Biomimetic capillary network coupled kidney function microfluidic chip and method of use

Abstract

The application discloses a kidney function microfluidic chip coupled with a bionic capillary network and a use method thereof. The chip comprises an upper chip layer and a lower chip layer which are arranged in a stack and a porous membrane therebetween; the lower chip layer is provided with a central groove, the top opening of the central groove extends to the upper chip layer, the central groove is divided into an organoid accommodating area and a fluid distribution area by the porous membrane, a micro column array is arranged in the groove corresponding to the fluid distribution area, the micro column array can support the corresponding part of the porous membrane on the upper side, the lower chip layer is further provided with an entering ball microartery channel which is in communication with the fluid distribution area and an exiting ball capillary network channel which comprises at least two levels of bifurcation structures; and the upper chip layer is provided with a renal tubule channel which is opposite to the exiting ball capillary network channel and is separated from the exiting ball capillary network channel by the porous membrane. The application realizes the simulation of the continuous physiological process of glomerular filtration and renal tubular reabsorption, highly reproduces the microenvironment of the blood flow dynamics of the kidney unit, and can be widely applied to drug screening, disease modeling and regenerative medicine research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and microfluidic chip technology, specifically relating to a biomimetic kidney microfluidic chip that integrates filtration and reabsorption functions, and is particularly suitable for medical research that simulates the filtration and reabsorption functions of the kidney in vitro. Background Technology

[0002] The kidneys are vital metabolic and excretory organs in the human body. Their core functions include glomerular filtration and tubular reabsorption, which depend on the complex three-dimensional structure of the nephron and the exchange of substances between the blood and epithelium. Glomerular filtration relies on the unique hemodynamic microenvironment formed between the afferent and efferent arterioles. The afferent arterioles have high resistance characteristics to maintain high pressure in the glomerular capillaries, while the efferent arterioles branch to form a peritubular capillary network that surrounds the renal tubules, providing the structural basis for the exchange of substances for tubular reabsorption. These vascular structures and hemodynamic characteristics together constitute the structural and functional basis of the normal physiological function of the nephron.

[0003] Traditional research methods, such as animal models and two-dimensional cell culture, have promoted the development of kidney-related research to some extent, but they still have significant limitations. Animal models suffer from problems such as large species differences, high costs, and ethical restrictions, making it difficult to reflect the true physiological and pathological processes in humans. Two-dimensional cell culture systems lack realistic three-dimensional spatial structures and fluid microenvironments, failing to simulate the fluid shear forces and material exchange conditions experienced by cells in vivo, and thus making it difficult to accurately reproduce the physiological functions of the kidney. In recent years, while organoid-based three-dimensional culture technology has been able to reconstruct the three-dimensional structure of kidney tissue to some extent, it still has shortcomings in terms of long-term functional maintenance and maturity due to the lack of a stable and controllable perfusion system.

[0004] With the development of microfluidic technology, organ-on-a-chip (OABC) based on materials such as PDMS has provided a new technical approach for simulating complex organ functions in vitro. Its core advantage lies in its ability to precisely control fluid flow at the micrometer scale, construct cellular microenvironments, and achieve co-culture of multiple cell types. Existing kidney chips typically achieve cell co-culture and fluid shear force simulation by constructing microchannels, and some progress has been made in renal tubular models or filtration barrier models. For example, some studies have constructed single-channel chips with renal tubular epithelial cells to simulate reabsorption function, while others have constructed filtration barrier models by culturing glomerular endothelial cells and podocytes on both sides of a porous membrane.

[0005] However, existing technologies still have the following shortcomings:

[0006] First, there is a lack of continuous functional coupling between filtration and reabsorption. Most existing kidney chips focus on the construction of single functional modules, such as simulating only tubular reabsorption or only simulating glomerular filtration. They have not yet achieved continuous simulation of the complete physiological process chain of "glomerular filtration → tubular reabsorption" on the same chip, resulting in insufficient functional integrity and making it difficult to comprehensively assess the impact of drugs on the function of the entire nephron.

[0007] Secondly, the biomimicry of vascular structures is insufficient. Existing microfluidic chips mostly employ simple straight or single-bend channel structures for their vascular channels, failing to simulate the high-resistance blood flow characteristics of the afferent arterioles or reflect the complex three-dimensional network structure of the perivascular capillary network formed by the branches of the afferent arterioles. This simplified channel design leads to significant deviations between the fluid resistance distribution, shear force level, and flow patterns within the chip and the actual in vivo conditions, making it difficult to accurately reproduce the unique hemodynamic microenvironment of the nephron.

