Three-dimensional on-chip brain culture, photostimulation and signal processing method

Abstract

The invention discloses a culture, photostimulation and signal processing method of a three-dimensional on-chip brain. The three-dimensional on-chip brain is transfected by using adeno-associated viruses which can specifically express light-sensitive channel proteins, so that cells of the three-dimensional on-chip brain can express the light-sensitive channel proteins on cell membranes of the cells, and 470nm blue light emitted by a light source irradiates the three-dimensional on-chip brain expressing the light-sensitive channel proteins; according to the method, light-sensitive channel protein on a cell membrane is opened, ions in a culture medium enter cells, the ion concentration in the environment is transiently changed, the change of the ions can be detected through a multi-electrode array, and the change range is analyzed; the physiological characteristics of the three-dimensional on-chip brain can be obtained, the function network structure of the three-dimensional on-chip brain is analyzed, and the biological intelligent function of the three-dimensional on-chip brain is disclosed.

Description

Technical Field

[0001] The present invention relates to the fields of neuroscience and optogenetics, and in particular to a three-dimensional brain-on-a-chip culture, light stimulation and signal processing method. Background Art

[0002] Patterned neural optogenetic stimulation technology is mainly used in the fields of neuroscience research and neurological disease treatment. This technology plays an important role in studying the activity patterns of neurons, exploring the functions of neural networks, and developing new neuromodulatory treatment methods. It has been widely used in the development of brain disease models in basic scientific research and preclinical research stages.

[0003] Optogenetics uses the response of photosensitive proteins to light to regulate neuronal activity and has been widely used and developed in recent years. However, traditional light stimulation methods have difficulty in generating high-resolution patterns, which limits the ability to regulate complex neural networks. Existing optogenetic stimulation systems are often not synchronized with neural electrical signal recordings, affecting the accuracy and reliability of data.

[0004] As an important emerging branch of brain-computer interface technology, brain-computer interface on a chip couples in vitro cultured brain tissue with electrode chips to form a brain-on-a-Chip, and uses encoding and decoding technology and stimulation feedback systems to achieve information interaction between the brain and the outside world.

[0005] As a cross-disciplinary field of medicine and engineering, brain-computer interface technology on a chip involves multiple disciplines such as neuroscience, computer science, automation, intelligent medical engineering, biomedical engineering, etc., covering multiple research directions. The current research directions mainly cover the development of electrophysiological acquisition systems, multimodal information interaction, intelligent closed-loop feedback control, and adaptive stimulation paradigms, such as in vitro intelligent unmanned control and brain-like computing based on brain-computer interface technology on a chip. Previously, researchers have used closed-loop systems and Multichannel systems MEA systems to drive a car based on a brain-on-chip to complete obstacle avoidance tasks and realize the control of ping-pong games in virtual environments.

[0006] Brain-computer interface technology on a chip provides a new way to explore the fusion and interaction between the new generation of living and non-living things. With the continuous advancement of this technology, it will have broad application prospects in the diagnosis and treatment of brain diseases, neurorehabilitation, and the development of human-machine fusion intelligent bodies. In particular, it has shown great application potential in the fields of hybrid intelligence, brain-like computing, and medical rehabilitation, opening up a new path for neuroscience research and intelligent medicine. Summary of the invention

[0007] In order to solve the deficiencies of the above technical solutions, the object of the present invention is to provide a method for culturing a three-dimensional brain-on-a-chip.

[0008] Another object of the present invention is to provide a three-dimensional brain-on-a-chip obtained by the above culture method.

[0009] Another object of the present invention is to provide a method for light stimulation and signal processing of the above-mentioned three-dimensional brain-on-chip.

[0010] The purpose of the present invention is achieved through the following technical solutions.

[0011] A method for culturing a three-dimensional brain-on-a-chip comprises the following steps:

[0012] Step 1, culturing human induced pluripotent stem cells in mTeSR medium, removing differentiated cells during the culturing process, and digesting the cells with accutase digestion solution when the cell confluence is close to 70-80%, and then terminating the digestion with mTeSR medium to obtain a cell suspension;

[0013] Step 2, after blowing the cell suspension, centrifuge, discard the supernatant, resuspend the cells, and blow the resuspended cells to obtain a single cell suspension;

[0014] Step 3: Take the single cell suspension for cell counting and dilute it with neural induction medium until the concentration of single cell suspension reaches 5-7*10 4 viable cells / mL;

[0015] Step 4, planting cells: adding the diluted single cell suspension in step 3 to the neural induction medium, mixing to obtain a planting solution, adding an equal amount of the planting solution to each well of the well plate, and culturing the cells until a central embryoid body is formed in each well, and then changing half of the medium in each well;

[0016] Step 5, the central embryoid body is transferred to a neural differentiation medium for further culture. During the culture process, half of the medium is replaced with a neural differentiation medium containing CHIR, and the medium is further replaced with a neural differentiation medium containing Metrgel until the central embryoid body differentiates into a three-dimensional brain-on-chip.

