A method and apparatus for culture of reconfigurable modular electrode arrays for cell electrical stimulation.

Abstract

This invention relates to the field of basic biomedical experimental equipment technology, and discloses a method and apparatus for cultivating reconfigurable modular electrode arrays for cell electrical stimulation. The invention aims to solve the problems of existing devices having a single programmable control mode, making it difficult to flexibly construct the electric field, and the electrical stimulation dose being easily affected by environmental and electrolyte changes during long-term culture, leading to significant variations in experimental results. This invention establishes an electrical connection between a culture chamber containing cell fluid and an external multi-channel programmable stimulation center. It uses test electrical signals to poll and obtain the real-time impedance value of the conductive rail array to confirm the liquid phase environment status. When the environment is normal, the array is polarity-grouped according to requirements, and target electrical pulse parameters are issued. Subsequently, the target electrical pulse is output according to the parameters. During the intervention period, the impedance change rate is periodically monitored, and the abnormal output is cut off or the normal output is resumed based on the change rate.

Description

Technical Field

[0001] This invention relates to the field of basic biomedical experimental equipment technology, and more specifically, to a method and apparatus for culturing reconfigurable modular electrode arrays for cell electrical stimulation. Background Technology

[0002] With the widespread application of electrostimulation therapy in neuroscience, cancer treatment, and stem cell engineering, in vitro research on its mechanisms of action at the cellular level is continuously deepening. In existing basic experiments, researchers typically use in vitro electrostimulation cell culture devices. During cell resting, adhesion, and routine culture, specific electrical signals are applied to electrodes immersed in the culture medium via external devices. This artificially constructs and simulates electrical environments under different physiological or pathological conditions, allowing researchers to observe and analyze the biological behaviors and response mechanisms of cells under these environments, such as proliferation, differentiation, migration, and gene expression.

[0003] However, existing in vitro cell electric field experimental devices have revealed significant limitations in long-term use. On the one hand, most existing devices use conventional electrodes of fixed size and physical shape, with relatively simple programmable modes, making it difficult to flexibly construct large-area uniform electric fields or local dipole micro-electric fields according to the experimental objectives of different cell types, thus failing to meet the diverse needs of electrostimulation experiments. On the other hand, during constant temperature and high humidity culture cycles lasting tens of hours, the actual electrostimulation dose applied to both ends of the cells is easily subject to subtle fluctuations and attenuation due to a combination of factors, including the traditional, crude physical connection of wires, interference from the incubator environment, and the dynamic changes in the physicochemical properties of electrolytes in the culture medium over time. These deficiencies make it difficult to maintain a rigorous in vitro cell electrostimulation environment under the same experimental conditions, ultimately resulting in significant differences in experimental results and severely restricting the accuracy and data reproducibility of research on the mechanisms of electrostimulation in cells. Summary of the Invention

[0004] Given that the existing devices have a single programmable control mode, making it difficult to flexibly construct an electric field, and that the electrical stimulation dose is easily affected by environmental and electrolyte changes during long-term cultivation, leading to significant differences in experimental results, this invention provides the following technical solution to address these issues: This invention provides a method for culturing reconfigurable modular electrode arrays for cell electrical stimulation, comprising the following steps: S1. Establish an electrical connection between a culture chamber containing cell fluid and an external multi-channel programmed stimulation center, wherein a multi-channel conductive rail array is pre-embedded inside the culture chamber; S2. The multi-channel programmed stimulation center obtains the real-time impedance value of the multi-channel conductive rail array based on the test electrical signal, and compares the real-time impedance value with the reference impedance range to confirm the liquid phase environment state. S3. Under normal conditions in the liquid environment, the multi-channel conductive rail array is polarized according to the electric field reconstruction requirements, including the allocation of anode and cathode, and the target electric pulse parameters are sent out. S4. The multi-channel programmed stimulation center outputs the target electrical pulse according to the target electrical pulse parameters, and periodically pauses the output during the intervention period to monitor the impedance change rate, and determines abnormal output cut-off and normal recovery of the target electrical pulse based on the impedance change rate. S5. After the intervention cycle ends, disconnect the culture chamber from the multi-channel programmed stimulation center and extract target biological samples with different spatial distribution states in the culture chamber.

[0005] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step S1 of establishing an electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center specifically includes: the culture chamber having a physically sealed structure, the multi-channel conductive rail array being pre-embedded in the inner bottom surface of the culture chamber, and the lead-out cables of the multi-channel conductive rail array penetrating the chamber wall and extending to the outside of the culture chamber to form a sealed connection socket, thereby connecting with the multi-channel programmed stimulation center through the sealed connection socket, and establishing a physically isolated and electrically conductive connection channel between the inside and outside of the culture environment while keeping the inside of the culture chamber completely sealed to prevent external contamination.

[0006] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step S1, before establishing the electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center, includes a resting adhesion process. This resting adhesion process specifically includes: before injecting cell fluid into the culture chamber, a bio-adhesive coating is pre-applied to the inner bottom surface of the culture chamber and the surface of the multi-channel conductive rail array; after injecting cell fluid, resting culture is continuously performed; and when the spread area of ​​the target cells reaches a preset ratio, the adhesion state of the target cells is confirmed as having reached the preset adhesion state. Then, the operation of establishing the electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center is performed to prevent large-scale detachment of target cells that are not firmly adhered due to fluid flow fluctuations caused by energization.

