Separated organ-like chip model and use method thereof

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a separated type organ-like chip model and a use method thereof.The model comprises a chip, an organ-like unit module and a fluid control system, and the use method comprises the steps that firstly, the chip is assembled and prepared; step 2, cell inoculation and culture; step 3, establishing and operating a fluid circulation system; step 4, organ-like unit function detection and data acquisition; 5, analyzing an experimental result and adjusting the model; the model is connected with a micro-fluidic channel through an independent cavity, and the characteristics, cell types and culture conditions of a cell culture bracket can be regulated and controlled according to the characteristics of simulated organs. Meanwhile, the micro-fluidic channel ensures stable and ordered material exchange among the organoid units, and is beneficial to more accurately screening out effective treatment schemes and drug targets.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a separate organoid chip model and a method for using the same. Background Art

[0002] With the development of technology, organoid-on-a-chip technology has emerged. As a microphysiological system with human tissue, it aims to simulate organoid-level functions in vitro, thereby achieving human physiological modeling and enabling researchers to deeply explore organoid functions and the mechanisms by which external factors influence organoid injury, disease, and treatment response. Based on three-dimensional cell culture techniques, this technology comprehensively considers cell-cell interactions, matrix properties, and biochemical and biomechanical properties to construct a three-dimensional organoid physiological microsystem on a chip, bridging the gap between traditional cellular-level research and whole-organism research.

[0003] However, current organoid-on-a-chips on the market present a series of challenges in practical applications. For one thing, it's difficult to simultaneously address key factors such as organoid physiological properties, high throughput, and connectivity between multiple organoids. Furthermore, mainstream culture methods involve direct co-culture of principal and auxiliary cells, which undoubtedly complicates the subsequent functional testing of specific principal cells and the need for cell separation and purification. This significantly limits the application of organoid-on-a-chips in a wider range of fields. Therefore, we propose a dissociated organoid-on-a-chip model and its application method. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a separated organoid chip model and a method for using the same, thereby solving the technical problems mentioned in the background technology.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A separate organoid chip model, comprising a chip, an organoid culture unit module, and a fluid control system; The chip is rectangular in shape and has a culture chamber inside. The culture chamber serves as a separate organoid culture unit simulation area. The side wall of the chamber is provided with an inlet and outlet with a silicone sealing gasket. The chambers are connected by cylindrical microfluidic channels. Each chamber of the organoid culture unit module contains a cell culture scaffold, the surface of which is modified with extracellular matrix protein. A microelectrode array is arranged around the scaffold and connected to an external data acquisition system via a wire; The fluid control system consists of a peristaltic pump and a syringe pump. The peristaltic pump uses a motor and a control algorithm. The syringe pump is connected to the chip microfluidic channel through a polytetrafluoroethylene tube. A special sealing joint and a pressure sensor are provided at the connection.

[0006] In a possible implementation, the chip material is polydimethylsiloxane, which has optical transparency, gas permeability and biocompatibility.

[0007] In a possible implementation, the cell culture scaffold is a three-dimensional porous structure, and the material is a gelatin-fibrin composite hydrogel that has been chemically and physically cross-linked.

[0008] In a possible implementation, the microelectrode array is composed of 16 microelectrodes distributed in a 4×4 matrix. The microelectrodes are made of platinum-iridium alloy and the surface is covered with insulating material.

[0009] In a possible implementation, the external data acquisition system includes a signal amplifier, a filter, and an analog-to-digital converter.

[0010] In a possible implementation, the peristaltic pump tube is made of silicone.