[0017] In the above technical solution, in step 1, digestion is performed with accutase digestion solution in a 37° C. incubator for 8-10 minutes.

[0018] In the above technical solution, in step 2, the cells are resuspended in a neural induction medium containing KSR but not FBS.

[0019] In the above technical solution, in step 2, centrifugation is performed at 1100 rpm for 3 minutes at room temperature.

[0020] In the above technical solution, in step 3, the neural induction medium contains KSR but does not contain FBS.

[0021] In the above technical solution, in step 4, the culture medium is aspirated from each well and replaced with the same amount of neural induction culture medium containing Y-27632.

[0022] Another aspect of the present invention also includes a three-dimensional brain-on-a-chip obtained by the above culture method.

[0023] Another aspect of the present invention also includes the above-mentioned three-dimensional brain-on-chip optical stimulation and signal processing method, comprising the following steps:

[0024] Step 1, after differentiating and maintaining the three-dimensional brain-on-chip, optogenetic virus transfection of the three-dimensional brain-on-chip is performed using adeno-associated virus;

[0025] Step 2: 4-6 days after the transfection, the multi-electrode array is coated and incubated, the coating solution is aspirated, and a differentiation maintenance medium is added, and the transfected three-dimensional brain-on-chip is placed on the upper surface of the electrode and cultured for 3-5 days until the three-dimensional brain-on-chip is stable on the upper surface of the electrode;

[0026] Step 3: First, within 200ms, the three-dimensional brain-on-chip in a stable state obtained in step 2 is randomly subjected to full dark treatment, or a pattern is input into a digital micromirror, and the digital micromirror reflects the pattern after being irradiated with blue light, and the reflected light is incident on the three-dimensional brain-on-chip from the lower surface of the electrode to perform light stimulation, followed by full dark treatment for 5s, which is one stimulation cycle, and the stimulation cycle is repeated 20 times;

[0027] Step 4, the multi-electrode array has multiple channels, each channel has multiple columns of signals, each column of signals is centered on the 200 ms described in step 3, and takes the 1 s signal before 200 ms and the 4 s signal after 200 ms to form a signal slice;

[0028] Step 5, selecting the first 14 signal slices as the training data set and the last 6 signal slices as the test data set to train and test the support vector machine, and then the support vector machine classifies the signal slices to verify the electrophysiological state of the three-dimensional brain-on-chip under two different states of light stimulation and complete darkness treatment;

[0029] Step 6, extract the high-frequency part of the signal frequency in the range of 300-3000 Hz in the signal slice, calculate the variance of this part of the signal in the time domain, when the peak value of the signal is greater than -5 times the variance, and the second peak value does not occur within 2ms, it means that the three-dimensional on-chip brain neuron on the electrode has generated an action potential, and the spike value at this moment is 1, otherwise the spike value at this moment is 0. The binary signal obtained through this process is a spike signal, and a rectangular coordinate system is established at the same time, the horizontal axis of the rectangular coordinate system represents time, and the vertical axis represents the channel. When the spike value at a certain time is 1, a point is drawn at the corresponding position, so that the spike signals of multiple channels are drawn into a neural signal raster diagram;

[0030] Step 7, extract the low-frequency part of the electrical signal with a frequency of 1-300 Hz in the signal slice, extract the delta wave of 1-4 Hz, theta wave of 4-11 Hz, the beta wave of 11-30 Hz, and the gamma wave of 30-55 Hz, and draw a waveform diagram of the spontaneous electrical signal of the brain on a three-dimensional chip.

[0031] In the above technical solution, in step 1, the gene of the adeno-associated virus is pAAV-hSyn-ChrimsonR-EGFP-WPRE, and the adeno-associated virus carries a protein that specifically expresses the light-sensitive channel.

[0032] In the above technical scheme, in step 1, after optogenetic virus transfection, 24 hours later, the entire amount of differentiation maintenance medium is replaced with maintenance medium.

