Retina-like organ internal electric signal detection method based on special base and multi-point signal acquisition

Abstract

The invention provides a retinal organ internal electric signal detection method based on a special base and multi-point signal acquisition, and relates to the technical field of retinal organ internal electric signal detection. Comprising the following six steps: preparing a retinal organ fixing base, fixing the retinal organ, installing and positioning a flexible electrode, inserting the electrode, performing long-term culture and processing and analyzing a signal, the flexible electrode is adopted to directly collect an electric signal of an RGC layer in the retinal organ, and the defect that traditional MEA equipment can only collect a surface signal is overcome; therefore, accurate electric signal acquisition in the retina-like organ can be realized, the effectiveness of an experiment and the integrity of data are greatly improved, meanwhile, the position stability of the retina-like organ with the diameter smaller than 1mm in the detection process is ensured by using the design of the special fixed base and the central constraint column, and the accuracy and repeatability of data acquisition are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of internal electrical signal detection in retinal organoids, and in particular to a method for internal electrical signal detection in retinal organoids based on a dedicated base and multi-point signal acquisition. Background Art

[0002] Retinal organoids are three-dimensional structures derived from stem cells induced to differentiate in vitro. They mimic the fundamental morphological, layered, and functional characteristics of the human retina and are widely used in a variety of fields, including research on the mechanisms of visual diseases, drug screening, toxicological evaluation, and tissue engineering and regenerative medicine. Retinal organoids have shown great potential in replacing animal models and enabling personalized medical testing.

[0003] Due to its unique internal tissue structure, there are many technical challenges in the actual functional detection process. Among them, retinal ganglion cells are the core neurons that output information from the retina, and they are responsible for transmitting nerve impulses converted from light signals to the brain. These RGC cells are not distributed on the surface of the organoids, but are located in deeper structures. Therefore, the existing commonly used multi-electrode array system can only collect electrical signals from the surface of retinal organoids, and cannot penetrate the tissue to capture the real signal from the RGC layer. This makes the current electrophysiological research results limited, and it is difficult to fully reflect the activity state of the neural network inside the organ.

[0004] In addition, the diameter of retinal organoids is usually less than 1 mm, and the overall structure is extremely fragile and easy to float. During experimental operations, especially when inserting electrodes and collecting signals, if there is a lack of a stable fixation mechanism, it is very easy for the organs to shift in position, which will not only seriously affect the accuracy and repeatability of signal acquisition, but may even cause damage to the tissue structure.

[0005] Although needle electrodes have been used to collect local neural signals in the field of animal neuroscience (such as the mouse cerebral cortex), these electrode systems are mostly designed for larger tissue structures. When faced with small, fine-layered three-dimensional structures such as retinal organoids, it is difficult to accurately locate and stably insert them into the target layer. The operation is difficult and there is a risk of damage. Therefore, the present invention proposes a method for detecting internal electrical signals of retinal organoids based on a dedicated base and multi-point signal acquisition to solve the problems existing in the prior art. Summary of the Invention

[0006] In response to the above problems, the purpose of the present invention is to propose a method for detecting internal electrical signals of retinal organoids based on a dedicated base and multi-point signal acquisition. The method for detecting internal electrical signals of retinal organoids based on a dedicated base and multi-point signal acquisition ensures the stability of the organ position during electrode insertion and signal detection by setting a dedicated fixed base, thereby improving the accuracy and repeatability of the detection data. Combined with flexible electrodes, it can achieve precise acquisition of electrical signals of ganglion cells (RGCs) in retinal organoids, which can solve the problems existing in the prior art.

