Kidney organoid culture and multifunctional detection integrated micro-fluidic chip

Abstract

The invention discloses a kidney organoid culture and multifunctional detection integrated micro-fluidic chip, which is characterized in that a first chip (top layer) is provided with six independent cell culture chambers, the bottom is provided with a porous membrane, glomerular cells differentiated by human induced pluripotent stem cells are inoculated, and a blood filtration function is simulated; the second chip (middle layer) is provided with a raw urine-like collecting chamber, and a porous membrane is used for receiving the filtrate and detecting final urine-like components; and the third chip (bottom layer) is inoculated with vascular endothelial cells to simulate renal tubule reabsorption and blood purification processes. According to the invention, glomerular filtration and renal tubule reabsorption full-function chains are coupled, and the limitation of single function simulation is broken through; the parallel culture room supports synchronous drug toxicity testing or disease modeling; a multi-valve dynamic control system can accurately simulate pathological microenvironments such as high glucose and renal toxicity; and in-situ multi-parameter real-time analysis is realized through three paths of detection outlets. The chip provides a highly bionic in-vitro platform for drug screening, diabetic nephropathy mechanism research and kidney injury model construction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, and relates to a kidney organoid culture and multifunctional detection integrated microfluidic chip, in particular to an iPSC culture kidney organoid microfluidic chip design and kidney function simulation and detection. BACKGROUND

[0002] The kidney is an important excretory organ of the human body, which has the functions of urine generation and discharge, maintenance of electrolyte acid-base balance, synthesis and secretion of various hormones, etc., and can maintain the stability of the internal environment of the body. When various causes lead to acute or chronic damage to the kidney, the kidney function will decrease sharply or gradually, and eventually will develop into end-stage renal failure. Chronic kidney disease has become a serious threat to human health in the global public health problem, and chronic kidney disease has universality and high incidence. The fatality of the disease is strong when it progresses to the end stage, and the prevention and control situation has not been effectively improved in the past decades. And with the change of population structure, the prevention and control pressure of chronic kidney disease will continue to increase, and more effective diagnosis and treatment means and intervention measures are urgently needed. However, since the developed and mature kidney tissue cannot repair itself after being damaged, kidney damage is difficult to recover or reverse. In the face of this situation, researchers have proposed that kidney organoids have good prospects in the field of kidney disease research.

[0003] Kidney organoids have great potential for kidney regeneration, which can be achieved by using human pluripotent stem cells or kidney progenitor cells to differentiate into kidney structures. Patent CN117070440A discloses a method for efficiently constructing human kidney organoids. Using hPSCs as raw materials, through steps such as matrix glue dilution, cell digestion, and induction culture, 2D and 3D kidney organoids can be obtained simultaneously, with a success rate of up to 90%. The advantages of this patent are simple and fast process, suitable for kidney disease research, drug screening, etc.; the organoids have kidney-like structures and can simulate some functions; however, the organoids constructed by this patent lack vascularization and immune system, which differ from real kidneys; the induction differentiation period is relatively long (about 18 days for 2D and about 25 days for 3D). Patent CN120249182A discloses a method for constructing matrix glue-free kidney organoids and evaluating drug safety using hepatobiliary-kidney organoids. Using iPSCs as raw materials, through specific culture medium sets, kidney organoids with complete structure and function can be obtained in 16 days without matrix glue, and can also be used with hepatobiliary organoids to evaluate drug safety. The advantages of this patent are no need for matrix glue, simplified operation and reduced cost; short construction period, success rate over 95%, long-term culture (2-3 months); can respond to drugs, suitable for drug screening, etc.; disadvantages include dependence on specific culture medium sets, complex formula; lack of blood vessels and immune system, which differ from real kidneys. Patent CN119015010A discloses a method for constructing a human kidney organoid xenotransplantation model. By screening immune-deficient mice that meet the signs as experimental carriers, after pretreatment and exposure of the kidney capsule, kidney organoids are implanted using a pipette gun, and then monitored after suturing. The advantages of this patent are that it reduces human error by using computer programs for screening and operation; it simplifies the process and shortens the time by not needing to prepare agarose rods and mix other cells, which is beneficial for analyzing the impact of kidney capsules alone; however, this patent relies on specific immune-deficient mice, which limits its scope of application; the monitoring environment after transplantation is strict and the operation is delicate.