[0011] In one possible implementation, the method for using the isolated organoid chip model comprises the following steps: Step 1: Chip assembly and preparation: Inspect the chip body, organoid culture unit module, fixture, and microfluidic channel components in a dust-free environment to ensure they are free of impurities and damage. Use a positioning tool to accurately place the organoid culture unit module into the corresponding chamber of the chip. Check the sealing of the connection parts. Use a magnetic pressure plate to fix the module. Use a syringe to inject filtered sterile air into the microfluidic channel to test connectivity. If it is blocked, use a microendoscope to check. Different methods are used to clear the blockage according to the blockage situation. Step 2: Cell inoculation and culture: sterilize the cell culture scaffold with ultraviolet light. Rotate or flip the scaffold during irradiation to ensure uniform irradiation. Rinse with sterile phosphate buffer. After counting and viability testing, inoculate at a predetermined density. Use a micropipette to evenly add the cell suspension during inoculation. Place the chip in a cell culture incubator for pre-culture. During this period, add an appropriate amount of organoid-specific culture medium regularly. After adding, gently shake the chip to evenly distribute the culture medium. Step 3: Establish and operate the fluid circulation system. Connect the culture medium storage bottle, peristaltic pump, chip, and waste liquid collection bottle in sequence, ensuring that the tubing is not twisted or blocked and the connection is tight, and secure it with a tube clamp. Set the peristaltic pump flow rate, add fluorescent markers to the culture medium after starting, observe the fluid flow and check the sealing with a fluorescence microscope. Prepare the drug or bioactive substance solution according to the experimental requirements, and use a flow sensor to monitor the injection speed in real time to ensure stability. Step 4: Functional testing and data acquisition of the organoid culture unit. Before using the microelectrode array to monitor cell electrophysiological activity, its performance is calibrated and tested. The data acquisition system records the microelectrode signals and converts them into digital signals for storage. Analysis software is used to denoise the signals and extract action potential characteristic parameters to analyze the cell physiological state. Culture medium samples are regularly collected from the chip outlet, and metabolite concentrations are measured using a biochemical analyzer. The concentration-time curve is then plotted. Step 5: Analyze experimental results and adjust the model. Use statistical methods to analyze electrophysiological and biochemical data to determine the parameter differences and interaction relationships of organoid culture units under different conditions. If the metabolic function of a certain organoid unit is insufficient, adjust the cell seeding density or extend the pre-culture time through preliminary experiments, and monitor the cell status during this period. Conversely, if the drug metabolism rate is abnormal, adjust the peristaltic pump flow rate or drug injection rate.

[0012] Beneficial effects compared with existing technologies: 1. In this scheme, multiple chips are connected in parallel through independent chambers and microfluidic channels. By adjusting the peristaltic pump, the physiological environment of different organoid units can be accurately simulated. Each chip can regulate the characteristics of the cell culture scaffold, cell type and culture conditions according to the characteristics of the simulated organoid. At the same time, the microfluidic channel ensures stable and orderly material exchange within each organ unit, allowing various organs to influence and interact with each other. It provides a reliable in vitro model for the disease mechanisms of multiple organoids and the metabolic processes of drugs between multiple organoids in the body, greatly improving the fit between research results and the actual situation of the human body, and helping to more accurately screen effective treatment plans and drug targets; 2. In this protocol, by using a detachable separation culture unit, researchers can simplify the isolation and purification of organoids during subsequent functional testing, allowing direct observation and testing of organoids. Furthermore, this reduces the potential damage and interference to organoids caused by the separation process, improving the accuracy of experimental results. 3. In this scheme, by designing independent organoid unit simulation areas, experiments with different organoids or the same organoid under different culture conditions can be conducted simultaneously on the same microfluidic system, achieving high-throughput operations. Researchers can set multiple experimental variables in a single experiment, greatly improving experimental efficiency and shortening research cycles. In addition, this model can adjust the number, type, and connection method of organoid units according to different research needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings.

