Organ chip capable of realizing parallel precise time-sequence administration and application of organ chip

Abstract

The invention belongs to the technical field of organ chips, and discloses an organ chip capable of realizing parallel precise time-sequence drug delivery and application of the organ chip. The organ chip comprises an organ chip main body, a perfusion culture main channel is arranged in the organ chip main body, a plurality of medicine preloading cavities are arranged beside the organ chip main body, and the organ chip main body and the medicine preloading cavities are communicated through a micro-fence structure; and the medicine preloading chamber is provided with micropores and is sealed by a manual or automatic sealing element. Based on the principle of airlock effect, through the synergistic effect of the micro-fence structure, the sealing piece and the negative pressure environment, stable pre-storage and controllable release of the medicine are achieved. The application method comprises the steps of drug preloading, cell culture and drug administration triggering in sequence. The airlock can be removed by sequentially removing or simultaneously removing the sealing element, so that the medicine is accurately guided into the main channel in a controllable laminar flow state, and parallel and high-flux medicine testing is realized. According to the scheme, a passive flow control mechanism is used for replacing complex external equipment, and the system has the remarkable advantages of being simple in structure, convenient to operate, accurate in control and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of organ-on-a-chip and microfluidics technology, and particularly relates to an integrated organ-on-a-chip based on the airlock effect and passive flow control principle, which enables parallel and precise time-sequential drug delivery and its applications. Background Technology

[0002] Organ-on-a-chip technology represents a significant advancement in biomedical engineering. By constructing a three-dimensional cell culture microenvironment on a microfluidic chip, it simulates the tissue structure and physiological functions of human organs, providing a novel in vitro research platform for drug development, toxicity testing, and disease modeling. Compared to traditional in vitro cell cultures and animal models, organ-on-a-chip can more accurately simulate the human body's response to drugs, significantly improving the reliability of predictions, thus demonstrating broad application potential in pharmaceuticals and toxicology.

[0003] When using organ-on-a-chip for pharmacological or toxicological evaluation, it is usually necessary to add the test drug into the chip at specific time points to simulate complex drug administration scenarios where the human body is exposed to different drugs or drug concentrations at different times. Currently, most existing in-chip drug delivery methods rely on complex external fluid control systems (such as multi-channel injection pumps and valve arrays) for drug switching. This method is not only expensive and complex to build, but also makes it difficult to avoid the problems of dead volume and cross-contamination of drugs in the tubing, thus affecting the accuracy of the administered concentration. In addition, this type of externally driven method is difficult to achieve parallel triggering of multiple independent drug delivery events, which cannot meet the efficiency requirements of high-throughput screening. Although some integrated chip designs attempt to store drugs inside the chip, they usually lack flexible, simple and reliable triggering mechanisms, making it difficult to conveniently start drug delivery at preset time points during the experiment, which limits their application in complex drug delivery protocols. Furthermore, the current drug delivery method of preloading drugs into the chip either introduces too many air bubbles if the drug is added too quickly, or the drug delivery is too slow to achieve effective drug loading, affecting the drug delivery effect. Therefore, developing an integrated passive drug delivery device that is simple in structure, low in cost, can flexibly and accurately control the timing of drug delivery, and supports high-throughput parallel testing has become a key issue in promoting the further development of organ-on-a-chip technology. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an organ-on-a-chip capable of parallel and precise time-sequential drug delivery and its applications. This chip, through its unique internal structure design, eliminates the need for complex external active flow control systems, thus solving problems such as inflexible timing control, difficulties in high-throughput parallel testing, susceptibility to bubble introduction, and high equipment costs.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: An organ-on-a-chip capable of parallel and precise time-sequential drug delivery includes: an organ-on-a-chip body having a main perfusion culture channel for culturing cells inside, multiple drug preloading chambers located beside the main perfusion culture channel, the main perfusion culture channel and the drug preloading chambers being connected by a microgrid structure, micropores being formed on the drug preloading chambers, and a sealing element for sealing the micropores, wherein the sealing element is a manually removable sealing element or an automatically removable sealing element.

