A method for culturing, light stimulation and signal processing of a three-dimensional on-chip brain

Abstract

The application discloses a culture, light stimulation and signal processing method of a three-dimensional on-chip brain. The three-dimensional on-chip brain is transfected by an adeno-associated virus carrying a light-sensitive channel protein which can be specifically expressed, so that the cells of the three-dimensional on-chip brain can express the light-sensitive channel protein on the cell membrane. Blue light with a wavelength of 470 nm is emitted by a light source and irradiated on the three-dimensional on-chip brain expressing the light-sensitive channel protein, so that the light-sensitive channel protein on the cell membrane is opened, ions in the culture medium enter the cells, the ion concentration in the environment is transiently changed, the change of the ions can be detected by a multi-electrode array, the range of the change is analyzed, and the physiological characteristics of the three-dimensional on-chip brain can be obtained, the functional network structure of the three-dimensional on-chip brain can be analyzed, and the biological intelligent function of the three-dimensional on-chip brain can be revealed.

Description

Technical Field

[0001] This invention relates to the fields of neuroscience and optogenetics, and more specifically to a method for culturing, photostimulating, and processing signals of a three-dimensional brain slice. Background Technology

[0002] Patterned optogenetic stimulation (OES) technology is primarily used in neuroscience research and the treatment of neurological diseases. This technology plays a crucial role in studying neuronal activity patterns, exploring the function of neural networks, and developing novel neuromodulation therapies. It has been widely applied in basic scientific research and the development of brain disease models in preclinical research phases.

[0003] Optogenetics, a technique that utilizes the light-sensitive response of photoproteins to regulate neuronal activity, has seen widespread application and development in recent years. However, traditional light stimulation methods struggle to generate high-resolution patterns, limiting their ability to regulate complex neural networks. Existing optogenetic stimulation systems are often out of sync with neural electrical signal recordings, affecting the accuracy and reliability of the data.

[0004] Brain-on-a-chip (B-Chip) is an important emerging branch of brain-computer interface technology. It couples brain tissue cultured in vitro with an electrode chip to form a brain-on-a-chip, and uses encoding and decoding technology and a stimulus feedback system to realize information interaction between the brain and the outside world.

[0005] As an interdisciplinary field in medicine and engineering, brain-computer interface (BCI) technology involves multiple disciplines such as neuroscience, computer science, automation, intelligent medical engineering, and biomedical engineering, covering various research directions. Current research focuses primarily on electrophysiological acquisition systems, multimodal information interaction, intelligent closed-loop feedback control, and the development of adaptive stimulation paradigms. Examples include in vitro intelligent unmanned control and brain-like computing based on BCI technology. Previously, researchers have used closed-loop systems and multichannel systems (MEA) to drive a small vehicle that completed obstacle avoidance tasks and implemented ping-pong game control in a virtual environment.

[0006] Brain-computer interface (BCI) technology offers a new avenue for exploring the integration and interaction between living and non-living entities. With its continuous advancement, this technology holds broad application prospects in areas such as brain disease diagnosis and treatment, neurorehabilitation, and the development of human-machine integrated intelligent agents. It demonstrates immense application potential, particularly in hybrid intelligence, brain-like computing, and medical rehabilitation, paving new paths for neuroscience research and intelligent healthcare. Summary of the Invention

[0007] To address the shortcomings of the above-mentioned technical solutions, the present invention aims to provide a method for cultivating 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-described culture method.

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

[0010] The objective of this invention is achieved through the following technical solution.

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

[0012] Step 1: Culture human induced pluripotent stem cells in mTeSR medium. During the culture process, remove differentiated cells. When the cell confluence is close to 70-80%, digest with acutase digestion solution, and then stop digestion with mTeSR medium to obtain a cell suspension.

[0013] Step 2: After pipetting the cell suspension, centrifuge, discard the supernatant, resuspend the cells, and pipet the resuspended cells to obtain a single-cell suspension.

[0014] Step 3: After counting the cells in the single-cell suspension, dilute the single-cell suspension with neural induction medium until the concentration of the single-cell suspension reaches 5-7*10. 4 live cells / mL;

[0015] Step 4, cell seeding: Add the diluted single-cell suspension from step 3 to the neural induction medium, mix well to obtain the seeding solution, add an equal amount of seeding solution to each well of the plate, and culture the cells until a central embryoid is formed in each well. Then, replace half of the medium in each well.