[0007] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step S2 of obtaining the real-time impedance value of the multi-channel conductive rail array based on polling the test electrical signal specifically includes: the test electrical signal is a weak high-frequency AC signal and the amplitude of the test electrical signal is limited to a safe threshold range that does not cause the target cells in the cell fluid to generate action potentials; any two conductive rails in the multi-channel conductive rail array are sequentially and non-repeatedly connected to the test circuit through the electronic switch matrix integrated inside the multi-channel programmed stimulation center to obtain the real-time impedance value between the connected conductive rails.

[0008] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation according to the present invention, the step S2 of comparing the real-time impedance value with the reference impedance range to confirm the state of the liquid phase environment specifically includes: if the obtained real-time impedance value is higher than the upper limit of the reference impedance range, the multi-channel programmed stimulation center determines that a physical disconnection abnormality has occurred in the liquid phase environment; if the obtained real-time impedance value is lower than the lower limit of the reference impedance range, the multi-channel programmed stimulation center determines that a metal short circuit abnormality has occurred in the liquid phase environment; if all real-time impedance values ​​are within the reference impedance range, the multi-channel programmed stimulation center determines that the liquid phase environment is in a normal state and allows the subsequent parameter setting steps to proceed.

[0009] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step S3 of performing polarity grouping of the multi-channel conductive rail array according to the electric field reconstruction requirements, including anode and cathode allocation, specifically includes: connecting multiple selected conductive rails in the multi-channel conductive rail array in parallel on the underlying electrical circuit to form an integral anode output array through the configuration interface of the control terminal, and connecting and connecting multiple other selected conductive rails in the multi-channel conductive rail array in parallel to form an integral cathode output array, so as to realize the on-demand reconstruction of the spatial electric field morphology.

[0010] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step of issuing target electrical pulse parameters in step S3 specifically includes: the target electrical pulse parameters are limited to a constant current output mode; during the output of the target electrical pulse, the multi-channel programmed stimulation center updates the output voltage in real time according to the impedance value acquired in real time and the set target current amplitude, based on the real-time impedance value and the algorithm logic that the target output voltage is equal to the set target current amplitude multiplied by the real-time impedance value, thereby compensating for the decrease in liquid conductivity caused by electrolyte consumption in the liquid environment by continuously and adaptively increasing the output voltage, so as to ensure that the current amplitude that actually flows into the liquid environment and acts on the target cells always remains consistent with the initial set value.

[0011] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, step S4, which involves determining abnormal output cutoff based on the impedance change rate and determining normal recovery of the target electrical pulse, specifically includes: the impedance change rate is calculated using the formula that the change rate equals the difference between the real-time impedance value measured during the current pause and the initial impedance value, divided by the initial impedance value. If the impedance change rate is detected to show a positive impedance increase exceeding the upper limit of the safety threshold, the multi-channel programmed stimulation center determines that the liquid level evaporation is abnormal. If the impedance change rate is detected to show a negative impedance decrease below the lower limit of the safety threshold, the multi-channel programmed stimulation center determines that the target cells are abnormally dying and detaching due to electrical stimulation overload. In the event of an abnormality, the multi-channel programmed stimulation center immediately cuts off the target electrical pulse output and triggers an alarm. In the event of no abnormality, the multi-channel programmed stimulation center determines that the status is normal and seamlessly recovers the target electrical pulse after the pause ends.

[0012] As a preferred embodiment of the reconfigurable modular electrode array culture method for cell electrostimulation described in this invention, the step S5 of classifying and extracting target biological samples with different spatial distribution states in the culture chamber specifically includes: firstly, aspirating the liquid supernatant inside the culture chamber to separate and extract the free products secreted by the target cells after stimulation; then, injecting cell lysis reagent into the culture chamber and collecting the cell lysis products attached to the multi-channel conductive rail array and the bottom surface by physical scraping, so as to comprehensively obtain data reflecting the internal physiological functions and external interactions of the target cells.

[0013] The present invention also provides a reconfigurable modular electrode array culture device for cell electrical stimulation, used to perform the above-described method, specifically including: A culture isolation unit includes a cell culture flask. A flat plate electrode is placed on the bottom surface of the cell culture flask. The flat plate electrode is connected to a silver-plated wire. The connection between the flat plate electrode and the silver-plated wire, as well as the point where the silver-plated wire passes through the cell culture flask, are covered with insulating and sealing fasteners. The silver-plated wire extends through to the outside of the cell culture flask and is fixed with a crystal plug at its end. A testing stimulation unit, comprising a testing stimulator, wherein the testing stimulator is provided with a socket interface that matches the crystal plug; An external programming interaction unit, comprising a programmable tablet, wherein the programmable tablet is communicatively connected to an external programmable controller.