[0006] This invention ingeniously utilizes and enhances the principle of "airlock effect." Through the synergistic effect of the specific fluid resistance generated by the micro-fence structure, the reliable seal formed by the sealing element, and the pre-set negative pressure environment of the system, a stable gas-liquid interface and pressure balance are constructed between the drug preloading chamber and the main perfusion culture channel. In the sealed state, the system pressure difference is below this equilibrium value, and the drug is completely locked in. When the sealing element is removed, the chamber pressure changes instantaneously, and the system pressure difference exceeds the equilibrium value, thereby driving the drug solution to enter the main channel through the micro-fence structure at a controllable and predictable flow rate, achieving precise loading while effectively preventing the introduction of air bubbles.

[0007] In the aforementioned organ-on-a-chip capable of parallel and precise timing-based drug delivery, the manually removed seal is a PE film, special tape, or PDMS film, and the automatically removed seal is a temperature-sensitive or photosensitive hydrogel seal.

[0008] Furthermore, the length, width, and height of the main perfusion culture channel are 10-50 mm, 1-5 mm, and 0.1-0.5 mm, respectively; the length, width, and height of the drug preloading chamber are 1-10 mm, 1-10 mm, and 0.1-1 mm, respectively, with a volume of 0.1-100 μL; and the diameter of the micropores is 0.1-2 mm.

[0009] Furthermore, the microfence structure includes multiple rectangular microchannels with lengths of 0.1–5 mm, widths of 100–500 μm, and heights of 10–100 μm. This microfence structure is not only a connecting channel but also a multifunctional integrated unit. On one hand, it serves as a physical cell barrier; its precisely designed dimensions allow for the free diffusion of nutrients, cytokines, and drug molecules while effectively blocking cell migration within the main channel, preventing cross-contamination. On the other hand, it acts as a drug diffusion rate controller; by precisely designing the channel cross-sectional dimensions of the microfence, it can actively regulate the diffusion rate and loading time of drugs entering the main channel from the pre-loading chamber, thereby simulating the release kinetics of different drugs.

[0010] Furthermore, the main perfusion culture channel is externally connected to a negative pressure device through the negative pressure channel inlet and negative pressure channel outlet.

[0011] Furthermore, the organ-on-a-chip comprises, from top to bottom, an upper organ-on-a-chip body, a middle layer chip, and a lower layer chip; the middle layer chip and the lower layer chip are respectively provided with a middle layer cell culture chamber and a lower layer cell culture chamber, the shapes of the middle layer cell culture chamber and the lower layer cell culture chamber are consistent with the shape of the perfusion culture main channel and their positions correspond one-to-one; porous membranes are respectively provided on the upper and lower sides of the middle layer cell culture chamber, and the porous membranes divide the perfusion culture main channel, the middle layer cell culture chamber and the lower layer cell culture chamber into upper, middle and lower chambers.

[0012] Based on a general inventive concept, the present invention also provides an application of the organ-on-a-chip as described above in parallel and precise timing-based drug delivery for non-therapeutic purposes, comprising the following steps: (1) The drug solution is injected into the corresponding drug preloading chamber through the micropores respectively, and the micropores are sealed with the sealing element; (2) Inoculate cells in the main channel of the perfusion culture and perform perfusion culture; (3) At the predetermined drug administration time, all the seals on the micropores are removed manually or automatically or simultaneously, so that the drug solution in the drug preloading chamber can diffuse through the micro-fence structure into the perfusion culture main channel, thereby achieving parallel and precise timed drug administration.

[0013] In the above-described application, the cells inoculated in the main perfusion culture channel include at least one of the following cell types: hepatocytes, kidney cells, intestinal cells, nerve cells, and vascular endothelial cells.

[0014] Furthermore, during the perfusion culture, the negative pressure inside the chip is controlled to be -100 ~ -1 Pa by a negative pressure device.

[0015] Furthermore, the dimensions of the micro-grid structure, the negative pressure range, and the diameter parameters of the micro-holes opened on the preload chamber need to be set in combination to achieve the "airlock effect," while ensuring that the effective drug loading time is 3-15 minutes when the sealing device is removed for drug loading, and without introducing air bubbles into the chip.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The organ-on-a-chip of the present invention solves the problems of inflexible drug delivery timing control and difficulty in high-throughput parallel testing by integrating the internal design of the chip, such as the micro-fence structure and the micropores on the drug pre-loading chamber. Without increasing the complexity of external equipment, only the seals on the micropores need to be removed to achieve parallel loading and independent controlled release of different drugs or drugs of different concentrations. The overall solution has the significant advantages of simple structure, low cost, convenient operation and precise control. The highly integrated design also eliminates the need for expensive and complex external pump and valve control systems, which greatly reduces equipment costs and the threshold for use.