[0007] To achieve the purpose of the present invention, the present invention is implemented by the following technical solution: a method for detecting internal electrical signals of retinal organoids based on a dedicated base and multi-point signal acquisition, comprising the following steps:

[0008] Step 1: Prepare the retinal organoid mounting base

[0009] A retinal organoid fixation base is manufactured using 3D printing technology, and then the fixation base is pre-processed, wherein the fixation base includes a base body, and a plurality of restraining columns are evenly arranged on the base body;

[0010] Step 2: Fixing Retinal Organoids

[0011] Using a sterile operating table, place the fixed base into a sterile six-well plate, add organoid culture medium to the wells, and ensure that the fixed base is completely immersed. Then, place the mature cultured retinal organoids in the restraining column area to fix them;

[0012] Step 3: Flexible electrode installation and positioning

[0013] Using a stereotaxic apparatus, the flexible electrode was fixed to the manipulator arm of the stereotaxic apparatus. The precise insertion depth of the flexible electrode was then calculated based on the pre-measured size of the retinal organoid to ensure that the tip of the flexible electrode was positioned in the RGC layer.

[0014] Step 4: Electrode Insertion

[0015] Using a microscope and with the aid of a stereotaxic instrument, the flexible electrode is inserted into the retinal organoid. Once the electrode reaches the preset depth, it is fixed to a six-well plate and the stereotaxic instrument is removed.

[0016] Step 5: Long-term cultivation

[0017] The entire six-well plate was placed in a constant temperature incubator for long-term culture, and signals were collected regularly;

[0018] Step 6: Signal Processing and Analysis

[0019] The collected signals are converted into digital signals through a low-noise preamplifier, filtering module and analog-to-digital converter, and then subjected to time domain and frequency domain analysis and feature extraction to evaluate the functional status and response of retinal organoids.

[0020] A further improvement is that in step 1, the number of the restraining pillars is 4 to 6, and the spacing between each restraining pillar is 0.6 mm.

[0021] A further improvement is that in step 1, the height of the constraint column is 2 mm and the diameter is 1 mm, and the diameter of the base body is 35 mm and the height is 3 mm.

[0022] A further improvement is that in step one, the pretreatment method is to disinfect and sterilize the fixed base by high temperature sterilization or ultraviolet sterilization.

[0023] A further improvement is that in step 2, the diameter of the retinal organoid is less than 1 mm.

[0024] A further improvement is that in step 3, the specific method of pre-measuring the size of the retinal organoid and calculating the precise depth of flexible electrode insertion is:

[0025] Using a microscope, we obtained the dimensional data of the retinal organoids, including their diameter and thickness. Combined with the anatomical structure of the retinal organoids, we inferred the position of the RGC layer. Finally, combined with the size of the flexible electrode, we determined the depth at which the flexible electrode needed to be inserted.

[0026] A further improvement is that in step 4, the flexible electrode is fixed by glue or a fixing fixture.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention uses flexible electrodes to directly collect electrical signals from the RGC layer inside retinal organoids, thereby compensating for the defect that traditional MEA equipment can only collect surface signals, thereby enabling accurate electrical signal collection inside retinal organoids, greatly improving the effectiveness of the experiment and the integrity of the data.

[0029] (2) The present invention uses a dedicated fixed base and central restraint column design to ensure that retinal organoids with a diameter of less than 1 mm are positioned stably during the detection process, thereby improving the accuracy and repeatability of data collection.

[0030] (3) The present invention realizes the organic combination of electrical signals and organoid fixation, providing a complete and feasible technical solution for retinal organoid functional detection, drug screening and related disease research. This solution not only has good experimental operability and stability, but also can greatly improve the accuracy and repeatability of experimental results, providing strong technical support for retinal organoid related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic flow chart of the steps of the present invention.

[0032] Figure 2 It is a formal schematic diagram of the fixed base structure of the present invention.

[0033] Among them: 1. Base body; 2. Constraint column. DETAILED DESCRIPTION

[0034] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0035] Retinal organoids, as three-dimensional cell culture systems that mimic the structure and function of the human retina, are widely used in vision disease research, drug screening, and toxicology assessments. Because they possess a layered structure and neural networks similar to those of real retinal organoids, they are ideal models for studying retinal and neurodegenerative diseases. However, the acquisition and analysis of electrical signals from retinal organoids still face technical challenges, particularly in obtaining deep neural signals from the organ's internal layers.

