Microphysiological system sensor chip for real-time dynamic multi-parameter monitoring and its assembly method

Abstract

The present invention relates to a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring and its assembly method. The invention includes a cell positioning capture module, a multi-parameter cell sensing detection module, and a signal switching module. The cell positioning capture module includes multiple 3D cell or organoid capture and positioning microfluidic chips; the individual 3D cell or organoid capture and positioning microfluidic chips in the cell positioning capture module are combined and connected; the multi-parameter cell sensing detection module includes multiple sensors for real-time monitoring of 3D cells or organoids; the multi-parameter cell sensing detection module senses multiple parameters of multiple 3D cells or organoids in real time; and the signal switching module includes a switching circuit and an image acquisition area. The present invention performs high-throughput capture and positioning of different types of 3D cells or organoids based on the 3D cell or organoid capture and positioning microfluidic chip, constructs a microphysiological system composed of multiple 3D cells or organoids, and performs comprehensive evaluation of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of 3D cell or organoid microphysiological systems, and specifically relates to a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring and an assembly method thereof. Background Art

[0002] Before organoid research, drug discovery researchers often used 3D cells to address the shortcomings of 2D cell and animal models. Building on 3D cell technology, organoid models and the microphysiological systems comprised of these organoids have been developed to better mimic the in vivo environment of cells.

[0003] Although 2D cells are easier to culture and use, they lack an in vivo three-dimensional environment and tumor cell lines lose tumor specificity and heterogeneity during long-term culture, resulting in significant deviations between drug testing results and those in the in vivo environment, thereby increasing the time and cost of drug development. To obtain more accurate drug testing results, researchers have further adopted animal models or tumor xenograft animal models (PDX) for drug testing. Although they possess an in vivo three-dimensional environment and retain tumor heterogeneity and genomic stability, animal-related model experiments have a long cycle, high cost, and face animal ethics issues, which will ultimately affect the efficiency of drug development.

[0004] 3D cells, with their three-dimensional structure, can mimic the in vivo 3D microenvironment of cells, thus bridging the gap between 2D cells and animal models. Building upon 3D cells, combined with induced pluripotent stem cells or patient-derived tumor cells, normal organoids or patient-derived tumor organoids can be cultured to mimic organ microstructures and some functional properties. This further mimics the in vivo state and function of cells, leading to more accurate drug testing results. However, after oral administration or injection, active drug ingredients may travel through blood vessels and various normal organs before reaching the target lesion, potentially causing toxic side effects in normal organs. Furthermore, poor absorption of the active drug ingredients by normal organs can lead to reduced efficacy. Furthermore, some drugs require liver metabolism to be effective. Therefore, combining normal and tumor organoids with different drug administration methods to construct microphysiological systems can more effectively simulate the physiological communication between multiple organs, enabling more effective and comprehensive assessment of drug side effects and in vivo efficacy, improving drug development efficiency and promoting the development of customized medicine.

[0005] Microfluidics technology can manipulate tiny particles in liquids and solutions through micron-scale microchannels. It has the advantages of miniaturization, automation, low reagent consumption, high throughput, and low sample requirements. It is particularly suitable for combination with organoid culture to achieve the capture and positioning of organoids and the construction of multi-organoid models. Cell micro-nanosensor chips with interdigitated electrodes and microelectrode arrays can evaluate the effects of drugs on organoids by detecting changes in cell impedance and potential in real time. However, current cell micro-nanosensor chips are generally suitable for 2D cultured cells and need to be further combined with microfluidic chips to achieve three-dimensional support and dynamic culture of 3D cells and organoids. Therefore, in the field of drug screening, there is an urgent need for a microphysiological system sensor chip that can realize the construction of microphysiological systems and high-throughput drug screening models, and can detect changes in the physiological state of 3D cells or organoids in real time. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring and an assembly method thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] According to a first aspect of the present specification, there is provided a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring, comprising:

[0009] A cell positioning and capture module includes multiple 3D cell or organoid capture and positioning microfluidic chips, each of which includes a microstructure array for positioning and capturing corresponding 3D cells or organoids; the cell positioning and capture module simultaneously captures and positions multiple 3D cells or organoids while maintaining the three-dimensional structure of the 3D cells or organoids and positioning the 3D cells or organoids on the microphysiological system sensor chip; the various 3D cell or organoid capture and positioning microfluidic chips in the cell positioning and capture module are combined and connected to construct a physiological communication flow path between different 3D cell or organoid arrays;

[0010] A multi-parameter cell sensing detection module includes multiple cell sensing areas, each of which is composed of one or more sensors arranged for real-time monitoring of 3D cells or organoids. The multi-parameter cell sensing detection module senses multiple parameters of various 3D cells or organoids in real time to provide multi-angle feedback on the growth status of 3D cells or organoids.

[0011] a signal adapter module, which internally includes an adapter circuit for leading out each cell sensor detection circuit in the multi-parameter cell sensing detection module and an image acquisition area for observing cell status, wherein the image acquisition area corresponds to the cell sensing area in the multi-parameter cell sensing detection module; and

[0012] The cell positioning capture module, the multi-parameter cell sensing detection module, and the signal switching module are arranged in sequence from top to bottom in space.

[0013] Furthermore, the 3D cell or organoid capture and positioning microfluidic chip has a liquid inlet and a liquid outlet and a chamber;

[0014] All 3D cell or organoid capture and positioning microfluidic chips are connected to the liquid inlet and liquid outlet between different 3D cell or organoid capture and positioning microfluidic chips through a steel needle hose.

[0015] Furthermore, the position of the microstructure array in the 3D cell or organoid capture and positioning microfluidic chip corresponds to the sensor in the multi-parameter cell sensing detection module.

[0016] Furthermore, the multi-parameter cell sensing detection module has multiple cell sensing areas that correspond one-to-one to the 3D cell or organoid capture and positioning microfluidic chip in the cell positioning and capture module. The cell sensing areas of the multi-parameter cell sensing detection module include a multi-channel cell impedance sensor and a multi-channel microelectrode array. The cell impedance sensor is used for 3D cell or organoid activity and pulsation detection, and the microelectrode array is used for 3D cell or organoid electrical discharge detection.

[0017] The main body of the cell impedance sensor adopts an interdigitated electrode structure, wherein the interdigitated electrodes include a pair of excitation electrodes and working electrodes, each excitation electrode finger and working electrode finger includes multiple electrode regions connected in series, and the gap between a pair of excitation fingers and working fingers corresponds to the center of the microstructure in the 3D cell or organoid capture and positioning microfluidic chip;

[0018] The multi-channel microelectrode array includes a microelectrode array composed of multiple microelectrodes, which are arranged in the center of the microstructure in the 3D cell or organoid capture and positioning microfluidic chip, and each microstructure array corresponds to multiple microelectrodes.

[0019] Furthermore, the signal adapter module includes pads, leads and pin headers for externally connecting the sensor signals in the multi-parameter cell sensing detection module;

[0020] The signal switching module has an image acquisition area for observing changes in the growth state of the 3D cells or organoids that have completed capture and positioning in the cell positioning capture module.

[0021] Furthermore, at least two of the normal 3D cells or organoids and the 3D cells or organoids of the patient-derived abnormal tissue together constitute a microphysiological system;

[0022] Wherein, when the microphysiological system is entirely composed of 3D cells or organoids of abnormal tissues derived from patients, the microphysiological system is in a high-throughput drug evaluation model mode;

[0023] The microphysiological system sensor chip comprehensively evaluates the efficacy and side effects of the microphysiological system on the drug through impedance changes or electrical signal emission changes.