[0016] Step 5: Transfer the central embryonic body to the neural differentiation medium for further culture. During the culture process, replace half of the medium with CHIR neural differentiation medium and continue to replace half of the medium with Metrogel neural differentiation medium until the central embryonic body differentiates into a three-dimensional slice of brain.

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

[0018] In the above technical solution, in step 2, the cells are resuspended in a neural induction culture 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 culture 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 includes a three-dimensional brain-on-a-chip obtained by the above-described culture method.

[0023] Another aspect of the present invention includes the above-described 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 slice brain, the three-dimensional slice brain is transfected with optogenetic virus using adeno-associated virus;

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

[0026] Step 3: First, within 200ms, randomly perform full darkness processing on the three-dimensional brain-on-a-chip obtained in Step 2 to achieve a stable state, or input the pattern into the digital micromirror. The digital micromirror reflects the pattern after being irradiated with blue light. The reflected light enters the three-dimensional brain-on-a-chip from the lower surface of the electrode to stimulate it with light. Then, perform full darkness processing for 5s. This is one stimulation cycle. Repeat the stimulation cycle 20 times.

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

[0028] Step 5: Select the first 14 signal slices as the training dataset and the last 6 signal slices as the test dataset to train and test the support vector machine. Then, the support vector machine classifies the signal slices to verify the electrophysiological state of the brain on the three-dimensional slice under two different states of light stimulation and complete darkness.

[0029] Step 6: Extract the high-frequency portion of the signal from the signal slice, which is between 300-3000Hz. Calculate the variance of this portion of the signal in the time domain. If the peak value of the signal is greater than -5 times the variance, and no second peak value occurs within 2ms, it indicates that the three-dimensional on-slice brain neuron on the electrode has generated an action potential. 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 the spike signal. At the same time, a Cartesian coordinate system is established, with the horizontal axis representing time and the vertical axis representing the channel. When the spike value is 1 at a certain time, points are plotted at the corresponding positions, thus drawing the spike signals of multiple channels into a neural signal raster map.

[0030] Step 7: Extract the low-frequency portion of the electrical signal from the signal slice, which has a frequency of 1-300Hz. Extract the delta wave (1-4Hz), the theta wave (4-11Hz), the beta wave (11-30Hz), and the gamma wave (30-55Hz) respectively. Simultaneously, plot the waveform of the spontaneous brain signal on the three-dimensional slice.

[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 a light-sensitive channel.

[0032] In the above technical solution, in step 1, after optogenetic virus transfection for 24 hours, the differentiation maintenance medium is completely replaced with maintenance medium.

[0033] In the above technical solution, in step 1, the brain on the three-dimensional slice is differentiated and maintained using a neural maintenance culture medium. The neural maintenance culture medium is a maturation culture 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, light stimulation occurs 10 times and complete darkness processing occurs 10 times throughout the entire cyclic stimulation process.

[0037] The advantages and beneficial effects of this invention are as follows:

[0038] 1. This invention utilizes adeno-associated virus carrying a protein that specifically expresses light-sensitive channels to transfect a three-dimensional brain slice, enabling the cells of the three-dimensional brain slice to express light-sensitive channels on their cell membranes. This invention irradiates the three-dimensional brain slice expressing light-sensitive channels with 470nm blue light from a light source, causing the light-sensitive channels on the cell membrane to open, allowing ions in the culture medium to enter the cells. This causes a transient change in the ion concentration in the environment, which can be detected by a multi-electrode array. By analyzing the range of this change, the physiological characteristics of the three-dimensional brain slice can be obtained, its functional network structure can be analyzed, and its biological intelligent functions can be revealed.

[0039] 2. In terms of hardware, this invention employs optocouplers. A laser generator emits a laser beam, which is reflected by a digital micromirror device (DMD) and patterned onto a three-dimensional brain slice. Furthermore, a multi-electrode array (MEA) records the electrophysiological changes of the three-dimensional brain slice in real time, thereby achieving real-time optical stimulation and recording of its electrical signals. The use of digital micromirror devices (DMD) technology allows for high-resolution pattern design and flexible, precise application of patterned optical stimulation to different locations on the brain slice. Attached Figure Description

[0040] Figure 1 This is a flowchart of the culture process of a three-dimensional brain slice with light stimulation, as described in Example 1.