[0014] Figure 1 Schematic diagram of the overall structure of the isolated organoid chip model of the present invention; Figure 2 Schematic diagram of the method for using the isolated organoid chip model of the present invention. DETAILED DESCRIPTION

[0015] Preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention can be implemented in various forms, and therefore the present invention is not limited to the embodiments described below. In addition, in order to more clearly describe the present invention, components that are not related to the present invention will be omitted from the drawings. The technical solution in the embodiments of the present application is to solve the problems of the above-mentioned background technology, and the overall idea is as follows: Example 1: The isolated organoid chip model is a highly sophisticated and complex microfluidic device designed to simulate the functions and interactions of human organoids. It consists primarily of a chip body, organoid unit modules, and a fluid control system. It measures 7.5 cm long, 7 cm wide, and 1.5 cm high, forming a rectangular parallelepiped.

[0016] The chip model is in the shape of a rectangular parallelepiped, with precise dimensions of 7.5 cm long, 7 cm wide, and 1.5 cm high. It is like a small transparent brick, which is convenient for experimenters to operate and observe. Five independent chambers are designed inside it, each of which is a separate organoid unit simulation area with a diameter of 3.2 cm and a height of 1.2 cm. This enables the simulation of different organoids to be carried out in relatively independent yet interrelated environments. The side walls of each chamber are equipped with sealed inlets and outlets, and silicone sealing gaskets are installed at the inlets and outlets. The silicone sealing gaskets are soft and elastic, mostly black or dark in color, and fit tightly to the edges of the inlets and outlets, effectively preventing fluid leakage and ensuring the independence and stability of each organoid unit simulation area.

[0017] They are interconnected through microfluidic channels. The inner diameter of the microfluidic channels is 500 microns, the length is 2.5 cm, and the channel shape is cylindrical. The channel walls are smooth and uniform. When observed under a microscope, the inner walls are like polished mirrors. The roughness of the channel walls is less than 0.1 microns, which ensures that the culture medium and other fluids can flow stably in the chip. In order to achieve efficient material exchange simulation, the design of the microfluidic channels has undergone multiple fluid mechanics simulation analyses to ensure that the uniformity error of the fluid flow rate in the chip is less than 5%, creating a stable nutrient supply and metabolic waste discharge environment for the cells.

[0018] The chip is made of polydimethylsiloxane (PDMS), which exhibits excellent optical transparency, gas permeability, and biocompatibility. Optical transparency allows researchers to observe the growth and morphological changes of cells within the chip directly through a microscope; gas permeability facilitates gas exchange between cells and the external environment, meeting their respiratory needs; and excellent biocompatibility ensures that cells can grow and differentiate normally within the chip, unaffected by the toxicity of the material. During the fabrication process, photolithography and etching techniques are used to control the positional accuracy of each microfluidic channel to within ±50 microns, and the dimensional accuracy is also within a ±50 micron error range, ensuring consistent chip fabrication and the authenticity of experimental results.

[0019] The core of each organoid unit module is the cell culture scaffold, which features a three-dimensional porous structure with 100-micron pores and a porosity of 70%. Microscopically, it resembles an intricately arranged porous sponge, providing cells with a growth environment similar to the extracellular matrix in the body, conducive to cell attachment, growth, and differentiation. The 100-micron pore size ensures ample space for cells to expand while allowing nutrients and metabolic waste to pass smoothly through the pores. Extensive experimental verification has shown that a porosity of 70% provides optimal cell growth and activity.

[0020] The material of the cell culture scaffold is a gelatin-fibrin composite hydrogel, which has been cross-linked to improve its stability. The cross-linking process uses a combination of chemical cross-linking and physical cross-linking. First, a stable chemical bond is formed between gelatin and fibrin by a chemical cross-linking agent, and then the network structure of the hydrogel is further enhanced by a physical freeze-thaw method. The treated hydrogel can remain stable for at least 7 days under a physiological environment of 37°C, meeting the needs of most experiments. Its surface is modified with extracellular matrix proteins, specifically collagen I and laminin. These proteins are covalently linked to the hydrogel surface, greatly increasing the adhesion of cells. Studies have shown that after protein modification, the initial adhesion rate of cells on the scaffold increased by more than 30%, promoting the subsequent growth and differentiation of cells.