[0014] The beneficial effects of this invention are as follows: By pre-embedding a multi-channel conductive rail array inside the culture chamber and extending it outward to form a sealed connection socket, a stable electrical channel is established while ensuring a sterile and contamination-free culture environment. Relying on the parallel assembly control of the underlying electrical circuitry, the multi-dimensional spatial electric field morphology can be flexibly reconstructed without replacing physical hardware, significantly improving the versatility of the equipment in experimental scenarios. During the intervention cycle, weak high-frequency test signals that do not induce cell action potentials are used for impedance polling, achieving non-destructive screening of the liquid environment. In the long-cycle output phase, an adaptive constant current regulation mechanism is used to dynamically compensate for the conductivity decrease error caused by electrolyte consumption in the culture medium, ensuring that the current dose acting on the cells remains consistent. Furthermore, this invention, combined with anomaly monitoring logic based on impedance change rate, can accurately identify environmental anomalies caused by excessive liquid evaporation or large-scale cell shedding, and effectively maintain the objective constancy of the long-cycle electrical stimulation intervention conditions for in vitro cells by timely output cut-off, ultimately greatly improving the accuracy and reproducibility of experimental data. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of a reconfigurable modular electrode array culture method for cell electrical stimulation.

[0017] Figure 2 Logic diagram for troubleshooting topological impedance in liquid phase environments.

[0018] Figure 3 This is a closed-loop diagram for adaptive constant current and dynamic safety monitoring.

[0019] Figure 4 This is a diagram of the modular electrode array culture system architecture for cell electrical stimulation.

[0020] Figure 5 Hardware physical connection architecture diagram of a reconfigurable modular electrode array culture device for cell electrical stimulation.

[0021] Figure 6 A schematic diagram of the assembly of the electrode array and insulating sealing components inside a cell culture flask. Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0024] Secondly, the term "one embodiment" or "example" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The appearance of an embodiment in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments.

[0025] Example 1 Reference Figures 1-3 This is one embodiment of the present invention, which provides a method for culturing a reconfigurable modular electrode array for cell electrical stimulation, comprising the following steps: S1. Establish an electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center. The culture chamber is pre-embedded with a multi-channel conductive rail array. The culture chamber has a physically sealed structure. A multi-channel conductive rail array is embedded in the bottom of the culture chamber. The lead-out cables of the multi-channel conductive rail array run through the chamber wall and extend to the outside of the culture chamber to form a sealed connection socket. The sealed connection socket connects to the multi-channel programmed stimulation center. While keeping the inside of the culture chamber completely sealed to prevent external contamination from entering, a physically isolated and electrically conductive connection channel is established between the inside and outside of the culture environment.

[0026] Before injecting cell fluid into the culture chamber, a bio-adhesive coating is pre-applied to the inner bottom surface of the culture chamber and the surface of the multi-channel conductive rail array. After injecting cell fluid, the culture chamber is continuously quiescent. When the spread area of ​​the target cells reaches the preset ratio, the adhesion state of the target cells is confirmed as the preset adhesion state. Then, the operation of establishing an electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center is performed to prevent large-scale detachment of target cells that are not firmly attached due to fluid flow fluctuations caused by power.

[0027] In this embodiment, the physical sealing structure of the culture chamber can be specifically represented as a standard 75 cm² transparent polystyrene cell culture flask with an integrated leak-proof threaded sealing cap. The multi-channel conductive rail array specifically consists of eight parallel-arranged platinum-iridium alloy wires with a diameter of 50 micrometers (or an ITO transparent conductive film attached to the bottom surface using a sputtering process). The sealing connection socket is specifically an IP68 waterproof 8-pin aviation socket embedded in the side wall of the culture flask and sealed with polymer resin, or a magnetic Pogo-pin contact base. This physical design completely eliminates the problems of traditional experiments where loose wires are forcibly inserted into the flask opening, leading to loose caps, carbon dioxide leakage, and bacterial contamination.

[0028] At the biological operational level, the bioadhesive coating specifically refers to a poly-D-lysine solution with a concentration of 0.1 mg / mL, used to assist in the adhesion of cells that are extremely difficult to adhere to, such as neurons. The so-called observation and determination that the target cells have reached the preset adhesion state specifically refers to the operator observing under a microscope, between 12 and 24 hours after cell inoculation, that the fused area of ​​the cell population occupies 70% to 80% of the bottom area of ​​the culture chamber, and the cell morphology changes from spherical to extended branching. Only after reaching this specific fusion state can external cables be connected to provide electricity; otherwise, directly applying electricity to cells that are not firmly attached will cause the tiny water flow propulsion from the electric field to destroy all the cells.

[0029] S2. The multi-channel programmed stimulation center obtains the real-time impedance value of the multi-channel conductive rail array based on the test electrical signal and compares the real-time impedance value with the reference impedance range to confirm the liquid phase environment state. The test electrical signal is a weak high-frequency AC signal, and the amplitude of the test electrical signal is limited to a safe threshold range that does not cause the target cells in the cytosol to generate action potentials. The electronic switch matrix integrated inside the multi-channel programmable stimulation center sequentially and non-repeatedly connects any two conductive rails in the multi-channel conductive rail array into the test circuit to obtain the real-time impedance value between the connected conductive rails.

[0030] If the acquired real-time impedance value is higher than the upper limit of the reference impedance range, the multi-channel programmed stimulation center determines that a physical disconnection abnormality has occurred in the liquid phase environment. If the acquired real-time impedance value is lower than the lower limit of the reference impedance range, the multi-channel programmed stimulation center determines that a metal bridging short circuit abnormality has occurred in the liquid phase environment. If all real-time impedance values ​​are within the reference impedance range, the multi-channel programmed stimulation center determines that the liquid phase environment is in a normal state and allows the subsequent parameter setting steps to proceed.