[0041] Figure 2 This is a schematic diagram of light stimulation in Example 2.

[0042] Figure 3 This is a schematic diagram of the photostimulation process in Example 2.

[0043] Figure 4 This is a neural signal raster image in a fully dark processing state for Example 2.

[0044] Figure 5 This is a three-dimensional on-chip electroencephalogram of signal waveforms in a fully dark processing state, as shown in Example 2. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0046] Example 1

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

[0048] Step 1: Culture human induced pluripotent stem cells in mTeSR medium. During the culture process, remove differentiated cells with a Pasteur tube. When the cell confluence is close to 70-80%, digest with acutase digestion solution in a 37°C incubator for 8-10 minutes, and then stop digestion with mTeSR medium to obtain a cell suspension.

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

[0050] Step 3: After counting cells, take 10 μL of single-cell suspension and dilute the single-cell suspension with neural induction medium (containing KSR but excluding FBS) until the single-cell suspension concentration reaches 6*10⁻⁶. 4 live cells / mL.

[0051] Step 4, Cell Planting: 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 the planting solution, add 150 μL of planting solution to each well of a 96-well plate, plant the cells, 9000 live cells per well, and start timing, marked as day 0 (D0). On day 1 (D1), one central embryoid body (EB) is formed in each well of the plate. On day 2 (D2), aspirate 75 μL of medium from each well, then add 75 μL of neural induction medium (containing Y-27632) to each well. On days 4, 6, and 8, aspirate 75 μL of medium from each well, then add 75 μL of neural induction medium (containing Y-27632) to each well.

[0052] Step 5: On day 10 (D10), the central embryoid bodies are transferred to 6-well plates and cultured on a shaker. During the culture process, neural differentiation medium is added. The central embryoid bodies differentiate to form a three-dimensional slice brain, specifically including the following steps:

[0053] Step 5.1: Fill each well of a 6-well plate with 3 ml of neural differentiation culture medium. Transfer the central embryoids from the 96-well plate to the 6-well plate using a pipette tip or a Pasteur tube. Place 8 central embryoids in each 6-well plate and place them on a shaker for differentiation culture. This is recorded as day 1.

[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%). Depending on the state of the neurons, if the state is good, Metrgel can be omitted. The central embryoid body differentiates to form a three-dimensional slice brain.

[0055] Step 6: After the seventeenth day, the brain on the three-dimensional slice is differentiated and maintained using a neural maintenance culture medium containing vitamin A and 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-chip includes the following steps:

[0058] Step 1: After maintaining the differentiation of the three-dimensional slice brain from Example 1 for 102-132 days, the three-dimensional slice brain was transfected with adeno-associated virus (AAV) using optogenetic virus transfection. The AAV was H15951 from OBiO (OBiO), packaged as AAV2 / 1 (AAV2 genome, AAV1 capsid protein), with the gene pAAV-hSyn-ChrimsonR-EGFP-WPRE: pAAV stands for plasmid AAV, i.e., the adeno-associated virus plasmid vector; hSyn stands for human synapsin, i.e., a human synapse-specific neuronal marker gene, which encodes synaptic vesicle proteins and is mainly expressed in neurons, acting as a promoter in the plasmid to drive gene expression; ChrimsonR represents the gene encoding a light channel protein in this example, which opens the channel under blue light (470nm) irradiation, allowing sodium or calcium ions from the culture medium to enter the cell; EGFP stands for Enhanced Green Fluorescent. Protein, namely enhanced green fluorescent protein, produces green fluorescence when excited by green light; WPRE is an enhancer derived from the woodlouse hepatitis virus, used to improve transcription efficiency and mRNA stability. In this embodiment, since it is located downstream of the light channel protein on the plasmid, the expression of the light channel protein can be estimated by observing the expression of green fluorescent protein, and thus the transfection status of adeno-associated virus can be estimated. The specific steps include:

[0059] Add 100-150 μL of differentiation maintenance medium to each well of a 96-well plate, then add a three-dimensional brain slice from Example 1 that has been maintained for 102-132 days of differentiation, and then add 4-6 x 10⁻⁶ μL of medium. 12One adeno-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 transfection, observe the green fluorescence of the brain on the 3D slice under a confocal fluorescence microscope, indicating that the adeno-associated virus gene transfection was successful. Coat the multi-electrode array with matrix gel solution dissolved in DF12 and incubate at 37°C and 5% CO2. After one hour, aspirate the entire coating solution and add 1-2 mL of differentiation maintenance medium. Place the brain on the 3D slice on the electrode surface and continue culturing for 3-5 days until the brain on the 3D slice is stable on the electrode surface.