[0021] A microelectrode array is set up around the cell culture scaffold to monitor the electrophysiological activities of cells in the organoid unit in real time. The microelectrode array consists of 16 microelectrodes distributed in a 4×4 matrix with an electrode spacing of 500 microns. The microelectrodes are made of platinum-iridium alloy, which has good conductivity and chemical stability. To ensure that the microelectrodes only detect the weak electrical signals generated by the cells, their surface is covered with a layer of insulating material, leaving only an opening with a diameter of 10 microns at the top. The microelectrodes are very small and require the help of a microscope to see clearly. They are like tiny "antennae" that monitor the electrophysiological changes of cells at all times.

[0022] The microelectrode array is connected via wires to an external data acquisition system, which includes a signal amplifier, filter, and analog-to-digital converter. The signal amplifier amplifies the extremely weak electrical signals detected by the microelectrodes by 1000 times, bringing them within a detectable and processable range. The filter uses a 100Hz-10kHz bandpass filter to remove external interference signals and improve signal quality. The analog-to-digital converter, sampling at a 10kHz frequency, quickly and accurately converts analog signals into digital signals for computer storage and subsequent analysis. The entire data acquisition system has been calibrated and optimized to achieve microvolt-level detection accuracy, ensuring the monitoring and recording of cellular electrophysiological activity.

[0023] The peristaltic pump and syringe pump connected to the outside of the chip together constitute a fluid control system, which plays a key role in maintaining the growth environment of cells within the chip and simulating changes in substances in the body.

[0024] The peristaltic pump is used to maintain the continuous circulation of the culture medium in the chip, ensuring that the cells grow in a stable nutrient environment and with an adequate oxygen supply. The flow rate of the peristaltic pump can be adjusted in the range of 0.01-10 ml / min, and the flow rate is precisely controlled by adjusting the rotation speed of the peristaltic pump. The peristaltic pump uses a motor and control algorithm inside, and the flow accuracy can reach ±0.001 ml / min. When running continuously for 24 hours, the flow rate fluctuation error is less than 1%, ensuring the stability of the culture medium supply. The pump tube of the peristaltic pump is made of silicone, which has good elasticity and wear resistance. It is a transparent or translucent hose connected between the peristaltic pump and the chip. It can maintain stable performance under long-term extrusion and friction and will not contaminate the fluid. After screening and testing a variety of silicone materials, the silicone material finally selected will not release any harmful substances after long-term contact with the culture medium, ensuring the safety of the cell growth environment.

[0025] Syringe pumps are used to control the infusion rate of drugs or other bioactive substances to simulate the changes in the body during drug metabolism and disease progression. With an injection accuracy of up to 0.1 μL, syringe pumps enable accurate administration of minute doses. The injection rate ranges from 0.01 to 100 μL / minute, adjustable to suit different experimental requirements. For drug metabolism studies, a lower injection rate can be used for drugs that require slow infusion to simulate in vivo physiological processes; however, a higher injection rate can be used for experiments that require rapid concentration. A syringe pump typically contains a syringe for loading the drug or bioactive substance. This syringe is connected to the chip via polytetrafluoroethylene tubing with an inner diameter of 100 μm and a length of 50 cm. This connection is sealed with a specialized connector designed to ensure a tight connection without affecting fluid flow. During the injection process, a pressure sensor monitors the injection pressure in real time to ensure no leakage.

[0026] Example 2: A method for using a dissociated organoid chip model comprises the following steps: Step 1: Chip assembly and preparation Before chip assembly, all components must be carefully inspected and cleaned. The chip itself, culture unit modules, fixtures, and microfluidic channels must all be inspected in a dust-free environment to ensure they are free of impurities and damage. To prevent fluid leakage, during installation, each connection must undergo a preliminary inspection for sealing performance. A small amount of sealant may be applied to enhance the seal, but care must be taken to prevent the sealant from entering the microfluidic channels or affecting the cell culture environment.