[0031] In this preparatory scanning step, the parameter settings for the test electrical signal are based on strict electrophysiological principles. The resting potential of a normal neuron is approximately -70 mV. When the depolarization amplitude reaches 15 mV (i.e., the membrane potential rises to around -55 mV), an action potential is triggered. To ensure that the scanning process does not cause additional electrophysiological interference to the cells, the test electrical signal emitted by the system is specifically limited to an amplitude of 10 millivolts (10 mV), which is strictly below the 15 mV depolarization threshold. At the same time, the frequency is set to 1 kilohertz (1 kHz), and the high-frequency characteristics can effectively penetrate the double-layer capacitance of the electrode surface to obtain extremely pure solution ohmic impedance.

[0032] The reference impedance range is the absolute benchmark for judging the quality of the environment. Considering the inherent conductivity of standard cell culture media (such as saline DMEM), the lower limit of the reference impedance range in this embodiment is specifically set at 0.01 kΩ, and the upper limit is specifically set at 6.40 kΩ. The lower limit of 0.01 kΩ is set because the impedance of a conventional liquid conductor cannot be lower than this value; if it falls below 0.01 kΩ, it indicates that internal metal debris has detached, causing a purely physical metal overlap between the two rails. The upper limit of 6.40 kΩ is set because even if... The impedance of the two pairs of track furthest apart within the culture flask will not exceed this value when filled with culture medium. If it exceeds 6.40 kΩ, the system determines it to be a physical open circuit, meaning the track was not effectively covered by liquid or the lead is broken. Only when all impedance values ​​measured by polling fall within this reasonable range will the system proceed to the next step.

[0033] S3. Under normal conditions in the liquid phase environment, the multi-channel conductive rail array is polarized according to the electric field reconstruction requirements, including the allocation of anode and cathode, and the target electric pulse parameters are issued. By using the configuration interface of the control terminal, multiple selected conductive rails in the multi-channel conductive rail array are connected in parallel on the underlying electrical circuit to form a whole anode output array, and multiple other selected conductive rails in the multi-channel conductive rail array are connected in parallel to form a whole cathode output array, so as to realize the on-demand reconstruction of the spatial electric field pattern.

[0034] The target electrical pulse parameters are limited to include a constant current output mode. During the output of the target electrical pulse, the multi-channel programmed stimulation center updates the output voltage in real time based on the real-time impedance value and the set target current amplitude. This is achieved by using an algorithm logic that the target output voltage equals the set target current amplitude multiplied by the real-time impedance value. This continuously and adaptively increases the output voltage to compensate for the decrease in liquid conductivity caused by electrolyte consumption in the liquid environment, ensuring that the actual current amplitude flowing into the liquid environment and acting on the target cells always remains consistent with the initial set value.

[0035] The control terminal's configuration interface is physically a tablet computer with a dedicated app, displaying eight channels (0 to 7) on the screen. If the experiment requires large-scale extraction of exosomes from mesenchymal stem cells, the operator can select channels 0, 1, 2, and 3 as anodes and channels 4, 5, 6, and 7 as cathodes, creating a large-area, uniform electric field. If the experiment simulates deep brain stimulation (DBS) intervention of local neurons, the operator only needs to set channel 1 as an anode and channel 2 as a cathode, leaving the others untouched. This demonstrates how the electric field polarity can be reconfigured through parallel circuitry at the software level.

[0036] For the target electrical pulse parameters, in the simulated deep brain stimulation experiment scenario, the specific values ​​are set as follows: amplitude 2.0 mA, pulse width 120 μs, and frequency 90 Hz. These specific values ​​are used because they completely replicate the FDA-standard pacing parameters for clinical treatment of neurological diseases such as Parkinson's disease, ensuring that the results of in vitro cell experiments have extremely high clinical translational value. The forced activation of the constant current output mode is to address the problem of degradation and deterioration of the saline solution when energized. Under tens of hours of electric shock, the conductive salt ions in the culture medium will be electrolyzed and consumed; if a fixed voltage is applied, the actual current delivered to the cells will be significantly reduced. In this embodiment, the system's underlying layer strictly executes the following adaptive constant current regulation algorithm formula: in, This represents the target voltage that the system is currently updating and outputting in real time. This represents the set target current amplitude (fixed at 2.0mA). This represents the current liquid phase environment impedance value captured by the machine through millisecond-level polling. Whenever the system detects an increase in impedance, it automatically and synchronously raises the voltage to precisely maintain a constant actual current intensity flowing into the cells.