[0061] Step 3, as follows Figures 2-3 As shown, within 200ms, the three-dimensional brain slice that is stable in step 2 is randomly subjected to complete darkness (without shining light on the three-dimensional brain slice), or the pattern is input into a digital micromirror. The digital micromirror reflects the pattern after being irradiated with 470nm blue light. Two prisms are used to change the light path of the reflected light. The light enters the three-dimensional brain slice from the lower surface of the electrode (because the three-dimensional brain slice tissue is relatively thick, the light path is difficult to penetrate the tissue to irradiate the three-dimensional brain slice neurons in contact with the electrode). The entire three-dimensional brain slice is light-stimulated, followed by a 5s complete darkness treatment. This is one stimulation cycle. The stimulation cycle is repeated 20 times. During the entire stimulation cycle, light stimulation occurs 10 times and complete darkness treatment occurs 10 times.

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

[0063] Step 5: Each signal column has 20 signal slices. Select the first 14 signal slices as the training dataset and the last 6 signal slices as the test dataset to train and test the support vector machine. Then, use the support vector machine to classify the signal slices to verify the electrophysiological state of the brain on the three-dimensional slice under two different states of light stimulation and complete darkness.

[0064] Step 6: Using a 4th-order Butterworth bandpass filter, extract the high-frequency portion of the signal slice between 300-3000Hz. Calculate the variance of this portion of the signal using time as the variable (i.e., average the signal over time, then square the difference between the average and all time-based signal values, and average the squared differences again to obtain the variance). When the peak value of the signal is greater than -5 times the variance, and no second peak value occurs within 2ms (the absolute refractory period of cortical neurons is approximately 2ms), it indicates that the three-dimensional on-slice brain neuron on that electrode has generated an action potential. The spike value at this moment is 1; otherwise, the spike value is 0. The data sequence obtained through this process belongs to the 0-1 binary signal and is called the spike signal. A program was written in Matlab to visualize spike signals. A Cartesian coordinate system was established, with the horizontal axis representing time and the vertical axis representing channels. When the spike value at a certain time is 1, a point was plotted at the corresponding location, thus creating a neural signal raster map of the spike signals from 60 channels (e.g., ...). Figure 4 (As shown)

[0065] Steps 5 and 6 involve peak extraction and signal classification of the electrical signals from the 3D brain-on-a-chip. If peaks occur within the network connections of the 3D brain-on-a-chip, it indicates that the network connections are mature. If the classification accuracy is higher than that of random sampling (in this example, the probability of random sampling is 50%), it indicates that the network connections of the 3D brain-on-a-chip have a specific response to light stimulation, exhibiting a certain degree of specificity for different light stimuli, and that its network recursive characteristics have potential for further exploration.

[0066] Step 7: Use a 400th-order Fir bandpass filter to extract the low-frequency portion of the electrical signal (1-300Hz) from the signal slice. Then, use a 20000th-order Fir bandpass filter to extract the delta wave (1-4Hz); a 10000th-order Fir bandpass filter to extract the theta wave (4-11Hz); a 10000th-order Fir bandpass filter to extract the beta wave (11-30Hz); and a 5000th-order Fir bandpass filter to extract the gamma wave (30-55Hz). Simultaneously, plot the waveform of the spontaneous brain-generated signal on the 3D slice (e.g.,...). Figure 5 As shown), from Figure 5 It can be seen that by observing the characteristics of the low-frequency signal of the brain in three-dimensional slices, it is proven that the brain in three-dimensional slices has a discharge function and 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. This MEAs has 59 TiN / SiN electrodes arranged in an 8*8 array, excluding the four vertices and the reference electrode. These 59 electrodes can be used to detect the extracellular potential of the neural network. This multi-electrode array can detect and record the extracellular potential of multiple sites in the in vitro biological neural network. This multi-electrode array is an in vitro electrophysiological signal acquisition and stimulation system with multiple channels.