[0027] The organoid culture unit module is secured using a custom fixture, a pressure plate powered by ultrasonic waves. Before use, the device must be inspected to ensure proper function. Ultrasonic heating presses the organoid culture unit module firmly against the bottom of the chamber. Ensure uniform force is applied to the pressure plate during securing to avoid deformation or damage to the organoid culture unit module due to excessive localized pressure.

[0028] At the same time, the connectivity of the microfluidic channel needs to be checked. Use a syringe to inject filtered sterile air into the channel and observe whether there are bubbles escaping from the outlet. When injecting air, it is important to control the injection pressure and speed to avoid damage to the channel due to excessive pressure. If bubbles escape, it means the channel is unobstructed; if there are no bubbles, it is necessary to use specialized channel detection equipment, such as a microendoscope, to check for blockages in the channel. Once a blockage is found, appropriate cleaning methods should be used based on the nature and location of the blockage, specifically flushing, micromechanical cleaning, etc., to ensure that the microfluidic channel is unobstructed.

[0029] Step 2: Cell seeding and culture Thoroughly disinfect the cell culture scaffolds of each organoid culture unit module. First, irradiate with ultraviolet light for 30 minutes. Ensure that all parts of the cell culture scaffold are fully exposed to the UV light. Rotate or flip the scaffold to achieve uniform irradiation. After irradiation, rinse three times with sterile phosphate-buffered saline (PBS), each rinse lasting at least 5 minutes, to completely remove any residual disinfectant.

[0030] Then, the corresponding cell types are inoculated onto the culture scaffolds of each organ culture unit. Before inoculation, the cells are counted and tested for viability to ensure that the number of inoculated cells is accurate and the activity is good. Specifically, when simulating the liver organoid culture unit, hepatocytes are inoculated at a density of 1×10 6To ensure uniform seeding, use a micropipette to evenly add the cell suspension onto the culture scaffold, and avoid creating bubbles during the addition process. When simulating kidney organoid culture units, glomerular cells and renal tubular epithelial cells were seeded at a seeding density of 8×10 5 cells / ml and 1.2×10 6 The order of inoculating different cell types has been experimentally verified to be better able to simulate the physiological structure and function of the kidney by first inoculating glomerular cells and then inoculating renal tubular epithelial cells after their initial attachment.

[0031] After the inoculation is completed, the serially connected organoid chips are placed in a cell culture incubator for pre-culture. The temperature of the incubator is set to 37°C, the humidity is maintained at 95%, and the carbon dioxide atmosphere is controlled at 5%. The pre-culture time is 24 hours. During this period, an appropriate amount of culture medium is added to the chip every 6 hours to maintain a moist environment for the cells. The culture medium uses a special culture medium corresponding to each type of organ unit. Specifically, the liver organoid unit uses a high-glucose DMEM / F12 culture medium containing serum substitutes and 1% penicillin-streptomycin solution. When adding culture medium, pay attention to sterile operation to avoid contamination. After each addition of culture medium, shake the chip gently to ensure that the culture medium is evenly distributed on the cell culture scaffold.

[0032] Step 3: Establishment and operation of fluid circulation system Connect the peristaltic pump and syringe pump to the microfluidic channels of the chip. Clean and disinfect the tubing and connectors before making any connections. Connect the culture medium reservoir to the inlet of the peristaltic pump through the tubing. Connect the outlet of the peristaltic pump to the inlet of the chip, and the outlet of the chip to the waste collection bottle. During the connection process, ensure that the tubing is free of twisting and blockage, and ensure that each section of tubing is tightly and securely connected to avoid leakage. Use tubing clamps to secure the tubing to prevent it from shifting during the experiment.