[0037] S4. The multi-channel programmed stimulation center outputs the target electrical pulse according to the target electrical pulse parameters. During the intervention period, the output is periodically paused to monitor the impedance change rate. The abnormal output is cut off and the normal recovery of the target electrical pulse is determined based on the impedance change rate. The impedance change rate is calculated using the formula: the change rate equals the difference between the real-time impedance value measured during the current pause and the initial impedance value, divided by the initial impedance value. If the impedance change rate shows a positive increase in impedance exceeding the upper limit of the safety threshold, the multi-channel programmed stimulation center determines it as an abnormal decrease in liquid level due to evaporation. If the impedance change rate shows a negative decrease in impedance below the lower limit of the safety threshold, the multi-channel programmed stimulation center determines it as an abnormal large-scale death and shedding of target cells due to electrical stimulation overload. In the event of an abnormality, the multi-channel programmed stimulation center immediately cuts off the target electrical pulse output and triggers an alarm. In the event of no abnormality, the multi-channel programmed stimulation center determines the status as normal and seamlessly resumes the target electrical pulse after the pause ends.

[0038] This embodiment is designed for unattended experimental cycles typically lasting 48 to 72 hours. The periodic output pause is specifically configured as follows: every hour of continuous pulse output, the machine will force a 3-second power outage. During these 3 seconds, the machine will immediately invoke a 10 millivolt test signal to remeasure the current impedance, strictly applying the following impedance change rate calculation formula: in, This represents the current rate of change of impedance upon which the system determines an anomaly. This represents the fresh, real-time impedance value measured within a 3-second pause time window; This represents the initial reference impedance value measured at the very beginning of the experiment.

[0039] There are clear numerical limits and biological basis for the safety threshold for alarm judgment: the upper limit of the safety threshold is specifically set at +30% (i.e. The reason for setting this value is that moisture evaporation in the thermostat is a slow, cumulative process. When the liquid volume decreases, causing the impedance to rise by 30%, the concentration of residual nutrients and osmotic pressure in the culture medium will reach the lethal limit, and the system must immediately alarm to save the experiment; the lower limit of the safety threshold is specifically set at -20% (i.e., This value is set because a healthy population of living cells acts like an insulating layer covering the conductive rails. If electrical overstimulation causes widespread cell apoptosis and detachment from the conductive rails, the rails will be directly exposed to the low-impedance nutrient solution, resulting in a bare-polarity effect. Experimental data shows that when the cell detachment area exceeds 50%, the overall impedance drop of the system will exceed 20%. Therefore, if the calculated drop falls below -20%, it indicates a serious cell detachment incident. If the calculated rate of change fluctuates within the normal range, the machine smoothly returns to operating status after a 3-second self-check, ensuring the continuity of the experimental process.

[0040] S5. After the intervention cycle ends, disconnect the culture chamber from the multi-channel programmed stimulation center and extract target biological samples with different spatial distribution states in the culture chamber. First, the supernatant from the liquid phase inside the culture chamber is aspirated to separate and extract the free products secreted by the target cells after stimulation. Then, cell lysis reagent is injected into the culture chamber and the cell lysis products attached to the multi-channel conductive rail array and the bottom surface are collected by physical scraping to comprehensively obtain data reflecting the internal physiological functions and external interactions of the target cells.

[0041] In the specific implementation process, when the electric shock experiment cycle of several tens of hours is completely over, the operator disconnects the external cables of the chamber, transfers the sealed culture chamber to the experimental table, and unscrews the chamber cover.

[0042] The target biological samples to be obtained are divided into two spatial distribution states: The first part consists of substances suspended in the nutrient solution. The operator uses a pipette to remove all the supernatant from the liquid phase in the chamber and transfers it to an ultracentrifuge for centrifugation. The free products (such as exosomes) secreted and released into the water after the cells are stimulated are purified and collected for the purpose of studying the external information interaction mechanism between cells. The second part consists of the cell body, still stably attached to the bottom surface and metal tracks. Operators directly inject chemical reagents, such as RIPA cell lysis buffer, into the culture chamber to disrupt the cell's lipid bilayer. Then, using a sterile plastic scraper, they scrape back and forth on the bottom surface, removing the intracellular material and collecting it in centrifuge tubes. These cell lysis products will be used to analyze the residual adenosine triphosphate (ATP) content and reactive oxygen species (ROS) levels within the cells. This will allow for precise quantitative assessment of whether this specially designed electric field stimulation promotes mitochondrial repair or exacerbates oxidative stress damage.

[0043] Example 2 Reference Figure 4These are two embodiments of the present invention. This embodiment provides a reconfigurable modular electrode array culture system for cell electrical stimulation. This system is typically deployed in a biosafety laboratory containing a 37°C carbon dioxide incubator to automate the method steps in Embodiment 1.

[0044] The system's hardware and control architecture consists of a set of highly coordinated functional modules, specifically including: Physical isolation and sealing modules are used to construct sterile and leak-proof culture chambers to isolate liquid cell culture media from the external environment; The array embedding and docking module is used to pre-place multiple parallel conductive tracks in the culture chamber and to seamlessly extend the wiring terminals of the conductive tracks to the outside of the chamber to form a unified electrical interface. The topological impedance self-test module is used to release a weak high-frequency test signal before formal power-on intervention to measure the resistance value between conductive tracks in order to check for open circuit or short circuit anomalies. The electric field polarity reconfiguration module is used to perform parallel or suspended operation on the bottom layer of the conductive track according to external control commands, so as to freely combine the array arrangement of anodes and cathodes. The adaptive constant current drive module is used to continuously output electrical pulse energy according to the set parameters, and dynamically adjust the output voltage according to the real-time impedance changes to maintain a constant intervention current. The abnormal interception and recovery module is used to periodically detect the rate of environmental change during long-term power-on cycles and intercept serious accidents caused by liquid level drying or cell shedding. The in-situ sample retention module is used to preserve biological samples in both suspended and adherent states within the original chamber for extraction after the entire intervention cycle has ended.