[0033] In the above technical scheme, in step 1, the three-dimensional brain-on-chip is differentiated and maintained using a neural maintenance medium, wherein the neural maintenance medium adopts a maturation medium containing vitamin A, supplemented with brain-derived neurotrophic factor, cyclic adenosine monophosphate and ascorbic acid to promote long-term neural maturation.

[0034] In the above technical solution, in step 2, the multi-electrode array is coated with a matrix gel solution dissolved in DF12 and incubated at 37° C. and 5% CO2.

[0035] In the above technical solution, in step 3, a laser generator is used to generate blue light for blue light irradiation, and the wavelength of the blue light is 470nm.

[0036] In the above technical solution, in step 3, during the entire stimulation cycle, light stimulation occurs 10 times in total, and full dark treatment occurs 10 times in total.

[0037] The advantages and beneficial effects of the present invention are:

[0038] 1. The present invention uses adeno-associated viruses that can specifically express channelrhodopsin to transfect a three-dimensional brain-on-chip, so that cells of the three-dimensional brain-on-chip can express channelrhodopsin on their cell membranes. The present invention uses a light source to emit 470nm blue light to illuminate the three-dimensional brain-on-chip expressing channelrhodopsin, so that the channelrhodopsin on its cell membrane is turned on, ions in the culture medium enter the cells, and the ion concentration in the environment changes transiently. The change of such ions can be detected by a multi-electrode array, and the range of the change can be analyzed to obtain the physiological characteristics of the three-dimensional brain-on-chip, analyze its functional network structure, and reveal the biological intelligence function of the three-dimensional brain-on-chip.

[0039] 2. The present invention adopts a photoelectric coupling method in terms of hardware. The laser is emitted by a laser generator, reflected by a digital micromirror, and patterned onto the three-dimensional brain-on-chip. The electrophysiological changes of the three-dimensional brain-on-chip are recorded in real time through the MEA multi-electrode array, thereby achieving real-time light stimulation of the three-dimensional brain-on-chip and recording of its electrical signals. The use of digital micromirror devices (DMD) technology can achieve high-resolution pattern design, and flexibly and accurately irradiate patterned light stimulation to different positions of the brain-on-chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow chart of the light-stimulated three-dimensional brain-on-a-chip culture in Example 1.

[0041] Figure 2 Schematic diagram of light stimulation in Example 2.

[0042] Figure 3 Schematic diagram of the light stimulation process of Example 2.

[0043] Figure 4 This is a grating diagram of the neural signal in Example 2 under full dark processing.

[0044] Figure 5 This is a waveform diagram of the three-dimensional on-chip electroencephalographic signal in Example 2 in a completely dark processing state. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0046] Example 1

[0047] like Figure 1 As shown, a three-dimensional brain-on-a-chip culture method comprises the following steps:

[0048] Step 1, culture human induced pluripotent stem cells in mTeSR medium, use a Pasteur pipette to remove differentiated cells during the culture process, and when the cell confluence is close to 70-80%, digest with accutase digestion solution in a 37°C incubator for 8-10 minutes, and then terminate the digestion with mTeSR medium to obtain a cell suspension.

[0049] Step 2: Collect the cell suspension into a centrifuge tube, pipette and vortex 3-4 times with a 1 mL pipette tip, centrifuge at 1100 rpm for 3 minutes at room temperature, discard the supernatant, resuspend the cells with 1 mL of neural induction medium (containing KSR but not FBS), and pipette and vortex the resuspended cells to obtain a single cell suspension.

[0050] Step 3: Take 10 μL of the single cell suspension for cell counting and dilute it with neural induction medium (containing KSR but not FBS) until the concentration of the single cell suspension reaches 6*10 4 viable cells / mL.

[0051] Step 4, planting cells: add 6.48 mL of neural induction medium to a centrifuge tube, then add 720 μL of the single cell suspension diluted in step 3, mix well to obtain a planting solution, add 150 μL of the planting solution to each well of a 96-well plate, plant cells, 9000 living cells per well, and start timing, recorded as day 0 (D0). On the first day (D1), a central embryoid body (EB) is formed in each well of the well plate. On the second day (D2), 75 μL of medium is aspirated from each well, and 75 μL of neural induction medium (containing Y-27632) is added to each well. On D4, D6, and D8, 75 μL of medium is aspirated from each well, and 75 μL of neural induction medium (containing Y-27632) is added to each well.