[0004] In addition, current researchers are still unable to construct complete or functional vascular networks due to technical limitations, which makes kidney organoids unable to be cultured and survive for a long time, and also hinders the application of kidney organoids. An ideal in vitro kidney model should have similar functions to in vivo, and also have similar physiological microenvironments and ways of material transfer.

[0005] Kidney organ chip can play a key role in the construction of in vitro kidney model, but the construction method of in vitro kidney model needs further study. The patent CN117586943A discloses a method for constructing an organ in vitro and an organ. Cells are mixed with Matrigel and then contacted with the outer surface of a hollow pipe to form a coagulum. Culture medium is injected into the pipe, and the micro-pores of the pipe are used to exchange the culture medium. The organ is obtained by culture. The advantages of the patent are that the organ grows fast, the cycle is short, the volume reaches centimeter level, the maturity is high, the size is uniform, the bionic ability is strong, and various organs such as kidney and lung cancer can be constructed. However, the patent relies on a hollow pipe system containing micro-pores, which requires high equipment; Matrigel needs to be used, which may increase the cost and operation complexity.

[0006] The existing kidney organ chip still has many shortcomings. Most chips only simulate a single function (such as filtration or reabsorption), and it is difficult to completely reproduce the complex physiological process of the kidney. The cell function is prone to degradation during long-term culture, and the stability is insufficient. The detection means mainly relies on end-point analysis, and lacks real-time and multi-parameter dynamic monitoring. In addition, the standardization degree of the chip is low, and the data comparability in different laboratories is poor. Therefore, the design of the chip still needs to be optimized, the maturity of the cells needs to be improved, and an integrated sensing system needs to be developed in the future. SUMMARY

[0007] Based on the shortcomings of the prior art, the purpose of the present application is to provide a kidney organ culture and multifunctional detection integrated microfluidic chip, which can accurately control the nutrient composition and flow rate, realize the detection of kidney filtration and reabsorption function, and simulate drug toxicity test, screening and disease pathological state.

[0008] The present application solves the above technical problems by the following technical solutions: Technical solution 1 provides a kidney organ culture and multifunctional detection integrated microfluidic chip, which comprises a first chip, a second chip and a third chip, which are tightly connected. The first chip is located at the uppermost layer of the chip and is used to simulate the glomerular filtration function. The second chip is located at the middle layer and is used to collect the primary urine and detect the composition of the final urine. The third chip is located at the lowermost layer and is used to simulate the renal tubular reabsorption function and detect the degree of blood purification. A porous membrane is arranged between each two chips, i.e. a porous membrane is arranged between the first chip and the second chip, and a porous membrane is arranged between the second chip and the third chip.

[0009] The core functional unit of the kidney is the nephron, which consists of the glomerulus and the renal tubule. The glomerulus is a cluster of capillaries that functions like a fine sieve: filtering blood under high pressure to form primary urine rich in water, electrolytes, glucose, amino acids, and waste products (such as urea and creatinine), while retaining blood cells and large protein molecules. The filtered primary urine then enters the renal tubule, where it actively reabsorbs approximately 70% of the water, sodium, and potassium, all of the glucose, amino acids, and most of the bicarbonate from the primary urine.

[0010] Furthermore, the first chip comprises at least six independent cell culture chambers, each with a porous membrane at its bottom. These cell culture chambers are used to seed human pluripotent stem cells (HPS cells) that are directed to differentiate into glomerular cells to mimic glomerular filtration function. Prior to use, HPS cells are seeded into these chambers and culture medium is added.