[0036] Traditional multi-electrode array systems mainly collect electrical signals from the surface of the organ and cannot effectively penetrate the deep retinal ganglion cell (RGC) layer. As a result, current electrophysiological studies often find it difficult to fully reflect the activity state of the neural network inside retinal organoids. In addition, retinal organoids are small in size, usually less than 1 mm in diameter, and their overall structure is extremely fragile. During experimental operations, especially when inserting electrodes and collecting signals, the lack of a stable fixation mechanism may cause the position of the organ to shift, thereby affecting the accuracy and repeatability of the signal.

[0037] according to Figure 1-Figure 2 As shown, this embodiment proposes a method for detecting internal electrical signals of retinal organoids based on a dedicated base and multi-point signal acquisition, including the following steps:

[0038] Step 1: Prepare the retinal organoid mounting base

[0039] A retinal organoid fixing base is made using 3D printing technology, and then the fixing base is pretreated, wherein the fixing base includes a base body 1, on which constraint columns 2 are arranged, and the constraint columns 2 are evenly arranged in several groups. Specifically, the number of constraint columns 2 is 4 to 6, and the spacing between each constraint column 2 is 0.6 mm. The height of the constraint column 2 is 2 mm and the diameter is 1 mm. The base body 1 has a diameter of 35 mm and a height of 3 mm. Furthermore, the pretreatment method is: the fixing base is disinfected and sterilized by high-temperature sterilization or ultraviolet sterilization.

[0040] In this embodiment, a dedicated fixed base structure is designed for the retinal organoid base, which includes base dimensions, base height, and central restraint column parameters to ensure the stability of the organ during detection. Accordingly, the position and height of the restraint column 2 are precisely designed to ensure that the periphery of the retinal organoid can be firmly fixed without exerting excessive pressure on the organ.

[0041] Furthermore, the 3D printed fixed base 1 is sterilized. Correspondingly, when high-temperature sterilization is performed, the fixed base 1 is placed in a high-temperature steam sterilization device at 120° C. for 15 to 30 minutes, which can effectively sterilize and remove possible microbial contamination.

[0042] When ultraviolet sterilization is performed, the fixed base 1 is placed in an ultraviolet sterilization box, and the microorganisms on the surface and inside are killed by the UV-C light source.

[0043] Furthermore, in order to reduce friction when the retinal organoid contacts the fixed base and further improve stability, the fixed base can be surface treated, such as using a surface polishing process to remove possible rough areas or minor defects to ensure a smooth surface of the base.

[0044] Step 2: Fixing Retinal Organoids

[0045] Before fixing retinal organoids, it is first necessary to ensure that the operating environment is sterile. Therefore, a sterile operating table is used to provide a sterile environment to prevent external contamination. Then the fixed base is placed in a sterile six-well plate. Each well must be cleaned and disinfected to prevent any microbial contamination. Then, organoid culture medium is added to the well (the choice of culture medium should be based on experimental requirements. Usually, a specific culture medium is used to support the growth and survival of retinal organoids), and ensure that the fixed base is completely immersed. Then, the cultured mature retinal organoids (less than 1 mm in diameter) are placed in the constraint column 2 area, that is, sterile tweezers are used to carefully place the retinal organoids in the constraint column 2 area on the fixed base, ensuring that the retinal organoids are precisely placed between the constraint columns and do not shift. Due to the design of constraint column 2, the movement of retinal organoids can be effectively restricted, ensuring that the retinal organoids remain stable during subsequent electrical signal acquisition and experiments.