[0052] Step 5, on the 10th day (D10), the central embryoid body is transferred to a 6-well plate and placed on a shaker for culture. During the culture process, a neural differentiation medium is added, and the central embryoid body is differentiated into a three-dimensional brain-on-chip, which specifically includes the following steps:

[0053] Step 5.1, add 3 ml of neural differentiation medium to each well of the 6-well plate, transfer the central embryoid bodies from the 96-well plate to the 6-well plate using a pipette or a Pasteur tube, place 8 central embryoid bodies in a 6-well plate, place on a shaker, and perform differentiation culture, which is recorded as the first day.

[0054] Step 5.2, on the third day, half of the culture medium in each well of the 6-well plate was replaced. The neural differentiation medium replaced from the ninth to the thirteenth day contained CHIR (concentration of 3 μM), and the neural differentiation medium replaced from the fourteenth to the seventeenth day contained Metrgel (concentration of 10%). According to the state of the neurons, if the state is good, Metregl is not needed, and the central embryoid body differentiates to form a three-dimensional brain-on-chip.

[0055] Step 6: After the seventeenth day, the three-dimensional brain-on-chip is differentiated and maintained using a neural maintenance medium, wherein the neural maintenance medium adopts a maturation medium containing vitamin A and is supplemented with brain-derived neurotrophic factor, cyclic adenosine monophosphate and ascorbic acid to promote long-term neural maturation.

[0056] Example 2

[0057] A method for optical stimulation and signal processing of a three-dimensional brain-on-a-chip, comprising the following steps:

[0058] Step 1, after the three-dimensional brain-on-chip of Example 1 is differentiated and maintained for 102-132 days, the three-dimensional brain-on-chip is transfected with an adeno-associated virus for optogenetic virus. The adeno-associated virus is H15951 of OBiO, packaged as AAV2 / 1 (AAV2 genome, AAV1 capsid protein), and its gene is pAAV-hSyn-ChrimsonR-EGFP-WPRE: pAAV represents plasmid AAV, i.e., adeno-associated virus plasmid vector; hSyn represents human synapsin, i.e., human synaptic-specific neuron marker gene, which is a gene encoding synaptic vesicle protein, mainly expressed in neurons, and acts as a promoter in the plasmid to drive gene expression; ChrimsonR represents the gene encoding the optical channel protein in this example, and under the irradiation of blue light (470nm), the channel of this gene opens, and sodium ions or calcium ions in the culture medium enter the cells; EGFP represents Enhanced Green Fluorescent Protein, namely enhanced green fluorescent protein, produces green fluorescence under the excitation of green light; WPRE is an enhancer derived from wood pig hepatitis virus, which is used to improve transcription efficiency and mRNA stability. In this embodiment, since it is located downstream of the photochannel protein on the plasmid, the expression of the photochannel protein can be estimated by observing the expression of green fluorescent protein, and then the transfection status of the adeno-associated virus can be estimated, which specifically includes the following steps:

[0059] 100-150 μL of differentiation maintenance medium was added to each well of a 96-well plate, and a three-dimensional brain-on-chip prepared in Example 1 for differentiation maintenance for 102-132 days was placed in the well. 4-6*10 12Adeno-associated virus was used for optogenetic virus transfection. After 24 hours, the differentiation maintenance medium was completely replaced with maintenance medium.

[0060] Step 2. 4-6 days after the transfection, observe green fluorescence in the three-dimensional brain-on-chip under a confocal fluorescence microscope, indicating that the adeno-associated virus gene transfection is successful. Coat the multi-electrode array with a matrix gel solution dissolved in DF12 and incubate at 37°C and 5% CO2. After one hour, aspirate all the coating solution, add 1mL-2mL of differentiation maintenance medium, and place the three-dimensional brain-on-chip on the upper surface of the electrode for continued culture for 3-5 days until the three-dimensional brain-on-chip is stable on the upper surface of the electrode.

[0061] Step 3, such as Figure 2-Figure 3 As shown, first, within 200ms, the three-dimensional brain-on-chip that is stable in step 2 is randomly subjected to full dark processing (no light is directed to the three-dimensional brain-on-chip), or a pattern is input into a digital micromirror, which reflects the pattern after being irradiated with 470nm blue light, and two prisms are used to change the optical path of the reflected light, so that the light is incident on the three-dimensional brain-on-chip from the lower surface of the electrode (due to the thick tissue of the three-dimensional brain-on-chip, it is difficult for the light path to penetrate the tissue and illuminate the part of the three-dimensional brain-on-chip neurons in contact with the electrode), and the entire three-dimensional brain-on-chip is light-stimulated, followed by full dark processing for 5s, which is one stimulation cycle, and the stimulation cycle is repeated 20 times. During the entire stimulation cycle, light stimulation occurs 10 times and full dark processing occurs 10 times.