[0017] 2. The organ-on-a-chip of the present invention can be configured with multiple drug pre-loaded chambers, each of which is an independent unit, supporting the inoculation of multiple drugs or different concentrations of the same drug. By removing the seals on one or multiple micropores individually or simultaneously, parallel and controllable drug delivery can be achieved, and cross-contamination can be effectively avoided during drug delivery, meeting the requirements of high-throughput testing. The organ-on-a-chip has strong compatibility and is suitable for various single-organ-on-a-chip or multi-organ-on-a-chip applications. It can be applied to multiple non-diagnostic and therapeutic scenarios such as drug development, toxicity testing, and pharmacokinetic studies, providing an efficient tool for experimental research in related fields.

[0018] 3. The method of using the organ-on-a-chip of the present invention involves preloading drugs into a drug preloading chamber, combining a micro-fence structure and a closed design of micropores on the drug preloading chamber, and then using negative pressure treatment for perfusion culture to construct a stable "airlock effect" mechanism, thereby achieving precise time-sequential drug delivery. During use, the seals on the micropores are removed sequentially or simultaneously according to experimental requirements, allowing for the sequential introduction of different drugs or different concentrations of the same drug, thereby enabling parallel high-throughput drug testing or toxicity testing. The entire operation process is simple and convenient, requiring no complex external equipment, and is an integrated and efficient experimental solution. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Figure A is a schematic diagram of the organ-on-a-chip in Example 1; wherein, Figure A is a three-dimensional perspective view of the organ-on-a-chip structure and Figure B is a top view of the organ-on-a-chip structure. Figure 2 The dimensions of the organ-on-a-chip in Example 1 are as follows: Specifically, the main perfusion culture channel in the center of the chip body has dimensions of 37 mm (length) × 2 mm (width) × 0.2 mm (height); the drug preloading chamber has dimensions of 3.2 mm (length) × 3 mm (width) × 0.2 mm (height) and a volume of 1.92 µL, with a micropore of 1 mm in diameter at its top; the microgrid structure consists of 8 parallel square microchannels, each square microchannel having dimensions of 1 mm (length) × 0.2 mm (width) × 0.02 mm (height), and the effective drug delivery time under these dimensions is approximately 5 minutes. Figure 3Figure A shows the drug delivery effect analysis of the organ-on-a-chip after manual removal of the seal in Example 1; Figure B shows the results of the simulation calculation of fluid dynamics inside the chip using Comsol Multiphysics; Figure B shows the measured drug delivery flow analysis results before and after removal of the seal. Figure 4 This illustrates the relationship between organ-on-a-chip drug delivery time and negative pressure value in Example 1. Figure 5 The results of the organ-on-a-chip evaluation of the toxicity of cisplatin and doxorubicin combination therapy in Example 1 are shown. Figure A shows the live / dead cell fluorescence staining after treatment with 50 µM cisplatin and 1 mM doxorubicin in the HK-2 kidney-on-a-chip model, with a scale bar of 100 μm. Figure B shows the quantitative analysis of cell viability and mortality in Figure A before and after drug administration. Figure 6 This is a schematic diagram of the organ-on-a-chip in Example 2; Figure 7 The results of the drug delivery effect analysis of the organ-on-a-chip in Example 2 after manual removal of the seal are shown in Figure 2. Figure A shows the results of the fluid dynamics simulation of the micro-fence channel (AA' section) using Comsol Multiphysics after manual removal of the seal; Figure B shows the measured drug delivery flow analysis before and after the seal removal. Figure 8 The results of ALT secretion with increasing dose of amiodarone, as measured by organ-on-a-chip in Example 2; Figure 9 This is a schematic diagram of the official chip in Example 5.

[0021] Legend: 1. Organ-on-a-chip main body; 2. Intermediate layer chip; 3. Lower layer chip; 4. Porous membrane; 1-1. Main channel for perfusion culture; 1-2. Drug preloading chamber; 1-3. Micro-fence structure; 1-4. Micropores; 1-5. Sealing element; 1-6. Inlet of negative pressure channel; 1-7. Outlet of negative pressure channel; 2-1. Intermediate layer cell culture chamber; 3-1. Lower layer cell culture chamber. Detailed Implementation

[0022] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0023] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0025] PE film: polyethylene film; PDMS film: polydimethylsiloxane elastomer film; PC porous film: polycarbonate porous film.