[0033] Set the peristaltic pump flow rate to 0.1 ml / min, which is determined based on the metabolic needs of the cells and the fluid dynamics of the microfluidic channel. After starting the peristaltic pump, closely observe the flow of the culture medium within the chip. You can add a small amount of fluorescent marker to the culture medium and use a fluorescence microscope to observe the flow path and velocity distribution of the fluid. Ensure that the culture medium can circulate within the chip to provide cells with a continuous supply of nutrients and discharge of metabolic waste. At the same time, check the sealing of each connection. If there is any liquid leakage, stop the peristaltic pump immediately and readjust the connection to ensure the stability of the experimental environment.

[0034] According to the experimental requirements, specific drugs or bioactive substances are injected into the chip through a syringe pump to simulate the changes in substances under physiological or pathological conditions in the body. Before injection, the solution of drugs or bioactive substances must be accurately prepared to ensure its concentration is correct. Specifically, when studying the metabolic effects of drugs on the liver, the drug solution is prepared into a solution with a concentration of 100 micrograms / ml. The chip is injected at a rate of 0.1 microliters / minute through a syringe pump, and the changes in metabolites of the liver organoid culture unit are monitored at the same time. During the injection process, a flow sensor is used to monitor the actual injection speed of the syringe pump in real time to ensure the stability of the injection speed and avoid uneven drug concentration due to speed fluctuations. If a deviation in the injection speed is found, the parameters of the syringe pump must be adjusted in time to ensure the accuracy of the experimental data.

[0035] Step 4: Functional testing and data collection of organoid culture units Microelectrode arrays are used to monitor the electrophysiological activities of cells in various organ culture units in real time. Before monitoring, the microelectrode arrays must be calibrated and performance tested to ensure that they can accurately detect the weak electrical signals generated by the cells. Electrophysiological signals are collected through an external data acquisition system and analyzed and processed. Specifically, for neural organ culture units, the excitability of neurons and the neural signal transmission function are evaluated by analyzing the changes in the frequency and amplitude of the action potentials of nerve cells. The data acquisition system records the signal detected by the microelectrode every 1 second, converts it into a digital signal, and stores it in a computer.

[0036] Analytical software is used to denoise and extract features from the signal. Denoising utilizes advanced algorithms such as wavelet transforms to effectively remove background noise and interfering signals, improving the signal-to-noise ratio. Parameters such as the peak value, width, and frequency of the action potential are calculated. Analysis of these parameters provides insights into the physiological state and functional changes of nerve cells. Specifically, an increase in action potential frequency indicates increased neuronal excitability, while changes in action potential amplitude reflect changes in synaptic function during neural signal transmission.

[0037] At the same time, culture medium samples are collected regularly from the liquid outlet of the chip. Before collection, the sampling instruments must be strictly disinfected and cleaned to avoid contamination of the samples. A biochemical analyzer is used to detect the concentration of metabolites in the culture medium, specifically the changes in the content of glucose, lactate, urea, etc., to evaluate the metabolic functions and interactions of various organ culture units. For example, in the multi-organoid chip serial model, effluent samples are collected every 2 hours, and an enzyme-linked immunosorbent assay (ELISA) kit is used to detect the albumin concentration secreted by the liver organoid culture unit and the urea concentration excreted by the kidney organoid culture unit. During the detection process, the operating instructions of the kit must be strictly followed to ensure the accuracy of the test results. Based on the test results, curves showing the changes in albumin and urea concentrations over time are drawn. By analyzing the curves, the functional status of the liver and kidney organoid culture units and their mutual influence can be intuitively understood.

[0038] Step 5: Result analysis and model adjustment A variety of statistical methods were used to conduct in-depth analysis of the collected electrophysiological data and biochemical analysis results. For the electrophysiological parameters of different organoid culture units, such as action potential frequency and amplitude, analysis of variance (ANOVA) was used to determine whether there were significant differences in these parameters under different experimental conditions (e.g., before and after administration, at different time points, etc.). For metabolite concentration data, such as the concentration of albumin secreted by the liver and the concentration of urea excreted by the kidneys, t-tests or non-parametric tests were used (selecting the appropriate test method based on the distribution characteristics of the data) to determine the changes in the metabolic functions of various organ culture units under different conditions. At the same time, correlation analysis, such as Pearson correlation coefficient calculation, was used to explore the degree of correlation between changes in metabolite concentrations between different organoid culture units, thereby revealing the interactive relationship between them.