[0046] Step 3: Flexible electrode installation and positioning

[0047] Use a brain stereotaxic instrument to fix the flexible electrode on the manipulator arm of the brain stereotaxic instrument. Then, based on the pre-measured size of the retinal organoid, calculate the precise depth of the flexible electrode insertion to ensure that the tip of the flexible electrode is positioned in the RGC layer. The specific method for calculating the precise depth of the flexible electrode insertion based on the pre-measured size of the retinal organoid is as follows:

[0048] Use a microscope to obtain the dimensional data of retinal organoids, including the diameter and thickness of the retinal organoids, and the thickness distribution of different layers. Based on the measured thickness data of the retinal organoids, combined with the anatomical structure of the retinal organoids described in the literature, the position of the RGC layer is estimated (since retinal organoids cultured for different days may have different anatomical structures, existing literature provides descriptions of the anatomical structure of retinal organoids at multiple different time points. Based on these literature reports, the position of the RGC layer in the spherical retinal organoids can be estimated). Finally, combined with the size of the flexible electrode, the depth at which the flexible electrode needs to be inserted is determined.

[0049] Furthermore, in addition to depth, the angle of electrode insertion also needs to be determined. Because the structure of retinal organoids may not be completely flat, the electrode insertion angle needs to be adjusted based on the actual position of the retinal organoid to ensure precise contact between the electrode and the RGC layer. This angle is typically adjusted within a range of several degrees to ensure that the electrode is inserted from the intended direction.

[0050] Combined with the obtained dimensional data, the target position is input through the control system of the brain stereotaxic instrument to ensure that the insertion path of the electrode fully matches the calculated depth and angle. Before insertion, a final calibration is performed to ensure that the electrode will accurately reach the target position. The operating parameters of the brain stereotaxic instrument (including the insertion angle, etc.) are adjusted to ensure smooth operation while reducing mechanical damage to the organ.

[0051] Step 4: Electrode Insertion

[0052] Use a microscope (specifically a stereo microscope), and with its assistance, insert the flexible electrode into the retinal organoid through a brain stereotaxic instrument. During the process, ensure that the microscope has been calibrated and can observe the contact between the electrode and the retinal organoid in real time. During the entire insertion process, the microscope will serve as an auxiliary tool to help the operator confirm the exact depth and position of the electrode. After reaching the preset depth, fix the flexible electrode (fix the flexible electrode by glue or a fixing clamp) on the six-well plate. Specifically, non-toxic glue or professional fixing clamps suitable for biological experiments are usually used to firmly fix the electrode in place to prevent the electrode from moving or displacing during subsequent experiments. Then withdraw the brain stereotaxic instrument to ensure that the position of the electrode is not affected by external forces;

[0053] Step 5: Long-term cultivation

[0054] Place the entire six-well plate in a constant temperature incubator for long-term culture, and collect signals regularly. Specifically, the temperature in the incubator must be maintained at 37°C ± 2°C. The incubator must be sterile to prevent external contamination from affecting experimental results. Regularly change the organoid culture medium based on experimental needs and cell growth. Maintain a stable composition and pH value of the culture medium to prevent nutrient deficiency or waste accumulation from affecting the health of the retinal organoids.

[0055] Furthermore, electrical signals are regularly collected from retinal organoids, i.e., flexible electrodes are used for signal monitoring to capture the neural activity of retinal organoids, especially the electrical signals of the RGC layer.

[0056] Step 6: Signal Processing and Analysis

[0057] The collected signals are converted into digital signals through a low-noise preamplifier (since the electrical activity signals of retinal organoids are very weak, the role of the preamplifier is to enhance the strength of the signal while reducing the impact of electrical noise to ensure clear and accurate signal quality), a filtering module (removing high-frequency noise and low-frequency interference in the signal to ensure the purity of the electrical signal) and an analog-to-digital converter (the analog signal after the wave is converted into a digital signal through an analog-to-digital converter (ADC)), and then time domain and frequency domain analysis and feature extraction are performed to monitor the functional status of retinal organoids and their response to external drugs and environmental changes.