[0026] Example 1: A kidney chip capable of parallel and precise timing-based drug delivery (e.g.) Figure 1 As shown), for evaluating drug nephrotoxicity, it includes: an organ-on-a-chip body 1, with a perfusion culture main channel 1-1 in the center with dimensions of 37×2×0.2 mm (length×width×height); on each side of the perfusion culture main channel 1-1, there is a drug preloading chamber 1-2 with dimensions of 3.2×3×0.2 mm (length×width×height) and a volume of 1.92 µL; each drug preloading chamber 1-2 has a micropore 1-4 with a diameter of 1 mm in the center of its top, and a PE film is attached above the micropore 1-4 as a manual seal 1-5 to keep it sealed; the perfusion culture main channel 1-1 and the drug preloading chamber 1-2 are connected by a micro-fence structure 1-3, which includes a row (a total of 8) of square microchannels with dimensions of 1×0.2×0.02 mm (length×width×height). Figure 2 ).

[0027] The method for using this liver microarray for nephrotoxicity evaluation includes the following steps: (1) The drug solution (50 µM cisplatin and 1 mM doxorubicin solution) is injected into the two corresponding drug preloading chambers 1-2 through micropores 1-4 respectively, and the micropores 1-4 are sealed with sealing element 1-5; (2) Cells (human renal tubular epithelial cells HK-2) were seeded in the main perfusion culture channel 1-1. A negative pressure of -10 Pa was introduced through the negative pressure channel inlet 1-6 and negative pressure channel outlet 1-7 of the main perfusion culture channel 1-1 to form a flow culture environment, and perfusion culture was carried out for 48 h. (3) At the same time, manually remove the seals 1-5 on all micropores 1-4, so that the drug solution in the drug preloading chamber 1-2 can diffuse through the micro-barrier structure 1-3 into the perfusion culture main channel 1-1 and time it for 4 min. Then, attach the seals 1-5 to the top of the micropores 1-4 to keep them sealed and continue culturing for 12 h. Introduce live / dead dye (calcein / pyridine iodide, AM / PI) into the perfusion culture main channel 1-1 for live / dead staining. Evaluate the nephrotoxicity of the combination of cisplatin and doxorubicin based on the survival of HK-2 cells.

[0028] Using Comsol Multiphysics to calculate and experiment with the fluid dynamics within the chip, it was found that after manually removing the seals 1-5, the flow field distribution was uniform and in a laminar state; the flow velocity in the drug preloading chamber 1-2 was low, and the flow velocity in the micro-fence structure 1-3 and the perfusion culture main channel 1-1 region was also low, and the overall flow conditions did not introduce air bubbles. Figure 3 (Figure A); Before manually removing the seals 1-5, the flow rate was almost zero, but after manually removing the seals 1-5, the flow rate of the drug entering the main perfusion culture channel 1-1 was approximately 0.4 μL / min. Figure 3 (Figure B).

[0029] Depend on Figure 4 It is evident that when the negative pressure value does not match the chip's geometric parameters, the drug delivery time changes significantly, greatly increasing the risk of air introduction. At a negative pressure of -1 Pa, manually removing seals 1-5 resulted in a flow rate of approximately 0.04 μL / min, increasing the delivery time from approximately 5 min to approximately 50 min; this slow delivery affects the drug delivery effect. At a negative pressure of -100 Pa, manually removing seals 1-5 resulted in a flow rate of approximately 4 μL / min, reducing the delivery time from approximately 5 min to approximately 30 s, significantly increasing the risk of air bubble introduction. Therefore, the drug solution loading time can be controlled to around 5 min, ensuring effective drug loading while avoiding excessively rapid loading that could introduce air bubbles.

[0030] By combining the 4-minute manual removal time of seals 1-5, this embodiment achieved precise loading of 1.6 μL cisplatin + 1.6 μL doxorubicin. Furthermore, by analyzing live and dead cell staining, the toxicity of 50 µM cisplatin combined with 1 mM doxorubicin in the HK-2 kidney microarray model could be determined, showing that the HK-2 cell survival rate decreased to approximately 70% after drug treatment. Figure 5 ).