[0011] Inducing human pluripotent stem cells to differentiate into glomerular cells requires the addition of specific components in stages: initially, Activin A, BMP4, and CHIR99021 are used to induce mesoderm; then, FGF9, BMP7, and LDN193189 are used to promote the formation of metanephrotic mesenchymal cells; next, VEGF-A, retinoic acid, and DAPT are used to induce glomerular progenitor cells; finally, low concentrations of TGF-β1 and angiotensin II are used to promote maturation. The effect can be enhanced by using a 3D culture environment. Ultimately, the cells will express specific markers such as podocytes and glomerular endothelial cells.

[0012] Furthermore, each cell culture chamber is equipped with two parallel inlets, each connected to a first inflow channel arranged side by side; the first inflow channel is connected to a peristaltic pump for pumping the liquid to be tested into the cell culture chamber; each parallel inlet is equipped with a front-end flow rate control valve; each cell culture chamber is equipped with an outlet, which is equipped with a third flow rate control valve and connected to a third outflow channel; the third outflow channel has two branches, one end connected to a third chip and the other end connected to a detection device; the branch of the third outflow channel connected to the detection device is equipped with a terminal flow rate control valve; the portion of the third outflow channel connected to the third chip is connected to a second outflow channel.

[0013] Furthermore, the second chip includes a number of primary urine collection and culture chambers, the number of which is equal to the number of cell culture chambers; the bottom of the primary urine collection and culture chamber is a porous membrane; a first flow rate control valve is provided at the outlet of the primary urine collection and culture chamber, and the outlet is connected to a first outflow channel; the first outflow channel has two branches, one of which is connected to the second outflow channel and communicates with the third chip, and the other branch is connected to the detection device at its end.

[0014] Furthermore, the third chip includes a vascular endothelial cell culture chamber, with a second inflow channel at the inlet of the vascular endothelial cell culture chamber connected to a second outflow channel; a fourth outflow channel at the end of the vascular endothelial cell culture chamber is connected to a detection device. Before use, vascular endothelial cells are inoculated into the vascular endothelial cell culture chamber and culture medium is added.

[0015] Furthermore, the first inflow channel and the second outflow channel intersect to form a cross shape. In addition to connecting to the inlet of the cell culture chamber, the first inflow channel also connects to the second outflow channel.

[0016] Furthermore, a second flow rate control valve is provided at the end of the second outflow channel.

[0017] Furthermore, the first, second, and third chips are made of transparent materials to facilitate observation of cell growth.

[0018] Furthermore, the detection device is used to achieve in situ, real-time, and multi-dimensional analysis of kidney function: 1) The device type of the glomerular filtration function detection end (third outflow channel outlet) integrates a micro flow sensor (such as a piezoresistive flow meter) + a miniature fluorescence spectrometer to detect indicators; 2) The final urine component analysis end (first effluent channel branch outlet) is equipped with an in-situ electrochemical microelectrode array (real-time monitoring of pH, sodium, potassium, and chloride); externally connected to a liquid chromatography-mass spectrometry (LC-MS) instrument or a microfluidic chip electrophoresis instrument. Detection indicators: Electrolyte balance: sodium / potassium concentration ratio (electrode array); Metabolic waste: creatinine, blood urea nitrogen (LC-MS); Pathological markers: early diabetic nephropathy microalbumin (detection limit 5 mg / L), drug metabolites (such as cisplatin derivatives).

[0019] 3) The equipment type for the blood purification level detection end (fourth outflow channel outlet) is a miniature colorimetric sensor (urea nitrogen / creatinine colorimetric method); impedance analyzer (endothelial cell barrier function assessment). Detection indicators: Purification efficiency: urea nitrogen clearance rate (dynamic monitoring using colorimetric method); Endothelial function: transendothelial resistance (TEER value, reflecting the integrity of tight junctions); Inflammatory factors: external ELISA detection of secretion levels of TNF-α, IL-6, etc.