[0008] Third, the design of the substance exchange interface is unreasonable. Most existing chips employ a planar, parallel channel layout, resulting in a large distance or limited contact area between vascular channels and renal tubular channels. This fails to achieve tight spatial coupling and efficient substance exchange between peritubular capillaries and renal tubules. Furthermore, in some chips, substance transfer between the two channels relies solely on diffusion, lacking sufficient convection, further limiting the efficiency of substance exchange.

[0009] Fourth, organoid culture and perfusion systems are decoupled. In existing systems, organoid culture and perfusion are usually separate operational stages or modules, and organoids lack stable physical support and perfusion interfaces within the chip. Some designs directly expose organoids to a fluid-fluid-fluid environment, which can easily cause cell damage or organoid disintegration; while other designs culture organoids under static conditions, which cannot achieve continuous perfusion supply, resulting in insufficient nutrient supply and accumulation of metabolic waste inside the organoids, making it difficult to maintain long-term functional activity.

[0010] Therefore, how to provide a renal function microfluidic chip coupled with a biomimetic capillary network and its usage method is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention provides a biomimetic capillary network coupled renal function microfluidic chip, which can realize multi-layer coupling of vascular network and renal tubular network in spatial structure, simultaneously cover the entire process of filtration and reabsorption in function, and has efficient material exchange capacity and organoid stable culture function. This microfluidic chip can more realistically simulate the physiological function of the kidney and provide a more reliable in vitro model platform for drug screening, disease modeling and regenerative medicine research.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: a biomimetic capillary network coupled renal function microfluidic chip, comprising: An upper chip layer and a lower chip layer are stacked together, and a porous membrane is disposed between the upper chip layer and the lower chip layer; The lower chip layer has a central groove, an afferent microartery channel, and an efferent capillary network channel. The top opening of the central groove extends to the upper chip layer. The central groove is divided by the porous membrane into an organoid receiving area above the porous membrane and a fluid distribution area below the porous membrane. The afferent microartery channel and the efferent capillary network channel are respectively connected to the fluid distribution area. The efferent capillary network channel includes at least two levels of bifurcated structures. The lower chip layer has a liquid collection groove downstream of the efferent capillary network channel. The upper chip layer is provided with renal tubular channels, which are arranged vertically opposite to the efferent capillary network channels and separated by the porous membrane.

[0013] The technical effects of this invention are as follows: By stacking upper and lower chips and combining them with a porous membrane for separation, the glomerular filtration and renal tubular reabsorption functions are integrated into the same chip, realizing the integrated simulation of the continuous physiological process of "filtration-reabsorption" and solving the defects of traditional chip functions being fragmented. At the same time, the lower chip has an efferent capillary network channel with at least two levels of branching structure, which, together with the renal tubular channels corresponding to the upper layer and separated by a porous membrane, forms a highly efficient material exchange interface between the vascular network and the tubular network. The design of the central groove being separated into an organoid containment area and a fluid distribution area by a porous membrane not only provides stable physical support and positioning for the organoid, but also realizes the directional material exchange between the perfusion fluid and the basal side of the organoid through the fluid distribution area. Thus, at the structural level, the complete hemodynamic path and material transport microenvironment of afferent-filtration-efferent branch capillary-peritumoral exchange in the nephron are systematically reproduced.

[0014] Preferably, the bottom of the central groove of the lower chip layer is provided with a micropillar array, and the porous membrane is disposed above the micropillar array and supported by the micropillar array.

[0015] The resulting technical effects are as follows: by setting a micropillar array at the bottom of the central groove and laying the porous membrane on it, a uniform and stable physical support is provided for the porous membrane in the central groove, which effectively prevents the porous membrane from deforming or collapsing under the gravity of the organoid and the pressure of fluid perfusion, and ensures the structural stability of the porous membrane when separating the organoid containing area and the fluid distribution area; the perfusion fluid can also flow unimpeded from the fluid distribution area through the gaps between the micropillar array to the lower surface of the porous membrane, and then uniformly pass through the entire porous membrane area to exchange substances with the upper organoid base side.

[0016] Preferably, the afferent microartery channel is a serpentine microchannel with a width of 80–120 μm and a height of 40–60 μm.