[0031] Example 2: A liver-on-a-chip capable of parallel and precise timing-based drug delivery (e.g.) Figure 6 As shown), for high-throughput drug concentration-hepatotoxicity studies, it includes: an organ-on-a-chip body 1, with a perfusion culture main channel 1-1 in the center with a length × width × height of 37 × 2 × 0.2 mm. On each side of the perfusion culture main channel 1-1, there are 5 drug preloading chambers 1-2 with a length × width × height of 3.2 × 3 × 0.2 mm and a volume of 1.92 µL. Each drug preloading chamber 1-2 has a microhole 1-4 with a diameter of 1 mm in the center of its top. A PE film is used as a manual seal 1-5 and is attached to the microhole 1-4 to keep it sealed. The perfusion culture main channel 1-1 and the drug preloading chambers 1-2 are connected by a micro-fence structure 1-3, which includes a row (a total of 8) of square microchannels with a length × width × height of 1 × 0.2 × 0.02 mm.

[0032] The application of this liver microarray in high-throughput drug concentration-hepatotoxicity studies includes the following steps: (1) The drug solution (amiodarone solution with concentration gradients of 0 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1 mM, 5 mM, 10 mM, 50 mM and 100 mM) is injected into the corresponding two drug preloading chambers 1-2 through micropores 1-4 respectively, and the micropores 1-4 are sealed with sealing element 1-5; (2) Cells (human primary hepatocytes, HPHs) were seeded in the main perfusion culture channel 1-1. A negative pressure of -10 Pa was introduced through the negative pressure channel inlet 1-6 and negative pressure channel outlet 1-7 of the main perfusion culture channel 1-1 to form a flow culture environment, and perfusion culture was carried out for 48 h. (3) Every 6 h, manually remove the seals 1-5 on the micropores 1-4 in order of increasing concentration, so that the drug solution in the drug preloading chamber 1-2 can diffuse through the micro-fence structure 1-3 into the perfusion culture main channel 1-1 and time for 3 min; collect the effluent from the chip during the period, detect the ALT content in the effluent, and evaluate the dose-related hepatotoxicity characteristics of the drug amiodarone.

[0033] The fluid dynamics within the chip before and after the manual removal of seals 1-5 were calculated and experimentally analyzed using Comsol Multiphysics. Figure 7 In the study, it was found that before manually removing seals 1-5, no drug was released from the chip, and the flow rate of the drug into the main channel was almost zero. However, after manually removing seals 1-5, the drug diffused into the main channel 1-1 of the perfusion culture in a laminar flow manner, with a flow rate of approximately 0.6 μL / min. The overall flow state did not introduce air bubbles, and the drug administration time could be controlled to be more than 3 minutes. Combined with the analysis of ALT content in the effluent, the dose-toxicity relationship of amiodarone in the human primary hepatocyte chip model was obtained. That is, as the concentration of amiodarone increases, the secretion of the hepatotoxic indicator ALT increases accordingly, and the maximum hepatotoxic effect tends to be reached at the concentration of amiodarone at 10 mM. Figure 8 ).

[0034] Example 3: An intestinal microarray capable of parallel and precise time-sequential drug delivery for studying drug-drug interactions in intestinal cells comprises: an organ-on-a-chip body 1, with a central perfusion culture main channel 1-1 measuring 25 × 1.5 × 0.2 mm in length × width × height; a drug preloading chamber 1-2 measuring 5 × 2 × 0.2 mm in length × width × height and with a volume of 2.0 µL on each side of the perfusion culture main channel 1-1; a micropore 1-4 with a diameter of 0.2 mm at the top center of each drug preloading chamber 1-2, with a PE film as a manual seal 1-5 attached above the micropore 1-4 to maintain a seal; the perfusion culture main channel 1-1 and the drug preloading chambers 1-2 are connected by a micro-fence structure 1-3, which includes a row (a total of 8) of square microchannels measuring 0.5 × 0.2 × 0.01 mm in length × width × height.