[0020] Technical solution 2 provides an application of a microfluidic chip integrating kidney organoid culture and multifunctional detection. The chip is used to simulate kidney function and further for establishing a cisplatin-induced kidney injury model, or for high-throughput screening of drug nephrotoxicity, or for establishing a hyperglycemic model of diabetic nephropathy, or for establishing an integrated physiological-pathological detection system.

[0021] Compared with the prior art, the present invention has at least the following improvements and beneficial effects: (1) Functional coupling innovation breaks through the limitation of existing kidney chips that only simulate a single function. The first chip simulates glomerular filtration, and the third chip simulates renal tubular reabsorption through vascular endothelial cells, completely replicating the "filtration-reabsorption" functional chain; the porous membrane layered interconnection structure realizes the dynamic transfer of urine-like material from filtration to reabsorption, which is closer to the physiological process of the kidney.

[0022] (2) This invention has the ability to simulate dynamic pathological microenvironments. It introduces multi-stage flow rate valves and cross-shaped flow channels to solve the problem of rigid flow rate / component control in traditional chips. The flow rate can be precisely adjusted through valve linkage to simulate pathological states such as hypertension and hypoperfusion. The dual-inlet design supports real-time switching of different culture media (such as high glucose and nephrotoxic drugs) to dynamically construct models such as diabetic nephropathy and cisplatin injury.

[0023] (3) High-throughput integrated detection, integrating three independent detection outlets (third outflow channel, first outflow channel, and fourth outflow channel), overcoming the shortcomings of traditional technologies that rely on endpoint analysis. Simultaneous real-time monitoring of filtration rate, final urine components, and blood purification indicators; multiple independent culture chambers support parallel drug concentration gradient testing, enabling the processing of more samples per day and higher efficiency.

[0024] (4) This invention uses PDMS / glass transparent material and modular flow channel design to solve the problems of non-standard chip structure and poor data comparability in traditional chips. The transparent chip supports in-situ microscopic observation and fluorescent labeling detection; the number of culture chambers can be expanded to 12 or more, and it is compatible with high-throughput screening platforms.

[0025] In summary, this invention, through a three-layer chip functional coupling design (upper layer glomerular filtration, middle layer primary urine collection, and lower layer renal tubular reabsorption), a multi-level dynamic control system (microfluidic valves for precise rate / component control), and three integrated detection outlets, achieves for the first time a biomimetic full-function chain of kidney "filtration-reabsorption-purification" within a single chip. It overcomes the bottlenecks of existing technologies' single function and lagging detection. Its core advantages are: the three chips synergistically simulate the physiological processes of a real nephron; dynamic adjustment of flow rate and components enables the construction of a highly biomimetic pathological model; at least six independent culture chambers support high-throughput parallel experiments, improving efficiency; and the integrated system provides real-time synchronous monitoring of multiple parameters, including filtration rate, reabsorption efficiency, and final urine composition, offering a highly efficient and accurate in vitro platform for research on kidney disease mechanisms and drug toxicity evaluation. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0027] Fig. 1 This is a side view of the overall structure of the integrated microfluidic chip for kidney organoid culture and multifunctional detection in Embodiment 1 of the present invention; Fig. 2 This is a top view of the first-layer chip structure in Example 1; Fig. 3 This is a top view of the second-layer chip structure in Example 1; Fig. 4 This is a top view of the third-layer chip structure in Example 1; Figure label: 1 First inflow channel, 2 Front flow rate control valve, 3 Cell culture chamber, 4: IPSC cells, 5 Porous membrane, 6 Primary urine collection culture chamber, 7 First flow rate control valve, 8 First outflow channel, 9 Second outflow channel, 10 Second flow rate control valve, 11 Vascular endothelial cell culture chamber, 12 Third flow rate control valve, 13 Third outflow channel, 14 Terminal flow rate control valve, 15 Second inflow channel, 16 Fourth outflow channel; P Peristaltic pump, X Detection equipment. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0029] Unless otherwise specified, all raw materials used in this invention are not subject to any particular restriction on their source; they may be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0030] Example 1: Chip Structure like Figs. 1 to 4 As shown, this embodiment provides a microfluidic chip integrating kidney organoid culture and multifunctional detection. The chip consists of a first chip, a second chip, and a third chip that are tightly connected. A porous membrane 5 is disposed between the first chip and the second chip, and between the second chip and the third chip.