[0039] Based on the experimental results, we made targeted adjustments and optimizations to the separated organoid chip model. If the metabolic function of a certain organoid culture unit is insufficient, specifically, it is found in the test that the concentration of albumin secreted by the liver organoid culture unit is significantly lower than the normal physiological level, indicating that its metabolic function is defective. At this time, we can try to appropriately increase the cell seeding density of the organoid culture unit. Before implementation, a series of preliminary experiments are needed to set up different cell seeding density gradients, increase the original seeding density by 10%, 20%, 30%, etc., and observe the growth status, metabolic function and impact of cells on other organoid culture units at different densities. Through comprehensive evaluation, the optimal seeding density is determined that can not only improve the metabolic function of the organoid culture unit, but also avoid problems such as insufficient nutrient supply and excessive accumulation of metabolic waste due to excessive cell density.

[0040] In addition, extending the pre-culture time is also a way to improve the functional stability of the organoid culture unit. Extending the pre-culture time allows cells more time to attach, stretch and proliferate on the culture scaffold, so that they can better adapt to the culture environment and exert their functions. In the process of extending the pre-culture time, it is necessary to closely monitor the growth status of the cells, including cell morphological changes, proliferation rate, vitality and other indicators. Use a microscope to observe the cell morphology every day. If abnormal phenomena such as shrinkage, deformation or shedding of cells are found, it may mean that the cell growth environment is not good and the culture conditions need to be adjusted in time. At the same time, test cell viability at regular intervals to ensure that the cells maintain high activity during the extended pre-culture time.

[0041] If it is found in the experiment that the metabolism rate of the drug in the chip is too fast or too slow, which is inconsistent with the actual metabolism in the body, we can optimize the model by adjusting the flow rate of the peristaltic pump or the drug injection rate. If the drug metabolism rate is too fast, it means that the culture medium is updated too quickly and the drug stays in the chip for too short a time. At this time, appropriately reduce the flow rate of the peristaltic pump, such as reducing the flow rate from 0.1 ml / min to 0.5 ml / min, slowing down the circulation rate of the culture medium, so that the drug has a longer time to interact with the cells in the chip, which is closer to the metabolic process in the body. After adjusting the flow rate, it is necessary to re-monitor the concentration changes of drug metabolites and the functional indicators of various organ culture units to evaluate the effect of the adjustment.

[0042] On the other hand, if the drug metabolism rate is too slow, the flow rate of the peristaltic pump can be appropriately increased to speed up the renewal of the culture medium and promote the metabolism and excretion of the drug; or the drug injection rate can be increased to make the drug reach an effective concentration in the chip more quickly, simulating a more realistic drug metabolism dynamic in the body. Similarly, after adjusting these parameters, it is necessary to re-experiment and compare the data before and after the adjustment to verify whether the adjusted model can more accurately simulate the organoid function and interaction under the physiological or pathological state of the human body. By continuously analyzing the experimental results and adjusting the model parameters, we can gradually optimize the separated organoid chip model and provide a more reliable and accurate in vitro model for subsequent drug development and disease research.

[0043] Finally, it should be noted that the above embodiments are merely examples for the purpose of illustrating the present invention and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to provide an exhaustive list of all embodiments. However, obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A separate organoid chip model, characterized in that: Includes chips, organoid unit modules and fluid control systems; The chip is rectangular in shape and has a culture chamber inside. The culture chamber serves as a separate organoid unit simulation area. The side walls of the chamber are provided with inlets and outlets with silicone sealing gaskets. The chambers are connected by cylindrical microfluidic channels. The organoid unit culture chamber comprises a cell culture scaffold, the surface of which is modified with extracellular matrix protein, and a microelectrode array is arranged around the scaffold and connected to an external data acquisition system via a wire; The fluid control system consists of a peristaltic pump and a syringe pump. The peristaltic pump uses a motor and a control algorithm. The syringe pump is connected to the chip microfluidic channel through a polytetrafluoroethylene tube. A special sealing joint and a pressure sensor are provided at the connection.