[0024] Furthermore, there are flow connections for physiological communication between the 3D cells or organoids of each important organ or tissue in the microphysiological system;

[0025] Each important organ or tissue module in the microphysiological system is a 3D cell or organoid with corresponding physiological function;

[0026] Among them, 3D cells are cell spheres with three-dimensional structures produced by three-dimensional culture of cell lines of important organs, and organoids are 3D cells with the basic functions and structures of organs that are induced by pluripotent stem cells or cultured with cells extracted from patient tissues;

[0027] The microphysiological system sensor chip combines a variety of externally cultured 3D cells or organoids and the physiological communication sequence can be dynamically adjusted.

[0028] According to a second aspect of the present specification, a method for assembling a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring is provided, comprising the following steps:

[0029] The master mold is prepared by photolithography, and a 3D cell or organoid capture and positioning microfluidic chip is prepared by casting to construct a cell positioning and capture module;

[0030] A multi-parameter cell sensing detection module consisting of a base layer, a sensing layer, and an insulating layer is prepared by micro-nano processing.

[0031] Arrange the signal adapter module and the external circuits of the multi-parameter cell sensing detection module accordingly, wherein the entire arrangement includes external leads, pads, positioning holes, and a 3D cell and organoid image acquisition area, and process the signal adapter module with the completed arrangement using a standard printed circuit production method to obtain a completed signal adapter module;

[0032] The cell positioning capture module, the multi-parameter cell sensing detection module and the final signal switching module are arranged in sequence from top to bottom in space to obtain a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring.

[0033] Furthermore, the multi-parameter cell sensing detection module is connected and assembled with the signal adapter module through leads made of gold or other conductive materials;

[0034] Combining the cell positioning capture module with the assembled assembly consisting of the multi-parameter cell sensing detection module and the signal adapter module by bonding or clamping;

[0035] When the microphysiological system sensor chip is assembled in a fixture manner, the cell positioning capture module is detachably arranged in the microphysiological system sensor chip.

[0036] The beneficial effects of the present invention are as follows: the present invention can perform high-throughput capture and positioning of different types of 3D cells or organoids through a microfluidic capture chip (i.e., a 3D cell or organoid capture and positioning microfluidic chip), and perform separate dynamic culture. After completing the stable culture of different types of 3D cells or organoids, the parallel microfluidic capture chips can be connected in series through a steel needle hose to freely construct a microphysiological system based on a variety of different 3D cells or organoids, through which drugs can be comprehensively evaluated or customized medical evaluations can be performed; in addition, the chip can construct a high-throughput drug evaluation model based on patient-derived tumor organoids for early screening of anti-cancer drugs. While constructing a microphysiological system and a high-throughput drug evaluation model, the present invention can perform real-time electrical evaluation of the constructed model through cell impedance and cell potential, filling the gap in dynamic monitoring of the microphysiological system. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 A schematic diagram of the overall structure of a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring provided by an exemplary embodiment;

[0039] Figure 2 A schematic diagram of the hierarchical structure of a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring provided by an exemplary embodiment;

[0040] Figure 3 A schematic structural diagram of a cell location capture module provided by an exemplary embodiment;

[0041] Figure 4 A schematic diagram of a cell sensing area of a multi-parameter cell sensing detection module provided by an exemplary embodiment;

[0042] Figure 5 A schematic diagram of a multi-channel impedance detection area of a multi-parameter cell sensing detection module provided by an exemplary embodiment;

[0043] Figure 6 A schematic diagram of an impedance-potential multifunctional detection area of a multi-parameter cell sensing detection module provided by an exemplary embodiment;

[0044] Figure 7 A schematic structural diagram of a signal switching module provided by an exemplary embodiment;

[0045] Figure 8 A schematic diagram of a multi-parameter microphysiological system sensor chip providing an exemplary embodiment for completing 3D cell capture, positioning and monitoring. DETAILED DESCRIPTION

[0046] In order to better understand the technical solution of the present application, the embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0047] It should be clear that the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0048] like Figure 1 — Figure 2 As shown, a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring is provided. The microphysiological system sensor chip for real-time dynamic multi-parameter monitoring includes: a cell positioning capture module 1, a multi-parameter cell sensing detection module 2 and a signal switching module 3.