[0017] The resulting technical effect is that by setting the afferent microartery channel to a serpentine tortuous structure and combining a narrow cross-sectional size of 80-120 μm width and 40-60 μm height, the total length of the flow channel is effectively extended and the fluid resistance is increased within a limited chip area. This creates a high-resistance microenvironment similar to that of the afferent arteriole in vivo before the perfusion fluid enters the central organoid region, providing physiologically appropriate pressure and flow input conditions for the subsequent capillary network.

[0018] Preferably, the bifurcation structure of the efferent capillary network channel includes at least a primary channel, a secondary channel, and a tertiary channel. The secondary channel is formed by bifurcation of the primary channel, and the tertiary channel is formed by bifurcation of the secondary channel. The bifurcation angle of the secondary channel and the tertiary channel is 40° to 75°.

[0019] The resulting technical effect is that by setting up a multi-level branching structure, a highly simulated tree-like fractal capillary network can be constructed on the chip, which effectively increases the total cross-sectional area and branch density of the vascular channel. This causes the flow rate of the perfusion fluid to gradually slow down and the pulsatile flow to attenuate when it flows through the network, thereby accurately reproducing the low-shear microenvironment in the perivascular capillary network after the branching of the efferent arteriole in vivo, where the blood flow tends to be stable.

[0020] Preferably, the renal tubular channel is a serpentine channel, located in the upper chip layer in the region corresponding to the efferent capillary network channel, and the width of the renal tubular channel is greater than the width of the efferent capillary network channel.

[0021] The resulting technical effects are: within a limited chip area, the length of the renal tubular flow channel is extended, and the spatial overlap area with the underlying capillary network is increased. At the same time, by utilizing the size difference between the width of the renal tubular channel and the efferent capillary network channel, the two sets of channels form a flow velocity difference and a transmembrane pressure gradient on both sides of the tightly coupled interface separated by a porous membrane. This effectively simulates the efficient reabsorption material exchange process between the renal tubules and peritubular capillaries in vivo based on a close anatomical adjacency.

[0022] Preferably, the porous membrane has a pore size of 0.4 to 10 μm, and the porous membrane at least covers the opening area of ​​the central groove and the area between the renal tubular channel and the efferent capillary network channel.

[0023] The resulting technical effects are: the porous membrane allows small molecule metabolites, drugs and nutrients to be transported freely across the membrane, while effectively blocking organoid tissues and macromolecules, thus achieving selective substance exchange; at the same time, by covering at least the central groove opening area and the area between the renal tubular channel and the efferent capillary network channel through the porous membrane, the integrity and continuity of the substance exchange interface in the entire process from filtration to reabsorption of the chip are ensured.

[0024] Preferably, the upper chip layer has an opening at the position corresponding to the organoid accommodating area, and the opening is provided with an openable and closable cover plate.

[0025] The resulting technical benefits are: the opening ensures that organoids can be conveniently and without damage implanted into the central organoid pool; after implantation, a closed culture environment is formed by the closed cover plate, which effectively prevents external contamination and avoids direct fluid scouring damage to the organoids by the perfusion fluid.

[0026] Preferably, the upper chip layer has an inlet and an outlet corresponding to both ends of the renal tubule channel, and the upper chip layer has a drainage hole that extends through to the collection tank, and the drainage hole is connected to an external drainage tube.

[0027] The resulting technical effect is that the independent inlet and outlet make the renal tubular channel form a complete flow path system with independent inflow and outflow, thereby enabling independent control of the perfusion flow rate and shear force conditions on the renal tubular side, achieving efficient directional perfusion of the renal tubular reabsorption function and collection and detection of outlet samples.

[0028] Preferably, the liquid collection tank is an arc-shaped tank, and the drainage holes are multiple and arranged at equal arc length intervals along the arc path.

[0029] The resulting technical effect is that by combining the arc-shaped groove with multiple drainage holes arranged at equal arc length intervals, the liquid collection volume is expanded and uniform drainage is achieved, effectively avoiding local blockage and flow field disturbance, and ensuring smooth and stable liquid discharge.

[0030] This invention also discloses a method of using a biomimetic capillary network coupled renal functional microfluidic chip, which employs the above-mentioned chip and includes: The kidney organoid is placed in the organoid receiving area so that the kidney organoid comes into contact with the porous membrane; Perfusion fluid is injected through the afferent microartery channel, allowing the perfusion fluid to pass through the porous membrane in the fluid distribution area and exchange substances with the renal organoids, before flowing out from the collection trough downstream of the efferent capillary network channel; Pulsating flow was applied to the ingressor microartery channel for perfusion culture.