[0031] The first chip is located on the top layer of the entire chip and includes six cell culture chambers 3. The base of each cell culture chamber 3 is a porous membrane 5. Human pluripotent stem cells (iPSCs) 4 are seeded in the cell culture chambers 3. These cells can differentiate into functional glomerular cells to simulate glomerular filtration function. Each cell culture chamber 3 is provided with two parallel inlets, which are respectively connected to the first inflow channels 1 arranged side by side. The first inflow channels 1 are connected to a peristaltic pump to pump the liquid to be tested into the cell culture chamber 3. The pathological state can be simulated by adjusting the flow rate, composition, or introducing pathogenic factors. Each parallel inlet is provided with a front-end flow rate control valve 2, that is, each cell culture chamber 3 has two front-end flow rate control valves 2, which are used to regulate the liquid flow rate and composition. Each cell culture chamber 3 is equipped with an outlet, which is equipped with a third flow rate control valve 12. The outlet is connected to a third outflow channel 13. The third outflow channel 13 has two branches, one end of which is connected to a third chip, and the other end is connected to a detection device to measure the glomerular filtration rate. The branch of the third outflow channel 13 connected to the detection device is equipped with a terminal flow rate control valve 14. The part of the third outflow channel 13 connected to the third chip is connected to the second outflow channel 9. The first chip is connected to the second chip below through the cell culture chamber 3 and the porous membrane 5.

[0032] The second chip, located in the middle layer, comprises six primary urine collection culture chambers 6 and a first outflow channel 8. The top of each culture chamber is connected to one of the six cell culture chambers 3 in the first chip via a porous membrane 5, for receiving the primary urine-like components filtered from the first chip. A first flow rate control valve 7 is installed at the outlet of the primary urine collection culture chamber 6, and the outlet is connected to the first outflow channel 8. The first outflow channel 8 has two branches: one connects to the second outflow channel 9 and communicates with the third chip, and the other branch connects to a detection device for real-time monitoring of metabolites, electrolytes, or drug residues in the final urine-like substance.

[0033] The third chip, located at the bottom layer, includes a vascular endothelial cell culture chamber 11. A second inflow channel 15 at the inlet of the vascular endothelial cell culture chamber 11 connects to a second outflow channel 9. Vascular endothelial cells are seeded inside the vascular endothelial cell culture chamber 11 to simulate renal tubular reabsorption function, and it is separated from the second chip by a porous membrane 5. A fourth outflow channel 16 at the end of the vascular endothelial cell culture chamber 11 connects to a detection device to monitor blood purification indicators in real time, including blood urea nitrogen, creatinine, or toxin clearance rate.

[0034] The first inflow channel 1 and the second outflow channel 9 intersect to form a cross shape. The first inflow channel 1 connects to the inlet of the cell culture chamber 3 and also to the second outflow channel 9. A second flow rate control valve 10 is located at the end of the second outflow channel 9. The first, second, and third chips are made of transparent material, facilitating microscopic observation of cell growth. The chips integrate microfluidic technology to dynamically regulate the culture media with different components, simulating the in vivo microenvironment. The cell culture chamber 3 of the first chip can be expanded to multiple chambers to support high-throughput experiments. The pore size of the porous membrane is adjustable to accommodate the filtration requirements of substances with different molecular weights. The chips can be used for drug toxicity testing, assessing the drug's impact on the kidneys by detecting filtration rate, reabsorption efficiency, and changes in final urine composition.