2. The isolated organoid chip model according to claim 1, wherein: The chip is made of polydimethylsiloxane, which has optical transparency, gas permeability and biocompatibility.

3. The isolated organoid chip model according to claim 1, wherein: The cell culture scaffold is a three-dimensional porous structure, and the material is a gelatin-fibrin composite hydrogel that has been chemically and physically cross-linked.

4. The isolated organoid chip model according to claim 1, wherein: The microelectrode array consists of 16 microelectrodes distributed in a 4×4 matrix. The microelectrodes are made of platinum-iridium alloy and the surface is covered with insulating material.

5. The isolated organoid chip model according to claim 1, wherein: The external data acquisition system includes a signal amplifier, a filter and an analog-to-digital converter.

6. The isolated organoid chip model according to claim 1, wherein: The peristaltic pump tube is made of silicone.

7. The method for using the isolated organoid chip model according to any one of claims 1 to 6, wherein: The following steps are involved: Step 1: Chip assembly and preparation: Inspect the chip body, organoid culture unit module, fixture, and microfluidic channel components in a dust-free environment to ensure they are free of impurities and damage. Use a positioning tool to accurately place the organoid culture unit module into the corresponding chamber of the chip. Check the sealing of the connection parts. Use a magnetic pressure plate to fix the module. Use a syringe to inject filtered sterile air into the microfluidic channel to test connectivity. If it is blocked, use a microendoscope to check. Different methods are used to clear the blockage according to the blockage situation. Step 2: Cell inoculation and culture: sterilize the cell culture scaffold with ultraviolet light. Rotate or flip the scaffold during irradiation to ensure uniform irradiation. Rinse with sterile phosphate buffer. After counting and viability testing, inoculate at a predetermined density. Use a micropipette to evenly add the cell suspension during inoculation. Place the chip in a cell culture incubator for pre-culture. During this period, add an appropriate amount of culture medium specifically for the organoid culture unit. After adding, gently shake the chip to evenly distribute the culture medium. Step 3: Establish and operate the fluid circulation system. Connect the culture medium storage bottle, peristaltic pump, chip, and waste liquid collection bottle in sequence, ensuring that the tubing is not twisted or blocked and the connection is tight, and secure it with a tube clamp. Set the peristaltic pump flow rate, add fluorescent markers to the culture medium after starting, observe the fluid flow and check the sealing with a fluorescence microscope. Prepare the drug or bioactive substance solution according to the experimental requirements, and use a flow sensor to monitor the injection speed in real time to ensure stability. Step 4: Functional testing and data acquisition of the organoid culture unit. Before using the microelectrode array to monitor cell electrophysiological activity, its performance is calibrated and tested. The data acquisition system records the microelectrode signals and converts them into digital signals for storage. Analysis software is used to denoise the signals and extract action potential characteristic parameters to analyze the cell physiological state. Culture medium samples are regularly collected from the chip outlet, and metabolite concentrations are measured using a biochemical analyzer. The concentration-time curve is then plotted. Step 5: Analyze experimental results and adjust the model. Use statistical methods to analyze electrophysiological and biochemical data to determine the parameter differences and interaction relationships of organoid culture units under different conditions. If the metabolic function of a certain organoid culture unit is insufficient, adjust the cell seeding density or extend the pre-culture time through preliminary experiments, and monitor the cell status during this period. Conversely, if the drug metabolism rate is abnormal, adjust the peristaltic pump flow rate or drug injection rate.

Images