[0049] like Figure 3As shown, the interior of the cell positioning and capture module 1 includes multiple 3D cell or organoid capture and positioning microfluidic chips for capturing, positioning and dynamically culturing 3D cells or organoids; in one embodiment, the position of the microstructure array in the 3D cell or organoid capture and positioning microfluidic chip corresponds to the sensor in the multi-parameter cell sensing detection module 2. In one embodiment, the interior of the cell positioning and capture module 1 includes four 3D cell or organoid capture and positioning microfluidic chips. The 3D cell or organoid capture and positioning microfluidic chips adopt PDMS microfluidic capture and positioning microfluidic chips. The 3D cell or organoid capture and positioning microfluidic chips have a liquid inlet 4, a liquid outlet 7 and a chamber 5. The liquid inlet 4 has a diameter of 1 mm, the chamber 5 is a fluid arc shape, and the liquid outlet 7 has a diameter of 1 mm. A total of 32 3D cell and organoid microstructure arrays are included, wherein the narrowest part of the microstructure array is 0.1 mm and the widest part is 0.3 mm; each 3D cell or organoid capture and positioning microfluidic chip in the cell positioning and capture module 1 is combined and connected to construct a physiological communication flow path between different 3D cell or organoid arrays; in one embodiment, all 3D cell or organoid capture and positioning microfluidic chips are connected to the liquid inlet and liquid outlet between different 3D cell or organoid capture and positioning microfluidic chips through a steel needle hose.

[0050] Each 3D cell or organoid capture and positioning microfluidic chip includes a microstructure array for positioning and capturing corresponding 3D cells or organoids; the cell positioning and capture module 1 simultaneously realizes the capture and positioning of multiple 3D cells or organoids while maintaining the three-dimensional structure of the 3D cells or organoids and positioning the 3D cells or organoids on the microphysiological system sensor chip;

[0051] By passing culture medium containing 3D cells or organoids into a single PDMS microfluidic capture and positioning microfluidic chip, high-throughput capture and enrichment of a single 3D cell or organoid can be achieved, and then positioned to the electrode sensing area; by connecting the liquid circuits of four microfluidic capture and positioning microfluidic chips in series, a microphysiological system consisting of up to four different 3D cells or organoids can be realized;

[0052] like Figure 3 As shown, the multi-parameter cell sensing detection module 2 includes a variety of sensors for real-time monitoring of 3D cells or organoids; the multi-parameter cell sensing detection module 2 senses multiple parameters of multiple 3D cells or organoids in real time to provide multi-angle feedback on the growth status of 3D cells or organoids;

[0053] In one embodiment, the multi-parameter cell sensing detection module 2 has multiple cell sensing areas that correspond one-to-one to the 3D cell or organoid capture and positioning microfluidic chip in the cell positioning and capture module. The sensing areas of the multi-parameter cell sensing detection module include a multi-channel cell impedance sensor and a multi-channel microelectrode array. The cell impedance sensor is used for 3D cell or organoid activity and pulsation detection, and the microelectrode array is used for 3D cell or organoid electrical discharge detection.

[0054] The main body of the cell impedance sensor adopts an interdigitated electrode structure, in which the interdigitated electrodes include paired excitation electrodes and working electrodes. Each excitation electrode finger and working electrode finger includes multiple electrode regions connected in series. The gap between a pair of excitation and working fingers corresponds to the center of the microstructure in the 3D cell or organoid capture and positioning microfluidic chip.

[0055] The multi-channel microelectrode array includes a microelectrode array composed of multiple microelectrodes, which are arranged in the center of a microstructure in a 3D cell or organoid capture and positioning microfluidic chip, and each microstructure corresponds to multiple microelectrodes.