[0035] Example 2: Chip Fabrication In the chip fabrication process, the microfluidic chip of this invention is prepared using biocompatible transparent materials such as PDMS (polydimethylsiloxane) or glass in terms of material selection and processing. PDMS material is formed using soft photolithography, while glass material is constructed using wet etching or laser processing to achieve microstructure construction. The specific steps are as follows: Mold fabrication: Microchannel molds are fabricated on silicon wafers using photolithography. The mold structure corresponds to the first inflow channel 1, cell culture chamber 3, front flow rate control valve 2, end flow rate control valve 14 and third outflow channel 13 of the first chip; the urine-like collection culture chamber 6, first outflow channel 8 and first flow rate control valve 7 of the second chip; and the vascular endothelial cell culture chamber 11, second inflow channel 15 and fourth outflow channel 16 of the third chip.

[0036] PDMS casting and curing: The PDMS prepolymer and curing agent are mixed in a ratio of 10:1, degassed, poured into a mold, and cured in an 80°C oven for 2 hours. After demolding, a chip body with microstructure is obtained.

[0037] Interlayer bonding: The PDMS chip surface is treated with oxygen plasma and bonded to the porous membrane 5 and another PDMS substrate to form a closed flow channel system, ensuring the sealing between each chip layer (first chip, second chip, third chip).

[0038] Integrated detection module and flow rate control valve installation: Two front-end flow rate control valves 2 are integrated at the front end of each cell culture chamber 3 of the first chip. A terminal flow rate control valve 14 is installed on the branch line of the third outflow channel 13 connected to the detection device. The flow rate (0.1-10μL / min) and composition are precisely controlled by external air pressure. Detection device docking: One end of the third outflow channel 13 of the first chip is connected to the glomerular filtration rate detection device, and the other end is connected to the second outflow channel 9 and connected to the third chip; the branch line end of the first outflow channel 8 of the second chip is connected to the final urine component detection device, and the fourth outflow channel 16 of the third chip is connected to the blood purification level detection device.

[0039] According to an embodiment of the present invention, in cell culture and functional simulation, glomerular filtration function is constructed through cell seeding: human pluripotent stem cells (iPSCs) 4 are induced to differentiate into podocytes and endothelial cells at a rate of 5 × 10⁻⁶ cells / cells. 4 cells / cm 2 The cells were seeded at a density on the porous membrane 5 of the substrate of the first chip cell culture chamber 3 and cultured for 3-5 days to form a filtration barrier. Physiological simulation: Culture medium containing 10% fetal bovine serum was introduced into the first inflow channel 1 by a peristaltic pump, and the flow rate was maintained at 1 μL / min by adjusting the front flow rate control valve 2 to simulate the glomerular filtration pressure (10-15 mmHg).

[0040] According to an embodiment of the present invention, the high-throughput advantage is achieved through parallel experimental design, including a first chip containing six independent cell culture chambers 3. Each culture chamber can independently control the flow rate and composition, supporting the simultaneous execution of six different experiments (such as different drug concentrations and different cell lines), with a daily sample processing capacity of more than 24 sets. In terms of modular expansion, the number of cell culture chambers 3 can be expanded to 12 or more as needed, and multiple sets of experiments can be operated in parallel by increasing the number of peristaltic pump channels, meeting the requirements of high-throughput drug screening.

[0041] According to an embodiment of the present invention, the dynamic regulation mechanism allows for real-time flow rate adjustment via a programmable peristaltic pump linked to the front-end flow rate control valve 2, enabling dynamic switching of the flow rate within the range of 0.1-10 μL / min to simulate changes in renal blood flow under different physiological conditions. The culture medium component switching is achieved by using a multi-channel peristaltic pump to input different culture medium components (such as solutions containing growth factors, toxins, or high glucose) into the first inflow channel 1. The front-end flow rate control valve 2 controls the switching time and proportion of the culture medium, dynamically simulating changes in the in vivo microenvironment.