[0062] Step 4, the multi-electrode array has 60 channels, each channel has 60 columns of signals, each column of signals is centered on the 200ms described in step 3, and takes the 1s signal before 200ms and the 4s signal after 200ms to form a signal slice.

[0063] Step 5, each column of signals has 20 signal slices. Select the first 14 signal slices as the training data set and the last 6 signal slices as the test data set. Train and test the support vector machine, and then use the support vector machine to classify the signal slices to verify the electrophysiological state of the three-dimensional brain-on-chip under two different states: light stimulation and complete darkness treatment.

[0064] Step 6, use a 4th-order Butterworth bandpass filter to extract the high-frequency part of the signal frequency in the range of 300-3000 Hz in the signal slice, and calculate the variance of this part of the signal with time as the variable (that is, find the average value of the signal according to time, then subtract the signal value of all time from the average value and square it, and then average all the squared values ​​after subtraction to get the variance). When the peak value of the signal is greater than -5 times the variance, and the second peak value does not occur within 2ms (the absolute refractory period of cortical neurons is about 2ms), it means that the three-dimensional on-chip brain neuron on the electrode has generated an action potential, and the spike value at this moment is 1, otherwise the spike value at this moment is 0. The data column obtained through this process belongs to a 0-1 binary signal, and the signal is called a spike signal. A program was written using Matlab to visualize the spike signals and establish a rectangular coordinate system. The horizontal axis of the rectangular coordinate system represents time and the vertical axis represents the channel. When the spike value at a certain time is 1, a point is drawn at the corresponding position, thereby drawing the spike signals of 60 channels into a neural signal raster diagram (such as Figure 4 (shown)

[0065] Steps 5 and 6 perform spike extraction and signal classification on the electrical signals of the 3D brain-on-chip. If a spike occurs in the network connection of the 3D brain-on-chip, it means that the network connection of the 3D brain-on-chip is mature. If the classification accuracy of the signal classification result is higher than that of random sampling (the probability of random sampling in this example is 50%), it means that the network connection of the 3D brain-on-chip has a specific response to light stimulation, and it has a certain specific response to different light stimulations. Its network recursive characteristics have potential to be explored.

[0066] Step 7, use a 400-order Fir bandpass filter to extract the low-frequency part of the electrical signal in the signal slice with a frequency of 1-300 Hz, and use a 20,000-order Fir bandpass filter to extract the 1-4 Hz delta wave; use a 10,000-order Fir bandpass filter to extract the 4-11 Hz theta wave; use a 10,000-order Fir bandpass filter to extract the 11-30 Hz beta wave; use a 5,000-order Fir bandpass filter to extract the 30-55 Hz gamma wave, and draw the waveform of the three-dimensional on-chip brain spontaneous electrical signal (such as Figure 5 shown), from Figure 5 It can be seen that by observing the characteristics of the low-frequency part of the signal of the three-dimensional brain-on-chip, it is proved that the three-dimensional brain-on-chip has the discharge function, it has electrophysiological activity, and has the potential to further explore its electrophysiological state and analyze its electrophysiological function.

[0067] In this embodiment, a multi-electrode array (MEAs) from Multi Channel Systems (MCS) is used. The MEAs has a total of 59 TiN / SiN electrodes arranged in an 8*8 array, excluding 4 vertices and reference electrodes. A total of 59 electrodes can be used to detect the extracellular potential of the neural network. The multi-electrode array detects and records the extracellular spike potentials of multiple sites of the in vitro biological neural network. The multi-electrode array is an in vitro electrophysiological signal acquisition and stimulation system with multiple channels.

[0068] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for culturing a three-dimensional brain-on-a-chip, characterized in that: The following steps are involved: Step 1, culturing human induced pluripotent stem cells in mTeSR medium, removing differentiated cells during the culturing process, and digesting with digestion solution when the cell confluence reaches 70-80%, and then terminating the digestion to obtain a cell suspension; Step 2, after blowing the cell suspension, centrifuge, discard the supernatant, resuspend the cells, and blow the resuspended cells to obtain a single cell suspension; Step 3: Take the single cell suspension for cell counting and dilute it with neural induction medium until the concentration of single cell suspension reaches 5-7*10 4 viable cells / mL; Step 4, planting cells: adding the diluted single cell suspension in step 3 to the neural induction medium, mixing to obtain a planting solution, adding an equal amount of the planting solution to each well of the well plate, and culturing the cells until a central embryoid body is formed in each well, and then changing half of the medium in each well; Step 5, the central embryoid body is transferred to a neural differentiation medium for further culture. During the culture process, half of the medium is replaced with a neural differentiation medium containing CHIR, and the medium is further replaced with a neural differentiation medium containing Metrgel until the central embryoid body differentiates into a three-dimensional brain-on-chip.