[0056] In one embodiment, the multi-parameter cell sensing detection module 2 has four sensing areas, each of which is a square with a side length of 2 cm. The total length of the chip is 8 cm and the width is 2 cm. It includes a No. 1 multi-channel cell impedance sensing area 8, a No. 2 multi-channel cell impedance sensing area 9, a cell impedance-potential multifunctional sensing area 10 and a No. 3 multi-channel cell impedance sensing area 11; Figure 5 As shown, a multi-channel cell impedance sensing area mainly uses interdigitated gold electrodes to perform real-time detection of cell impedance, wherein the diameter of a single circular gold electrode of the interdigitated electrode is 0.05 mm, the spacing between the interdigitated fingers is 0.03 mm, a single interdigitated finger contains a total of 100 circular gold electrodes, the excitation electrode array contains 7 gold electrodes, and the interdigitated electrode array is formed by alternating with 6 working electrodes. The multi-channel cell impedance sensing area has a total of 4 interdigitated electrode arrays for real-time detection of cell impedance, namely No. 1 impedance detection interdigitated electrode channel 12, No. 2 impedance detection interdigitated electrode channel 13, No. 3 impedance detection interdigitated electrode channel 14, and No. 4 impedance detection interdigitated electrode channel 15; the impedance detection interdigitated electrodes can be used to perform electrical real-time detection of the activity of 3D cells or organoids that have completed capture and positioning; as shown Figure 6 As shown, a multi-parameter cell sensing module impedance-potential multifunctional sensing area includes a No. 1 impedance detection interdigital electrode channel 16, a 4-channel cell potential reference electrode 17 (wherein the reference electrode is a quarter circle with a radius of 0.3 mm), an 18-channel cell potential detection electrode 18 (wherein the diameter of the potential detection microelectrode is 0.02 mm), an image observation area 19 and a No. 2 impedance detection interdigital electrode channel 20;

[0057] like Figure 7 As shown, the signal adapter module 3 internally includes an adapter circuit for leading out each cell sensor detection circuit in the multi-parameter cell sensing detection module 2 and an image acquisition area for observing cell status. The image acquisition area corresponds to the cell sensing area in the multi-parameter cell sensing detection module. In one embodiment, the signal adapter module includes pads, leads, and pin headers for externally connecting the sensor signals in the multi-parameter cell sensing detection module. The signal adapter module has an image acquisition area for observing changes in the growth status of 3D cells or organoids. The position of the image acquisition area of the signal adapter module corresponds to the cell sensing area in the multi-parameter cell sensing detection module. In one embodiment, the signal adapter module 3 includes four positioning holes 21, an adapter board lead external connection hole array 22 for externally connecting chip leads, and two image acquisition areas 23 for observing the growth status of 3D cells or organoids in the chip. The image acquisition area is 35 mm long and 10 mm wide.

[0058] The cell positioning capture module, the multi-parameter cell sensing detection module, and the signal switching module are arranged in order from top to bottom.

[0059] In one embodiment, the microphysiological system sensor chip and at least two of normal 3D cells or organoids and 3D cells or organoids of abnormal tissues derived from patients together constitute a microphysiological system;

[0060] Among them, when the microphysiological system is composed entirely of 3D cells or organoids of abnormal tissues from patients, the microphysiological system is in the high-throughput drug evaluation model mode;

[0061] The microphysiological system comprehensively evaluates the efficacy and side effects of drugs through changes in impedance or electrical signal emission.

[0062] In one embodiment, there are fluidic connections for physiological communication between 3D cells or organoids of various important organs or tissues in the microphysiological system;

[0063] Each important organ or tissue module in the microphysiological system is a 3D cell or organoid with corresponding physiological functions;

[0064] Among them, 3D cells are cell spheres with three-dimensional structures produced by three-dimensional culture of cell lines of important organs, and organoids are 3D cells with the basic functions and structures of organs that are induced by pluripotent stem cells or cultured with cells extracted from patient tissues;

[0065] The microphysiological system sensor chip combines multiple 3D cells or organoids cultured externally, and the physiological communication sequence can be dynamically adjusted.