[0042] Example 3: Application of the Chip (1) Used to establish a cisplatin-induced kidney injury model.

[0043] Model construction: Culture medium containing 10 μM cisplatin was introduced into the first inflow channel 1 of the first chip via a peristaltic pump, and the flow rate control valve 2 at the front end was adjusted to a flow rate of 5 μL / min, which was carried out continuously for 24 hours; at the same time, it flowed into the third chip through the second outflow channel 9.

[0044] Monitoring indicators: Glomerular filtration rate was measured by the detection device connected to the third outflow channel 13 of the first chip, and decreased by ≥30%; cell viability in the vascular endothelial cell culture chamber 11 of the third chip decreased by ≥50% as detected by the CCK-8 method; and final urine components were measured by the detection device connected to the first outflow channel 8 of the second chip, and protein content increased by ≥2 times to assess the nephrotoxicity of cisplatin.

[0045] (2) Used for high-throughput screening of drug nephrotoxicity.

[0046] Experimental design: Different concentrations of candidate drugs (such as 0.1 μM, 1 μM, and 10 μM) were added to the six cell culture chambers 3 of the first chip, while the control group was added with culture medium without drugs. The flow rate was uniformly controlled to 0.5 μL / min by the front-end flow rate control valve 2.

[0047] Indicator detection: After 48 hours of culture, the filtration rate (inulin clearance rate), reabsorption efficiency (glucose reabsorption rate), and concentration of drug metabolites in the final urine were monitored simultaneously in each group to screen out the drug concentration with the least impact on renal function.

[0048] (3) Used to establish a hyperglycemic model of diabetic nephropathy.

[0049] Stimulation input: Culture medium containing 30mM glucose is introduced into the first inflow channel 1 of the first chip through a peristaltic pump, and the flow rate is maintained at 1μL / min by adjusting the front flow rate control valve 2. This process is carried out for 72 hours to simulate the high-glucose environment of diabetes.

[0050] Pathological monitoring: The permeability of the filtration barrier in cell culture chamber 3 of the first chip (leakage of fluorescently labeled albumin increased by ≥1.5 times), the expression of tight junction protein (ZO-1) in endothelial cells in cell culture chamber 11 of the third chip (increased immunofluorescence intensity by ≥40%), and the glucose content in the final urine (increased by ≥3 times) were observed to verify the high glucose-induced kidney injury.

[0051] (4) Establishment of an integrated physiological-pathological detection system.

[0052] Real-time monitoring parameters: Glomerular filtration rate is measured in real time by the detection device connected to the third outflow channel 13 of the first chip, and the flow rate and creatinine concentration of the filtrate are measured in real time; Final urine components are analyzed in real time by the detection device connected to the first outflow channel 8 of the second chip, and indicators such as glucose, electrolytes, and drug residues are analyzed in real time; Blood purification level is monitored by the detection device connected to the fourth outflow channel 16 of the third chip, and indicators such as blood urea nitrogen and creatinine clearance rate are monitored.

[0053] Data synchronization and recording: Each detection module is connected to the computer via a USB interface to realize real-time acquisition and analysis of multi-parameter data, with a sampling frequency of ≥1 time / minute.

[0054] In summary, the three-layer chip of this invention corresponds to glomerular filtration, primary urine collection, and renal tubular-vascular reabsorption, respectively, completely simulating the functional chain of a nephron; multi-stage valves (front-end / terminal flow rate valves) precisely regulate the fluid path, supporting dynamic damage modeling; a 6-channel independent culture chamber enables parallel testing of multiple samples, significantly improving screening efficiency; and the transparent chip combined with a multi-outlet design supports in-situ observation and non-destructive sampling. This invention, through its three-chip integrated design and dynamic control system, overcomes the limitations of traditional kidney organoid on-chip simulations of single functions, providing an efficient and accurate in vitro model platform for kidney disease research and drug development.