[0045] The operation begins with the initial construction of a sterile environment by the physical isolation and sealing module. This module uses a standard transparent culture flask with a threaded cap, which is tightened after cell inoculation to achieve physical isolation.

[0046] The array embedding and docking module uses an integrated injection molding process to pre-embed multiple platinum-iridium alloy conductive rails inside the bottle bottom, in contact with the cell fluid. Simultaneously, the cables leading out are sealed with resin at the bottle wall, extending outwards to form a high-protection, sealed connection socket. This establishes the physical basis for communication with external devices without compromising internal airtightness. Based on this, a topological impedance self-test module establishes a safety verification channel. Through an externally connected multi-channel programmable stimulation center with a built-in electronic switch matrix, high-frequency AC probe signals of 10 mV and 1 kHz are sequentially released to the conductive rails. The system verifies based on the ohm values ​​returned by polling. Only when the impedance values ​​of all paired rails fall within the reference range of 0.01 kΩ to 6.40 kΩ is the liquid phase environment deemed completely normal, and subsequent operation permissions are granted.

[0047] After confirming environmental safety, the electric field polarity reconfiguration module responds to configuration commands from the terminal UI interface. Without replacing any physical culture flasks, it short-circuits and assembles designated conductive rails via an underlying relay array. For example, it maps the left half of the rails in parallel as the anode and the right half in parallel as the cathode, achieving flexible spatial reconfiguration from a macroscopic, large-area uniform electric field to a localized, micro-dipole electric field.

[0048] Subsequently, the adaptive constant current drive module takes over the energy output, continuously reading the real-time impedance value of the current array circuit and strictly following Ohm's law algorithm. The output voltage of the front-end power supply module is refreshed in real time. This mechanism can perfectly compensate for the electrolysis of culture medium salt ions caused by prolonged power supply, ensuring that the actual current flowing through the bottom wall cells is precisely maintained at the initial set value of, for example, 2.0 mA during interventions lasting tens of hours.

[0049] To ensure the absolute safety of long-term experiments, the abnormal cutoff and recovery module forms an independent security closed loop during the intervention process. This module forces the system to pause electrical pulse output for three seconds every hour of operation, and calls the impedance change rate calculation formula during this three-second monitoring window: Calculate the current state deviation. If the calculated rate of change is greater than 30%, it is determined that the incubator has lost water, causing severe evaporation of the culture medium; if the rate of change is less than 20%, it is determined that the stimulus intensity overload has caused a large number of cells to die and detach, resulting in a short circuit in the bare electrodes. In the face of these two extreme conditions, the module will immediately cut off the main circuit current and trigger an audible and visual alarm; if the rate of change fluctuates steadily within this safe range, the module will smoothly resume the original electrical pulse output after a three-second self-test.

[0050] Finally, after the preset cycle was completed, the system stopped working, and the operator disconnected the wiring at the sealed connector. Thanks to the in-situ sample retention module, the stimulated cell secretions were well preserved in the nutrient solution and could be directly aspirated and centrifuged, while the cells themselves remained firmly attached to the bottom track, awaiting scraping of the lysis buffer, thus ensuring the complete collection of experimental data.

[0051] Example 3 Reference Figure 5 This is the third embodiment of the present invention, which provides a reconfigurable modular electrode array culture device for cell electrical stimulation. The device is used to perform the method described in Embodiment 1 and serves as the hardware physical carrier of the system described in Embodiment 2.

[0052] Specifically, it includes: A culture isolation unit 100 includes a cell culture flask 101. A flat plate electrode 102 is placed on the bottom surface of the cell culture flask 101. The flat plate electrode 102 is connected to a silver-plated wire 103. The connection between the flat plate electrode 102 and the silver-plated wire 103, as well as the point where the silver-plated wire 103 passes through the cell culture flask 101, are covered with insulating sealing fasteners 104. The silver-plated wire 103 extends through the outside of the cell culture flask 101 and is fixed with a crystal plug 105 at its end. The test stimulation unit 200 includes a test stimulator 201, which is provided with a socket interface 202 that matches the crystal plug 105. An external programming interaction unit 300 includes a programmable tablet 301, which is communicatively connected to an external programmable controller 302.

[0053] During hardware assembly, the planar electrode 102 is first placed at the bottom of the cell culture flask 101, and then a silver-plated wire 103 with a crystal plug 105 is connected to the outside of the cell culture flask 101. To ensure the stability of the internal conductive structure and the safety of the culture environment, insulating and sealing fasteners 104 are applied to the junction of the planar electrode 102 and the silver-plated wire 103, as well as at the point where the silver-plated wire 103 penetrates the wall of the cell culture flask 101. The insulating sealing fastener 104 is specifically made of medical-grade biocompatible silicone or resin material. Its first function is to rigidly fix the flat electrode 102 to the bottom of the culture flask, preventing it from floating or shifting under the impact of the flowing culture medium. Its second function is to isolate the metal welding joints, preventing direct contact with the culture medium and the resulting electrochemical corrosion, which could lead to the precipitation of toxic metal ions into the liquid phase. Its third function is to completely fill the physical gaps left when the silver-plated wire 103 passes through the flask wall, ensuring the absolute airtightness and sterility of the entire culture isolation unit 100 during long-term constant-temperature culture. The crystal plug 105 is inserted into the socket interface 202, allowing the silver-plated wire 103 to physically connect to the test stimulator 201.