[0066] like Figure 8As shown, 3D cells or organoids can be introduced into the sensor chip along with the liquid, and the microstructure array in the PDMS capture and positioning microfluidic chip in the cell positioning capture module 1 will position them in the sensing area, thereby achieving three-dimensional support and positioning sensing of 3D cells or organoids.

[0067] The present invention provides a method for assembling a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring, which includes an assembly method for each layer in the chip and an overall assembly method, specifically comprising the following steps:

[0068] The chip assembly method is as follows:

[0069] (1) Preparation of cell positioning capture module: First, clean a 4-inch silicon wafer with compressed air, then apply 2-3 ml of negative photoresist SU8-2150 on the center of the silicon wafer, and spin at a speed of 500 rpm for 15 seconds and 1800 rpm for 40 seconds to achieve a photoresist coating thickness of 300 μm, pre-bake on a hot plate at 65°C for 8 minutes, then transfer to a hot plate at 95°C for 80 minutes of pre-bake, then transfer the silicon wafer to the chuck of the UV exposure mask aligner, expose through the designed mask, and the total UV exposure energy is 430 mJ, then pre-bake at 65°C for 5 minutes and post-bake at 95°C for 23 minutes, directly immerse the silicon wafer in SU-8 developer for development until obvious microstructures appear, and dry the sample to complete the preparation of the photolithography mold for the PDMS microfluidic capture positioning layer;

[0070] Next, 44g of PDMS prepolymer was prepared at a 10:1 curing agent ratio and poured onto the photolithographic mold of the PDMS microfluidic capture and positioning layer. The PDMS and mold were placed in an oven and baked at 80°C for 1.5 hours to fully cure. After curing, the PDMS was peeled off from the mold, and a 0.75mm diameter punch was then used to punch holes in the peeled and cut PDMS microfluidic capture and positioning microfluidic chip for subsequent chip assembly.

[0071] (2) Preparation of multi-parameter cell sensing detection module: Use Auto-CAD to complete the design of the sensor electrode arrangement and the insulation layer of the multi-parameter cell sensing detection module; process the 7-inch multi-parameter cell sensing detection module sensing layer and the insulation layer micro-nano processing chromium mask; use positive photoresist to perform photolithography development through the sensing layer mask, leaving the sensing layer pattern groove; use evaporation deposition to deposit a 10nm Cr layer and a 200nm gold layer on the sample surface respectively; use ultrasonic lift-off to peel off the excess metal layer; use PECVD plasma enhanced chemical vapor deposition to deposit a 200nm silicon oxide layer as the insulation layer; use negative photoresist to spin-coat and solidify and use the insulation layer mask to photolithography development to expose the cell contact area; use RIE reactive ion etching to remove the silicon oxide insulation layer in the sensing area; and complete the basic preparation of the multi-parameter cell sensing detection module by regional slicing.

[0072] (3) Preparation of signal transfer module: Using Altium Designer, the external circuits of the multi-parameter cell sensing detection module are arranged, and the 3D cell and organoid observation window and positioning hole design are designed. The original signal transfer module and the external circuits of the multi-parameter cell sensing detection module are arranged and configured accordingly. The entire arrangement configuration includes external leads, pads, positioning holes and 3D cell and organoid image acquisition areas. The completed signal transfer module design drawing is processed through standard printed circuit production methods to obtain the final signal transfer module.