[0055] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A microfluidic chip integrating kidney organoid culture and multifunctional detection, characterized in that, The chip consists of a first chip, a second chip, and a third chip: The first chip is located on the top layer of the overall chip and is used to simulate the glomerular filtration function. The second chip is located in the middle layer and is used to collect primary urine and to detect components of final urine. The third chip is located at the bottom layer and is used to simulate the reabsorption function of renal tubules and to detect the degree of blood purification. A porous membrane (5) is provided between the first chip and the second chip, and a porous membrane (5) is provided between the second chip and the third chip.

2. The chip according to claim 1, characterized in that, The first chip contains at least six independent cell culture chambers (3), and the bottom of the cell culture chambers (3) is a porous membrane (5).

3. The chip according to claim 2, characterized in that, The cell culture chamber (3) is used to inoculate human pluripotent stem cells (4), which are directed to differentiate into glomerular cells to simulate glomerular filtration function; before use, human pluripotent stem cells (4) are inoculated into it and culture medium is added.

4. The chip according to claim 3, characterized in that, Each cell culture chamber (3) is equipped with two parallel inlets, which are respectively connected to the first inflow channel (1) arranged side by side; the first inflow channel (1) is connected to a peristaltic pump, which is used to pump the liquid to be tested into the cell culture chamber (3); a front-end flow rate control valve (2) is set at each parallel inlet. Each cell culture chamber (3) is equipped with an outlet, and a third flow rate control valve (12) is installed on the outlet. The outlet is connected to a third outflow channel (13). The third outflow channel (13) has two branches, one end of which is connected to the third chip and the other end is connected to the detection device; the branch of the third outflow channel (13) connected to the detection device is equipped with an end flow rate control valve (14); the part of the third outflow channel (13) connected to the third chip is connected to the second outflow channel (9).

5. The chip according to claim 4, characterized in that, The second chip contains several primary urine collection culture chambers (6), the number of which is equal to the number of cell culture chambers (3); the bottom of the primary urine collection culture chamber (6) is a porous membrane (5); A first flow rate control valve (7) is installed at the outlet of the urine collection and culture chamber (6), and the outlet is connected to a first outflow channel (8); The first outflow channel (8) has two branches, one of which is connected to the second outflow channel (9) and connected to the third chip, and the other branch is connected to the detection device at its end.

6. The chip according to claim 5, characterized in that, The third chip includes a vascular endothelial cell culture chamber (11), with a second inflow channel (15) at the inlet of the vascular endothelial cell culture chamber (11) connected to a second outflow channel (9); a fourth outflow channel (16) at the end of the vascular endothelial cell culture chamber (11) is connected to a detection device; before use, vascular endothelial cells are inoculated into the vascular endothelial cell culture chamber (11) and culture medium is added.

7. The chip according to claim 6, characterized in that, The first inflow channel (1) intersects with the second outflow channel (9) to form a cross shape. In addition to connecting the inlet of the cell culture chamber (3), the first inflow channel (1) is also connected to the second outflow channel (9).

8. The chip according to claim 7, characterized in that, The second outflow channel (9) is equipped with a second flow rate control valve (10) at its end.

9. The chip according to claim 8, characterized in that, The first, second, and third chips are made of transparent materials to facilitate observation of cell growth.

10. The application of the chip as described in any one of claims 1-9, characterized in that, The chip is used to simulate kidney function and is further used to establish a cisplatin-induced kidney injury model, or for high-throughput screening of drug nephrotoxicity, or for establishing a hyperglycemic model of diabetic nephropathy, or for establishing an integrated physiological-pathological detection system.

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