[0068] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A method for optical stimulation and signal processing of a three-dimensional brain-on-chip, characterized in that, The three-dimensional slice brain was cultured using the following method: Step 1: Culture human induced pluripotent stem cells in mTeSR medium. During the culture process, remove differentiated cells. When the cell confluence reaches 70-80%, digest with digestion solution, then stop digestion to obtain a cell suspension. Step 2: After pipetting the cell suspension, centrifuge, discard the supernatant, resuspend the cells, and pipet the resuspended cells to obtain a single-cell suspension. Step 3: After counting the cells in the single-cell suspension, dilute the single-cell suspension with neural induction medium until the concentration of the single-cell suspension reaches 5-7 × 10⁻⁶. 4 live cells / mL; Step 4, cell seeding: Add the diluted single-cell suspension from step 3 to the neural induction medium, mix well to obtain the seeding solution, add an equal amount of seeding solution to each well of the plate, and culture the cells until a central embryoid is formed in each well. Then, replace half of the medium in each well. Step 5: Transfer the central embryonic body to the neural differentiation medium for further culture. During the culture process, replace half of the medium with CHIR neural differentiation medium and continue culturing with Matrigel neural differentiation medium until the central embryonic body differentiates into a three-dimensional slice of brain. The optical stimulation and signal processing method includes the following steps: Step a: After differentiating and maintaining the three-dimensional slice brain for 102-132 days, the three-dimensional slice brain is transfected with adeno-associated virus using optogenetic virus transfection. Step b: 4-6 days after transfection, the multi-electrode array is coated, incubated, the coating solution is aspirated, differentiation maintenance medium is added, and the transfected three-dimensional slice brain is placed on the electrode surface and cultured for 3-5 days until the three-dimensional slice brain is stable on the electrode surface. Step c: First, within 200ms, randomly perform full darkness processing on the three-dimensional brain-on-a-chip obtained in step b to achieve a stable state, or input the pattern into the digital micromirror. The digital micromirror reflects the pattern after being irradiated with blue light. The reflected light enters the three-dimensional brain-on-a-chip from the lower surface of the electrode to stimulate it with light. Then, perform full darkness processing for 5s. This is one stimulation cycle. Repeat the stimulation cycle 20 times. Step d: The multi-electrode array has multiple channels, each channel has multiple columns of signals, and each column of signals is centered on the 200ms mentioned in step c, taking the 1s signal before 200ms and the 4s signal after 200ms to form a signal slice. Step e: Select the first 14 signal slices as the training dataset and the last 6 signal slices as the test dataset to train and test the support vector machine. Then, the support vector machine classifies the signal slices to verify the electrophysiological state of the brain on the three-dimensional slice under two different states of light stimulation and complete darkness. Step f: Extract the high-frequency portion of the signal from the signal slice, which is between 300-3000Hz. Calculate the variance of this portion of the signal with time as the variable. When the peak value of the signal is greater than -5 times the variance, and no second peak value occurs within 2ms, the spike value at that moment is 1; otherwise, the spike value at that moment is 0. The binary signal obtained through this process is the spike signal. At the same time, establish a Cartesian coordinate system, with the horizontal axis representing time and the vertical axis representing the channel. When the spike value at a certain time is 1, plot the points at the corresponding positions to draw the spike signals of multiple channels into a neural signal raster map. Step g: Extract the low-frequency portion of the electrical signal from the signal slice, which has a frequency of 1-300Hz. Extract the delta wave of 1-4Hz, the theta wave of 4-11Hz, the beta wave of 11-30Hz, and the gamma wave of 30-55Hz respectively. At the same time, draw the waveform diagram of the spontaneous brain signal on the three-dimensional slice.

2. The optical stimulation and signal processing method according to claim 1, characterized in that, In step 1, digestion is performed for 8-10 minutes with accutase digestion solution in a 37°C incubator, followed by termination of digestion with mTeSR medium.

3. The optical stimulation and signal processing 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 optical stimulation and signal processing method according to claim 1, characterized in that, In step 2, centrifuge at 1100 rpm for 3 minutes at room temperature.

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

6. The optical stimulation and signal processing 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 culture medium containing Y-27632.

7. The optical stimulation and signal processing method according to claim 1, characterized in that, In step b, the multi-electrode array is coated with a matrix gel solution dissolved in DF12 and incubated at 37°C with 5% CO2.

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