[0073] (4) The overall assembly method is as follows:

[0074] The prepared multi-parameter cell sensing detection module is bonded to the signal adapter module through epoxy resin glue, wherein the sensing area in the multi-parameter cell sensing detection module needs to be aligned with the image detection area of the signal adapter module to facilitate subsequent observation of the growth status of 3D cells or organoids located in the sensing area; the sensor external pads in the multi-parameter cell sensing detection module are connected to the corresponding adapter pads in the signal adapter module through gold wires or other conductive material leads; the combined multi-parameter cell sensing detection module and the PDMS microfluidic capture and positioning microfluidic chip in the cell positioning and capture module are irreversibly bonded through oxygen plasma or the PDMS microfluidic capture and positioning chip and the multi-parameter cell sensing detection module are detachably assembled through a PMMA fixture that has been processed with corresponding arrangement; the signal adapter module welds components and external pin headers; the PDMS microfluidic capture and positioning microfluidic chip in the cell positioning and capture module is connected through steel needles and hoses to complete the overall flow path construction, wherein the cell positioning and capture module, the multi-parameter cell sensing detection module, and the signal adapter module are arranged in sequence from top to bottom.

[0075] In this example, normal intestinal, liver, and heart organoids derived from pluripotent stem cells cultured in multi-well plates, and abnormal intestinal, gastric, or lung cancer organoids derived from patients, were separately introduced into four PDMS microfluidic capture and positioning microfluidic chips in the sensing platform's cell positioning and capture modules. The cells were perfused and cultured at a flow rate of 1 μL / min using a syringe pump. During this period, the cell activity of the four different organoids was monitored in real time using the chip's multi-channel interdigitated electrodes. Furthermore, the chip's multi-channel microelectrode array was used to monitor the electrical discharge of 3D cells or organoids, such as 3D cardiomyocytes, olfactory organoids, and brain organoids, in real time. After three days of perfusion culture, the four PDMS microfluidic capture and positioning microfluidic chips were interconnected by flexible tubes to establish physiological communication pathways between the different organoids, thereby constructing a microphysiological system of intestinal, liver, heart, and intestinal cancer organoids. After the microphysiological system was constructed, different first-line anti-intestinal cancer chemotherapy drugs or targeted drugs were introduced into the chip, and changes in cell activity and electrical discharge of the four different organoids were simultaneously monitored. The combined side effects and actual drug efficacy of the drugs were analyzed.

[0076] In addition to building a microphysiological system for comprehensive drug evaluation, the present invention can also culture the same cancer 3D cells or organoids in the four PDMS microfluidic capture and positioning microfluidic chips in the cell positioning capture module to construct a high-throughput drug evaluation model, and reflect the drug's killing effect on cancer cells in real time through cell impedance detection; in addition, the constructed microphysiological system can freely select four different organoids and use a circulation pump to build a multi-organoid model for cutting-edge exploratory scientific research.

[0077] The present invention can use microfluidic capture chips to perform high-throughput capture and positioning of different types of 3D cells or organoids, and perform separate dynamic culture. After completing the stable culture of different types of 3D cells or organoids, the parallel microfluidic capture chips can be connected in series through a steel needle hose to freely construct a microphysiological system based on a variety of different 3D cells or organoids. The microphysiological system can be used to comprehensively evaluate drugs or perform customized medical evaluations. In addition, the chip can construct a high-throughput drug evaluation model based on patient-derived tumor organoids to perform early screening of anti-cancer drugs. While constructing a microphysiological system and a high-throughput drug evaluation model, the present invention can perform real-time electrical evaluation of the constructed model through cell impedance and cell potential, filling the gap in dynamic monitoring of the microphysiological system.

[0078] The above are only preferred embodiments of one or more embodiments of this specification and are not intended to limit one or more embodiments of this specification. Any modifications made within the spirit and principles of one or more embodiments of this specification are not intended to limit this specification.