[0035] The application of this intestinal microarray in the study of drug-drug interactions in intestinal cells includes the following steps: (1) The drug solution was injected into the corresponding two drug preloading chambers 1-2 through micropores 1-4 respectively (20 µM P-glycoprotein inducer rifampin was preloaded into the left drug preloading chamber 1-2, and 1 mMP-glycoprotein substrate drug digoxin was preloaded into the right drug preloading chamber 1-2), and micropores 1-4 were sealed with seals 1-5. (2) Cells (Caco-2 cells) were seeded in the main channel 1-1 of the perfusion culture to form a monolayer barrier. The negative pressure at -20 Pa was introduced through the negative pressure channel inlet 1-6 and negative pressure channel outlet 1-7 of the main channel 1-1 of the perfusion culture to form a flow culture environment, and perfusion culture was carried out for 24 h. (3) Manually remove the seal 1-5 of the left drug preloading chamber 1-2, so that the drug solution in the left drug preloading chamber 1-2 can diffuse through the micro-fence structure 1-3 into the perfusion culture main channel 1-1 and time it for 12 h; then manually remove the seal 1-5 of the right drug preloading chamber 1-2, so that the drug solution in the right drug preloading chamber 1-2 can diffuse through the micro-fence structure 1-3 into the perfusion culture main channel 1-1 and time it for 12 h; during this period, collect the effluent and detect the digoxin concentration in the effluent to analyze the drug interaction relationship.

[0036] Example 4: A neural chip capable of parallel and precise time-sequential drug delivery for drug neurotoxicity evaluation studies includes: an organ-on-a-chip body 1, with a central perfusion culture main channel 1-1 measuring 20×2.0×0.3 mm in length × width × height; a drug preloading chamber 1-2 measuring 8×5×0.5 mm in length × width × height and with a volume of 20 µL on each side of the perfusion culture main channel 1-1; a micropore 1-4 with a diameter of 1.0 mm at the top center of each drug preloading chamber 1-2, with a photosensitive gel phospholipid-polyethylene glycol-acrylate o-nitrobenzyl (DSPE-PEG-oNb) added above the micropore 1-4 to maintain a seal; the perfusion culture main channel 1-1 and the drug preloading chambers 1-2 are connected by a micro-fence structure 1-3, which includes a row (a total of 8) of square microchannels measuring 1.0×0.2×0.02 mm in length × width × height.

[0037] The application of this intestinal microarray in the study of drug-drug interactions in intestinal cells includes the following steps: (1) The drug solution (50 µM glutamic acid) was injected into the corresponding two drug preloading chambers 1-2 through micropores 1-4 respectively, and the micropores 1-4 were sealed with automatic sealing device 1-5; (2) Cells (SH-SY5Y cells) were seeded in the main channel 1-1 of the perfusion culture to form a monolayer barrier. The negative pressure at -20 Pa was introduced through the negative pressure channel inlet 1-6 and negative pressure channel outlet 1-7 of the main channel 1-1 of the perfusion culture to form a flow culture environment, and perfusion culture was carried out for 12 h. (3) The automatic sealing element 1-5 of the drug preloading chamber 1-2 was irradiated with 365-405 nm light to allow the glutamate solution in the drug preloading chamber 1-2 to diffuse through the micro-fence structure 1-3 into the perfusion culture main channel 1-1 and the time was 12 h. Then, the SH-SY5Y cells in the chip were stained with Hoechst / PI to evaluate the neurotoxicity of 50 µM glutamate.

[0038] Example 5: A biomimetic liver sinusoidal chip capable of parallel and precise timing-based drug delivery (such as...) Figure 9 As shown in the figure, it is used for drug hepatotoxicity evaluation and consists of an upper organ-on-a-chip body 1, a middle chip 2 and a lower chip 3 from top to bottom; The organ-on-a-chip body 1 has a perfusion culture main channel 1-1 with a length × width × height of 37 × 2 × 0.2 mm in the center. On each side of the perfusion culture main channel 1-1, there are 5 drug preloading chambers 1-2 with a length × width × height of 3.2 × 3 × 0.2 mm and a volume of 1.92 µL. Each drug preloading chamber 1-2 has a microhole 1-4 with a diameter of 1 mm in the center of its top. A PE film is used as a manual seal 1-5 and is attached to the microhole 1-4 to keep it sealed. The perfusion culture main channel 1-1 and the drug preloading chambers 1-2 are connected by a micro-fence structure 1-3. The micro-fence structure 1-3 includes a row (a total of 8) of square microchannels with a length × width × height of 1 × 0.2 × 0.02 mm. The intermediate layer chip 2 and the lower layer chip 3 are respectively provided with an intermediate layer cell culture chamber 2-1 and a lower layer cell culture chamber 3-1. The shapes of the intermediate layer cell culture chamber 2-1 and the lower layer cell culture chamber 3-1 are consistent with the shape of the perfusion culture main channel 1-1 and their positions correspond one-to-one. Porous membranes 4 (using PC porous membranes with pore sizes of 1-10 µm) are provided on the upper and lower sides of the intermediate layer cell culture chamber 2-1. The porous membranes 4 separate the perfusion culture main channel 1-1, the intermediate layer cell culture chamber 2-1 and the lower layer cell culture chamber 3-1 to form upper, middle and lower chambers.

