The left 2 wells of this electrode well plate are non-stimulated control wells, the upper right 2 wells are electric field stimulation well 1, and the lower right 2 wells are electric field stimulation well 2( Figure 2 A). Connect the electrode interface at the blue part on the right side of the electrode well plate to the MCS electrical stimulator. The left row of interfaces is connected to stimulation channel 1 and stimulation channel 2 of the MCS respectively, so that stimulation channel 1 can stimulate electric field stimulation well 1 and stimulation channel 2 can stimulate electric field stimulation well 2. The right row of interfaces is connected to the ground wire of the MCS( Figure 2 B). Place the entire device in the cell culture incubator; turn on the switch of the MCS stimulator, and at this time the power light of the MCS stimulator will light up; open the MCS stimulation software on the computer, click the download button under STG, and at this time the Prog light of the MCS stimulator will light up. In the early stage, parameter exploration was carried out. The results showed that when the electric field stimulation amplitude was 5 V and 10 V, it would cause obvious destructive changes in the morphology of brain organoids; under the stimulation of 1 V - 3 V, the brain organoids could achieve short-term maturation, and among them, long-term maturation of brain organoids could be achieved at 2 V; when the electric field stimulation time was 2 hours and 3 hours, the ability of the electric field to promote the maturation and differentiation of brain organoids was not good; the electric field stimulation time was set to 0.5 - 1 hour as the better stimulation duration. Set the optimal parameters of electric field stimulation: the electric field amplitude is 2 V, and it is processed for 1 h every day( Figure 2 C). Click the Start button, and at this time the Sweep light of the MCS stimulator will light up to start the stimulation. After the stimulation treatment is over, disconnect the connection interface between the electrode and the stimulator, close the computer software and the MCS stimulator, transfer the brain organoids to a 6-well plate containing mature medium, and place them back on the shaker for culturing at 80 rpm. The used electrodes are washed overnight with autoclaved distilled water, and the electric field intervention lasts for 7 days. On the 90th day of brain organoid development, the detection of the proportions of neurons (NeuN), synaptic density (SYN), and apoptotic cells (TUNEL) is carried out.

[0033] The time axis of the electric field intervention in the development of brain organoids is as Figure 1 shown, and the detection results are as Figure 3 shown. After the electric field stimulation treatment, the brain organoids showed a significant increase in the number of neurons (NeuN), a significant increase in the size of neuron cell bodies, a significant increase in synaptic density (SYN), and a significant decrease in the proportion of apoptotic cells (TUNEL), effectively promoting the structural development of brain organoids.

[0034] Example 3

[0035] Detection of the in vivo development and integration ability of brain organoids after electrical field stimulation intervention: The GFP-carrying brain organoids treated with electrical field on the 40th day in Example 2 were transplanted into the primary motor cortex of Nod-SCID mice. Two months after transplantation, the development ability of the brain organoids in the host and their integration ability with the host were detected. At the same time, the GFP-carrying brain organoids in the non-stimulated control group in Example 2 were transplanted into the primary motor cortex of Nod-SCID mice in the same treatment manner as a control experiment.

[0036] The results are shown in Figure 4 , after transplantation, the proportion of neurons (NeuN) in the brain organoids treated with electrical field stimulation was significantly increased, and the grafts were well integrated with the myelin sheaths (MBP) of host-derived neurons, and the integration ability with the host was significantly improved.

[0037] Example 4

[0038] Construction of a Rett syndrome brain organoid model with MECP2 knockout and electrical field treatment: Lentivirus carrying MECP2 knockout mediated by Crispr-cas9 was used to infect H9 embryonic stem cells. After two weeks of drug screening, single cell clones were selected for gene fragment verification and knockout effect identification. The results are shown in Figure 5 A, the results showed that the H9 embryonic stem cell line with MECP2 knockout was successfully constructed.

[0039] Cultivation and identification were carried out according to the method for constructing human brain organoids in Example 1. Then, treatment and detection were carried out according to the parameters and methods for electrical field intervention on brain organoids in Example 2. The experimental group of brain organoids infected with Crispr-cas9 control virus (lentiviral vector) was used as a control, the experimental group of MECP2 knockout brain organoids was used as the MECP2 knockout group, and the experimental group of MECP2 knockout brain organoids treated with electrical field was used as the MECP2 knockout + electrical field intervention group.

[0040] The specific results are shown in Figure 5 B, it can be seen that the Rett syndrome brain organoids after electrical field treatment showed a significant increase in the proportion of neurons, indicating that electrical field treatment can improve the symptoms of Rett syndrome.

[0041] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing three-dimensional brain organoids based on electric field stimulation, characterized in that, It includes the following steps: (1) Construct three-dimensional brain organoids: Cultivate embryonic stem cells or induced pluripotent stem cells and differentiate them into three-dimensional brain organoids; (2) Optimize the development of brain organoids with an electric field: Use an electric field device to perform stimulation intervention during the development of brain organoids and detect the structural changes of three-dimensional brain organoids; The embryonic stem cells mentioned above are commercial human embryonic stem cells or are obtained by isolating human embryos within 14 days of fertilization that have not undergone in vivo development.

2. The cultivation method according to claim 1, wherein The embryonic stem cells mentioned in step (1) are H9 human embryonic stem cells.

3. The cultivation method according to claim 1, wherein The specific process of constructing three-dimensional brain organoids in step (1) is as follows: Digest embryonic stem cells or induced pluripotent stem cells into single cells, resuspend them with a neural induction medium, plate them at a density of 5000 - 9000 cells / well in an ultra-low attachment U-bottom 96-well plate to form embryoid bodies, culture for 8 - 12 days, transfer them to a low-attachment 6-well plate, discard the neural induction medium, add a neural differentiation medium, and place them in a shaker for rotary culture. Culture for 16 - 20 days, discard the neural differentiation medium, and replace it with a maturation medium for continued rotary culture.

4. The cultivation method according to claim 3, characterized in that, The neural induction medium mentioned in step (1) is: DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100 μM β-Mercaptoethanol, 100 nM LDN-193189, 10 μM SB431542, 2 μM XAV939.

5. The culturing method according to claim 3, wherein, The neural differentiation medium mentioned in step (1) is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 50 μM β-Mercaptoethanol.

6. The cultivation method according to claim 3, characterized in that The maturation medium mentioned in step (1) is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, 20 ng / ml BDNF.

7. The cultivation method according to claim 1, wherein The parameters of the electric field mentioned in step (2) are a direct current electric field, with an electric field strength of 1 - 3 V, treated for 0.5 - 1 h per day, and treated for 2 - 20 days.

8. The cultivation method according to claim 1, characterized in that, In step (2), the starting time point of the stimulation intervention is the 30th to 40th day of the development of the brain organoid.

9. Use of the method for culturing a three-dimensional brain organoid based on electrical field stimulation according to any one of claims 1-8 in constructing a Rett syndrome brain organoid disease model.

10. The application according to claim 9, wherein In the Rett syndrome brain organoid disease model described above, human MECP2 has been knocked out.

Images