2. The culture method according to claim 1, characterized in that In the step 1, digestion is performed with accutase digestion solution in a 37° C. incubator for 8-10 minutes, and then digestion is terminated with mTeSR medium.

3. The culture method according to claim 1, characterized in that In step 2, the cells are resuspended in a neural induction medium containing KSR but not FBS.

4. The culture method according to claim 1, characterized in that In step 2, centrifuge at 1100 rpm for 3 minutes at room temperature.

5. The culture method according to claim 1, characterized in that In the step 3, the neural induction medium contains KSR but does not contain FBS.

6. The culture method according to claim 1, characterized in that In step 4, the culture medium is aspirated from each well and replaced with the same amount of neural induction medium containing Y-27632.

7. The three-dimensional brain-on-a-chip obtained by the culture method according to any one of claims 1 to 6.

8. A method for optical stimulation and signal processing of a three-dimensional brain-on-a-chip as claimed in claim 7, characterized in that: The following steps are involved: Step 1, after differentiating and maintaining the three-dimensional brain-on-chip, optogenetic virus transfection of the three-dimensional brain-on-chip is performed using adeno-associated virus; Step 2: 4-6 days after the transfection, the multi-electrode array is coated and incubated, the coating solution is aspirated, and a differentiation maintenance medium is added, and the transfected three-dimensional brain-on-chip is placed on the upper surface of the electrode and cultured for 3-5 days until the three-dimensional brain-on-chip is stable on the upper surface of the electrode; Step 3: First, within 200ms, the three-dimensional brain-on-chip in a stable state obtained in step 2 is randomly subjected to full dark treatment, or a pattern is input into a digital micromirror, and the digital micromirror reflects the pattern after being irradiated with blue light, and the reflected light is incident on the three-dimensional brain-on-chip from the lower surface of the electrode to perform light stimulation, followed by full dark treatment for 5s, which is one stimulation cycle, and the stimulation cycle is repeated 20 times; Step 4, the multi-electrode array has multiple channels, each channel has multiple columns of signals, each column of signals is centered on the 200 ms described in step 3, and takes the 1 s signal before 200 ms and the 4 s signal after 200 ms to form a signal slice; Step 5, selecting the first 14 signal slices as the training data set and the last 6 signal slices as the test data set to train and test the support vector machine, and then the support vector machine classifies the signal slices to verify the electrophysiological state of the three-dimensional brain-on-chip under two different states of light stimulation and complete darkness treatment; Step 6, extract the high-frequency part of the signal frequency in the range of 300-3000 Hz in the signal slice, calculate the variance of this part of the signal with time as the variable, when the spike value of the signal is greater than -5 times the variance, and the second spike value will not occur within 2ms, the spike value at this moment is 1, otherwise the spike value at this moment is 0. The binary signal obtained through this process is a spike signal, and a plane rectangular coordinate system is established at the same time, the horizontal axis of the plane rectangular coordinate system represents time, and the vertical axis represents the channel. When the spike value at a certain time is 1, a point is drawn at the corresponding position to draw the spike signals of multiple channels into a neural signal raster diagram; Step 7, extract the low-frequency part of the electrical signal with a frequency of 1-300 Hz in the signal slice, extract the delta wave of 1-4 Hz, theta wave of 4-11 Hz, the beta wave of 11-30 Hz, and the gamma wave of 30-55 Hz, and draw a waveform diagram of the spontaneous electrical signal of the brain on a three-dimensional chip.

9. The light stimulation and signal processing method according to claim 8, characterized in that: In step 1, the gene of the adeno-associated virus is pAAV-hSyn-ChrimsonR-EGFP-WPRE, and the adeno-associated virus carries a protein that specifically expresses the light-sensitive channel.

10. The light stimulation and signal processing method according to claim 8, characterized in that: In the step 2, the multi-electrode array is coated with a matrix gel solution dissolved in DF12 and incubated at 37° C. and 5% CO 2 .

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