[0054] The standardized plug-and-play connection mechanism constructed by the silver-plated wire 103 and the crystal plug 105 allows the test stimulator 201 to complete the underlying electrical closed-loop connection simply by inserting the crystal plug 105, eliminating the need to forcibly insert loose, exposed wires into the bottle opening and secure them with tape, as is done in traditional experimental environments. This significantly simplifies the wiring structure and hardware assembly logic of the experimental platform. While ensuring the stable and lossless transmission of weak electrical pulse signals along the silver-plated wire 103, it achieves low-cost, high-reliability physical wiring in a sterile operating environment.

[0055] After the hardware connections of the device were adjusted, for safety checks, the operator added an appropriate amount of 0.9% sodium chloride solution to the cell culture flask 101 and used the programmable control software on the programmable control panel 301 to test the underlying impedance. A normal resistance test indicates that the designed circuit is working properly; an open circuit indicates a problem with the circuit, requiring investigation of abnormalities such as a broken silver-plated wire 103 and a loose crystal plug 105.

[0056] After hardware verification, specific parameters are set in the programming software of the programmable controller tablet 301. These parameters include voltage, current, frequency, and pulse width. Continuous stimulation mode and intermittent stimulation mode are selected on the software interface. The electrical energy output from the test stimulator 201 precisely simulates the electrical environment experienced by cells under different physiological states and pathological regulation. The underlying physical mechanism of the device lies in the change in cell membrane potential of cells cultured on the tablet electrode 102 when subjected to the aforementioned electrical stimulation. This potential change activates ion channels on the cell membrane, promoting the activation of intracellular signal transduction pathways, thereby profoundly affecting various physiological functions of the cell, promoting cell proliferation, inducing cell differentiation, accelerating cell migration, and altering gene expression.

[0057] In summary, this invention establishes a stable electrical channel by pre-embedding a multi-channel conductive rail array inside the culture chamber and extending it outward to form a sealed connection socket, ensuring a sterile and contamination-free culture environment. Relying on the parallel assembly control of the underlying electrical circuitry, the multi-dimensional spatial electric field morphology can be flexibly reconstructed without replacing physical hardware, significantly improving the versatility of the equipment in experimental scenarios. During the intervention cycle, weak high-frequency test signals that do not induce cell action potentials are used for impedance polling, enabling non-destructive screening of the liquid environment. In the long-cycle output phase, an adaptive constant current regulation mechanism dynamically compensates for the conductivity decrease error caused by electrolyte consumption in the culture medium, ensuring a consistent current dose applied to the cells. Furthermore, this invention, combined with anomaly monitoring logic based on impedance change rate, can accurately identify environmental anomalies caused by excessive liquid evaporation or large-scale cell detachment, and effectively maintain the objective constancy of long-cycle in vitro cell electrical stimulation intervention conditions by timely output cut-off, ultimately significantly improving the accuracy and reproducibility of experimental data.

[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for culturing reconfigurable modular electrode arrays for cell electrical stimulation, characterized in that, Includes the following steps: S1. Establish an electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center, wherein a multi-channel conductive rail array is pre-embedded inside the culture chamber; S2. The multi-channel programmed stimulation center obtains the real-time impedance value of the multi-channel conductive rail array based on the test electrical signal, and compares the real-time impedance value with the reference impedance range to confirm the liquid phase environment state. S3. Under normal conditions in the liquid environment, the multi-channel conductive rail array is polarized according to the electric field reconstruction requirements, including the allocation of anode and cathode, and the target electric pulse parameters are sent out. S4. The multi-channel programmed stimulation center outputs the target electrical pulse according to the target electrical pulse parameters, and periodically pauses the output during the intervention period to monitor the impedance change rate, and determines abnormal output cut-off and normal recovery of the target electrical pulse based on the impedance change rate. S5. After the intervention cycle ends, disconnect the culture chamber from the multi-channel programmed stimulation center and extract target biological samples with different spatial distribution states in the culture chamber.

2. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step S1, which involves establishing an electrical connection between the culture chamber containing cell fluid and an external multi-channel programmed stimulation center, specifically includes: The culture chamber has a physically sealed structure. The multi-channel conductive rail array is embedded in the inner bottom surface of the culture chamber. The lead-out cables of the multi-channel conductive rail array pass through the chamber wall and extend to the outside of the culture chamber to form a sealed connection socket. The sealed connection socket is then connected to the multi-channel programmed stimulation center. This establishes a physically isolated and electrically conductive connection channel between the inside and outside of the culture environment while keeping the inside of the culture chamber completely sealed to prevent external contamination.

3. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, Before the step S1 of establishing the electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center, a resting adhesion process is included, which specifically includes: Before injecting cell fluid into the culture chamber, a bio-adhesive coating is pre-applied to the inner bottom surface of the culture chamber and the surface of the multi-channel conductive rail array. After injecting cell fluid, the culture chamber is continuously quiescent. When the spread area of ​​the target cells reaches a preset ratio, the adhesion state of the target cells is confirmed as reaching the preset adhesion state. Then, the operation of establishing an electrical connection between the culture chamber containing cell fluid and the external multi-channel programmed stimulation center is performed to prevent large-scale detachment of target cells that are not firmly attached due to fluid flow fluctuations caused by power supply.

4. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step S2, which involves obtaining the real-time impedance value of the multi-channel conductive rail array based on polling the test electrical signal, specifically includes: The test electrical signal is a weak high-frequency AC signal, and the amplitude of the test electrical signal is limited to a safe threshold range that does not cause the target cells in the cytosol to generate action potentials. The electronic switch matrix integrated inside the multi-channel programmable stimulation center sequentially and non-repeatedly connects any two conductive rails in the multi-channel conductive rail array into the test circuit to obtain the real-time impedance value between the connected conductive rails.

5. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step S2, which compares the real-time impedance value with the reference impedance range to confirm the state of the liquid phase environment, specifically includes: If the acquired real-time impedance value is higher than the upper limit of the reference impedance range, the multi-channel programmed stimulation center determines that a physical disconnection abnormality has occurred in the liquid phase environment. If the acquired real-time impedance value is lower than the lower limit of the reference impedance range, the multi-channel programmed stimulation center determines that a metal bridging short circuit abnormality has occurred in the liquid phase environment. If all real-time impedance values ​​are within the reference impedance range, the multi-channel programmed stimulation center determines that the liquid phase environment is in a normal state and allows the subsequent parameter setting steps to proceed.

6. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step S3, which involves polarity grouping of the multi-channel conductive rail array according to the electric field reconstruction requirements, including anode and cathode allocation, specifically includes: By using the configuration interface of the control terminal, multiple selected conductive rails in the multi-channel conductive rail array are connected in parallel on the underlying electrical circuit to form an integral anode output array, and multiple other selected conductive rails in the multi-channel conductive rail array are connected in parallel to form an integral cathode output array, so as to realize the on-demand reconstruction of the spatial electric field pattern.

7. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step of sending the target electrical pulse parameters in step S3 specifically includes: The target electrical pulse parameters are defined to include a constant current output mode. During the output of the target electrical pulse, the multi-channel programmed stimulation center updates the output voltage in real time according to the real-time impedance value and the set target current amplitude. This is achieved by using an algorithm logic that the target output voltage equals the set target current amplitude multiplied by the real-time impedance value. This continuously and adaptively increases the output voltage to compensate for the decrease in liquid conductivity caused by electrolyte consumption in the liquid environment, ensuring that the actual current amplitude flowing into the liquid environment and acting on the target cells always remains consistent with the initial set value.

8. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The steps in step S4, namely determining abnormal output cutoff based on the impedance change rate and determining normal recovery of the target electrical pulse, specifically include: The impedance change rate is calculated using the formula: the change rate equals the difference between the real-time impedance value measured during the current pause and the initial impedance value, divided by the initial impedance value. If the impedance change rate shows a positive increase in impedance exceeding the upper limit of the safety threshold, the multi-channel programmed stimulation center determines that the liquid level evaporation is abnormal. If the impedance change rate shows a negative decrease in impedance below the lower limit of the safety threshold, the multi-channel programmed stimulation center determines that the target cells are dying and detaching on a large scale due to electrical stimulation overload. In the event of an abnormality, the multi-channel programmed stimulation center immediately cuts off the target electrical pulse output and triggers an alarm. In the event of no abnormality, the multi-channel programmed stimulation center determines that the status is normal and seamlessly resumes the target electrical pulse after the pause ends.

9. The method for culture of reconfigurable modular electrode arrays for cell electrical stimulation according to claim 1, characterized in that, The step of classifying and extracting target biological samples with different spatial distribution states within the culture chamber in step S5 specifically includes: First, the liquid supernatant inside the culture chamber is aspirated to separate and extract the free products secreted by the target cells after stimulation. Then, cell lysis reagent is injected into the culture chamber and the cell lysis products attached to the multi-channel conductive rail array and the bottom surface are collected by physical scraping to comprehensively obtain data reflecting the internal physiological functions and external interactions of the target cells.

10. A reconfigurable modular electrode array culture device for cell electrical stimulation, characterized in that, The apparatus is used to perform the method according to any one of claims 1 to 9, specifically comprising: A culture isolation unit (100) includes a cell culture flask (101). A flat plate electrode (102) is placed on the bottom surface of the cell culture flask (101). The flat plate electrode (102) is connected to a silver-plated wire (103). The connection between the flat plate electrode (102) and the silver-plated wire (103) and the exit point of the silver-plated wire (103) through the cell culture flask (101) are covered with insulating sealing fasteners (104). The silver-plated wire (103) extends through to the outside of the cell culture flask (101) and is fixed with a crystal plug (105) at its end. The test stimulation unit (200) includes a test stimulator (201) and the test stimulator (201) is provided with a socket interface (202) that matches the crystal plug (105). An external programming interaction unit (300) includes a programmable tablet (301) and the programmable tablet (301) is communicatively connected to an external programmable controller (302).

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