[0058] Specifically, in time domain analysis, the waveform characteristics of the signal are observed to identify the electrical activity of retinal organoids. Thus, by analyzing the time series of the signal, the action potential, synaptic activity and response of the retinal organoids to external stimuli can be monitored; and by performing Fourier transform on the collected signal, the time domain signal is converted into a frequency domain signal. Frequency domain analysis can help reveal the frequency components in the signal and identify the electrical activity characteristics of the retinal organoids. Finally, by extracting time domain features (such as peak amplitude, waveform duration, discharge frequency, etc.), the electrical activity intensity and periodic changes of the retinal organoids can be evaluated, and extracting spectral features (such as power spectral density, frequency peak, etc.) from the frequency domain signal can help analyze the neural activity pattern of the retinal organoids, especially in response to external stimuli or drugs.

[0059] In this embodiment, a flexible electrode is involved, which is made of a biocompatible polymer (such as polylactic acid-polycaprolactone copolymer, polyurethane) and a metal composite material (including gold, silver, copper, platinum, etc.). The tip diameter is controlled between 10 and 30 microns to ensure that it can accurately reach the internal RGC (Retinal Ganglion Cells) layer after penetrating the surface layer of the retinal organoid.

[0060] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the framework and scope of application of the present invention. Such changes and improvements are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition, characterized by: The following steps are involved: Step 1: Prepare the retinal organoid mounting base A retinal organoid fixing base is manufactured using 3D printing technology, and then the fixing base is pre-processed, wherein the fixing base includes a base body (1), and a constraint column (2) is arranged on the base body (1), and the constraint column (2) is evenly arranged in a plurality of groups; Step 2: Fixing Retinal Organoids Using a sterile operating table, place the fixed base into a sterile six-well plate, add organoid culture medium into the wells, and ensure that the fixed base is completely immersed. Then, place the cultured mature retinal organoids in the restraint column (2) area to fix them; Step 3: Flexible electrode installation and positioning Using a stereotaxic apparatus, the flexible electrode was fixed to the manipulator arm of the stereotaxic apparatus. The precise insertion depth of the flexible electrode was then calculated based on the pre-measured size of the retinal organoid to ensure that the tip of the flexible electrode was positioned in the RGC layer. Step 4: Electrode Insertion Using a microscope and with the aid of a stereotaxic instrument, the flexible electrode is inserted into the retinal organoid. Once the electrode reaches the preset depth, it is fixed to a six-well plate and the stereotaxic instrument is removed. Step 5: Long-term cultivation The entire six-well plate was placed in a constant temperature incubator for long-term culture, and signals were collected regularly; Step 6: Signal Processing and Analysis The collected signals are converted into digital signals through a low-noise preamplifier, filtering module and analog-to-digital converter, and then subjected to time domain and frequency domain analysis and feature extraction to evaluate the functional status and response of retinal organoids.

2. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In the step 1, the number of the restraining columns (2) is 4 to 6, and the spacing between each restraining column (2) is 0.6 mm.

3. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In the step 1, the height of the restraining column (2) is 2 mm and the diameter is 1 mm, and the diameter of the base body (1) is 35 mm and the height is 3 mm.

4. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In the step 1, the pretreatment method is: disinfecting and sterilizing the fixed base by high temperature sterilization or ultraviolet sterilization.

5. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In step 2, the diameter of the retinal organoid is less than 1 mm.

6. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In step 3, the precise insertion depth of the flexible electrode is calculated based on the pre-measured size of the retinal organoid as follows: Using a microscope, we obtained the dimensional data of the retinal organoids, including their diameter and thickness. Combined with the anatomical structure of the retinal organoids, we inferred the position of the RGC layer. Finally, combined with the size of the flexible electrode, we determined the depth at which the flexible electrode needed to be inserted.

7. The method for detecting internal electrical signals in retinal organoids based on a dedicated base and multi-point signal acquisition according to claim 1, characterized in that: In the fourth step, the flexible electrode is fixed by glue or a fixing fixture.

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