Claims

1. A microphysiological system sensor chip for real-time dynamic multi-parameter monitoring, characterized in that: include: The cell positioning and capture module includes multiple 3D cell or organoid capture and positioning microfluidic chips, each of which has a liquid inlet, a liquid outlet, and a chamber. Each microfluidic chip includes a three-dimensional microstructure array for positioning and capturing corresponding 3D cells or organoids. The narrowest part of the microstructure in the microstructure array is 0.1mm, and the widest part is 0.3mm. The parallel microfluidic chips for dynamic culture are connected in series through a steel needle hose to construct a microphysiological system based on multiple different 3D cells or organoids. A multi-parameter cell sensing detection module has multiple sensing areas that correspond one-to-one to the 3D cell or organoid capture and positioning microfluidic chip in the cell positioning and capture module. Each sensing area is composed of one or more sensors arranged for real-time monitoring of 3D cells or organoids. The multiple sensing areas include a multi-channel cell impedance sensing area and a cell impedance-potential multifunctional sensing area; the multi-channel cell impedance sensing area has a total of 4 interdigital electrode arrays for real-time detection of cell impedance; the cell impedance-potential multifunctional sensing area includes a No. 1 impedance detection interdigital electrode channel, a 4-channel cell potential reference electrode, an 18-channel cell potential detection electrode, and a No. 2 impedance detection interdigital electrode channel; the 18-channel cell potential detection electrode includes a microelectrode array composed of multiple microelectrodes, and the microelectrodes are arranged in the center of the microstructure in the 3D cell or organoid capture and positioning microfluidic chip; A signal transfer module, which includes a transfer circuit, solder pads, and pin headers for leading out the sensor detection circuits in the multi-parameter cell sensing detection module, and an image acquisition area for observing cell status; The cell positioning capture module, the multi-parameter cell sensing detection module, and the signal switching module are arranged in sequence from top to bottom in space.

2. The microphysiological system sensor chip for real-time dynamic multi-parameter monitoring according to claim 1, characterized in that: The diameters of the liquid inlet and the liquid outlet are both 1 mm, and the chamber is in the shape of a fluid arc.

3. The microphysiological system sensor chip for real-time dynamic multi-parameter monitoring according to claim 1, characterized in that: The image acquisition area is 35 mm long and 10 mm wide, corresponding to the sensing area of the multi-parameter cell sensing detection module.

4. The microphysiological system sensor chip for real-time dynamic multi-parameter monitoring according to claim 1 is used for high-throughput drug evaluation, characterized in that: A microphysiological system is composed of 3D cells or organoids of abnormal tissues derived from patients. The microphysiological system sensor chip comprehensively evaluates the efficacy and side effects of the microphysiological system on drugs through impedance changes or changes in electrical signal emission.

5. The method for assembling a microphysiological system sensor chip for real-time dynamic multi-parameter monitoring according to any one of claims 1 to 3, characterized in that: The following steps are involved: The master mold is prepared by photolithography, and a 3D cell or organoid capture and positioning microfluidic chip is prepared by casting to construct a cell positioning and capture module; A multi-parameter cell sensing detection module consisting of a base layer, a sensing layer, and an insulating layer is prepared by micro-nano processing. Specifically, a sensing electrode mask in the sensing layer is prepared by photolithography development using a positive photoresist; the sensing electrode is prepared by evaporation deposition; the positive photoresist sensing electrode mask is stripped by ultrasound; the insulating layer is prepared by PECVD plasma enhanced chemical vapor deposition; a patterned insulating layer mask is prepared by negative photoresist; and a patterned insulating layer is prepared by RIE reactive ion etching. Arrange and configure the signal adapter module and the external circuit of the multi-parameter cell sensing detection module accordingly, including external leads, pads, positioning holes and image acquisition areas; process the signal adapter module with the completed arrangement and configuration using a standard printed circuit production method to obtain a completed signal adapter module; The multi-parameter cell sensing detection module is connected and assembled with the signal adapter module through external leads made of gold or other conductive materials; The cell positioning capture module is combined with the assembled combination of the multi-parameter cell sensing detection module and the signal adapter module by means of oxygen plasma irreversible bonding or detachable PMMA clamp fixation.

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