Claims

1. An organ-on-a-chip capable of parallel and precisely timed drug delivery, characterized in that, include: The organ-on-a-chip body (1) has a perfusion culture main channel (1-1) inside. Multiple drug preloading chambers (1-2) are provided on the side of the perfusion culture main channel (1-1). The perfusion culture main channel (1-1) and the drug preloading chambers (1-2) are connected by a micro-grid structure (1-3). The drug preloading chambers (1-2) are provided with micropores (1-4) and sealing elements (1-5) for sealing the micropores (1-4). The sealing elements (1-5) can be manually removed or automatically removed.

2. The organ-on-a-chip with parallel and precise timing-based drug delivery according to claim 1, characterized in that, The manually removed seal is a PE film, special tape, or PDMS film, while the automatically removed seal is a temperature-sensitive or photosensitive hydrogel seal.

3. The organ-on-a-chip with parallel and precise timing-based drug delivery according to claim 1, characterized in that, The length, width, and height of the main perfusion culture channel (1-1) are 10-50 mm, 1-5 mm, and 0.1-0.5 mm, respectively; the length, width, and height of the drug preloading chamber (1-2) are 1-10 mm, 1-10 mm, and 0.1-1 mm, respectively, and the volume is 0.1-100 μL; the diameter of the micropores (1-4) is 0.1-2 mm.

4. The organ-on-a-chip capable of parallel and precise timing-based drug delivery according to claim 1, characterized in that, The microfence structure (1-3) includes multiple rectangular microchannels with lengths of 0.1~5 mm, widths of 100~500 μm, and heights of 10~100 μm.

5. The organ-on-a-chip capable of parallel and precise timing-based drug delivery according to claim 1, characterized in that, The main perfusion culture channel (1-1) is connected to a negative pressure device through the negative pressure channel inlet (1-6) and negative pressure channel outlet (1-7).

6. The organ-on-a-chip with parallel and precise timing-based drug delivery according to any one of claims 1 to 5, characterized in that, The organ-on-a-chip consists of an upper organ-on-a-chip body (1), an intermediate chip (2), and a lower chip (3) from top to bottom. The intermediate chip (2) and the lower chip (3) are respectively provided with an intermediate cell culture chamber (2-1) and a lower cell culture chamber (3-1). The shapes of the intermediate cell culture chamber (2-1) and the lower cell culture chamber (3-1) are consistent with the shape of the perfusion culture main channel (1-1) and their positions correspond one-to-one. Porous membranes (4) are provided on the upper and lower sides of the intermediate cell culture chamber (2-1). The porous membranes (4) separate the perfusion culture main channel (1-1), the intermediate cell culture chamber (2-1), and the lower cell culture chamber (3-1) to form upper, middle, and lower chambers.

7. The application of an organ-on-a-chip as described in any one of claims 1 to 6 in parallel and precise timing-based drug delivery for non-therapeutic purposes, characterized in that, Includes the following steps: (1) The drug solution is injected into the corresponding drug preloading chamber (1-2) through the micropores (1-4) respectively, and the micropores (1-4) are sealed with the sealing element (1-5). (2) Inoculate cells in the main perfusion culture channel (1-1) and perform perfusion culture; (3) At the predetermined drug administration time, the seals (1-5) on all micropores (1-4) are removed manually or automatically or simultaneously, so that the drug solution in the drug preloading chamber (1-2) diffuses into the perfusion culture main channel (1-1) through the micro-fence structure (1-3), thereby achieving parallel and precise time-sequential drug administration.

8. The application according to claim 7, characterized in that, The cells seeded in the main perfusion culture channel (1-1) include at least one of the following cell types: hepatocytes, kidney cells, intestinal cells, nerve cells, and vascular endothelial cells.

9. The application according to claim 7, characterized in that, During the perfusion culture, the negative pressure inside the chip is controlled to be -100 ~ -1 Pa by a negative pressure device.

10. The application according to any one of claims 7 to 9, characterized in that, When removing the seals (1-5), ensure that the effective loading time of the drug solution is 3-15 minutes and do not introduce air bubbles into the chip.

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