[0031] The resulting technical effects are as follows: placing the kidney organoid in the organoid containment area and contacting it with the porous membrane creates a stable and controllable material exchange interface between the organoid basal side and the perfusion fluid; the perfusion path design, where the perfusion fluid flows from the afferent microartery channel through the fluid distribution area through the porous membrane to exchange substances with the organoid and then flows out through the efferent capillary network channel, completely replicates the hemodynamic path and transmembrane exchange process of "afferent arteriole → glomerular filtration → efferent arteriole → perivascular capillary" in vivo; and applying pulsatile flow to the afferent microartery channel for perfusion culture further uses physiological pulsatile flow as a mechanical signal to drive the maturation of vascular endothelial cells inside the organoid and the directional invasion of the vascular network into the organoid. At the same time, the pulsatile flow naturally attenuates into a stable laminar flow after passing through the high-resistance afferent channel and the bifurcation capillary network, thus enabling the entire chip to highly replicate the in vivo microenvironment of the nephron in terms of key hemodynamic indicators such as fluid shear force, transmembrane flux, and pulsatile attenuation.

[0032] Preferably, before perfusing the afferent microartery channel with perfusion fluid, a vascular partitioning inoculation step is further included: inoculating the afferent microartery channel with first vascular endothelial cells and inoculating the efferent capillary network channel with second vascular endothelial cells; the first vascular endothelial cells and the second vascular endothelial cells are phenotypically different vascular endothelial cells, used to simulate the arterial microenvironment and venous microenvironment respectively in perfusion culture.

[0033] The resulting technical effect is that, by utilizing the spatial isolation between the chip's entry and exit channels, xenogeneic vascular endothelial cells can be in situ seeded in different regions before perfusion, thereby constructing a functional vascular network with arteriovenous heterogeneity phenotype, significantly improving the physiological biomimicry and experimental controllability of the renal organoid microenvironment simulation. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of a biomimetic capillary network coupled renal function microfluidic chip structure according to the present invention; Figure 2 This is a diagram showing the internal flow channels of a biomimetic capillary network coupled renal function microfluidic chip according to the present invention. Figure 3 This is an exploded view of a biomimetic capillary network coupled renal function microfluidic chip according to the present invention. Figure 1 ; Figure 4 This is an exploded view of a biomimetic capillary network coupled renal function microfluidic chip according to the present invention. Figure 2 ; Figure 5 This is a diagram of the lower-layer chip structure of a biomimetic capillary network coupled renal functional microfluidic chip according to the present invention.

[0035] Figure 6 for Figure 5 A magnified view of part A.

[0036] 1 Upper chip layer, 2 Lower chip layer, 3 Porous membrane, 4 Central groove, 5 Inlet microartery channel, 51 Inlet microartery channel opening, 6 Outlet capillary network channel, 61 Capillary network channel pore, 7 Renal tubule channel, 71 Inlet, 72 Outlet, 8 Microcolumn array, 9 Cover plate, 10 Collection tank. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] See the appendix of this invention. Figures 1 to 6 According to an embodiment of the present invention, a biomimetic capillary network coupled renal function microfluidic chip includes: The upper chip layer 1 and the lower chip layer 2 are stacked together, and a porous membrane 3 is disposed between the upper chip layer 1 and the lower chip layer 2. The porous membrane is fan-shaped. The lower chip layer 2 has a central groove 4, an afferent microartery channel 5, and an efferent capillary network channel 6. The top opening of the central groove 4 extends to the upper chip layer 1. The central groove 4 is divided by a porous membrane 3 into an organoid receiving area above the porous membrane 3 and a fluid distribution area below the porous membrane 3. The fluid distribution area corresponds to the "glomerular region". The afferent microartery channel 5 and the efferent capillary network channel 6 are respectively connected to the fluid distribution area. The efferent capillary network channel 6 includes at least two levels of bifurcated structures. The lower chip layer 2 has a collection groove 10 downstream of the efferent capillary network channel 6. The upper chip layer 1 is provided with a renal tubular channel 7, which is arranged vertically opposite to the efferent capillary network channel 6 and separated by a porous membrane 3.

[0039] In some other embodiments, the bottom of the central groove 4 of the lower chip layer 2 is provided with a micropillar array 8, and the porous membrane 3 is disposed above the micropillar array 8 and supported by the micropillar array 8.

[0040] In other embodiments, the afferent microartery channel 5 is a serpentine microchannel with a width of 80–120 μm and a height of 40–60 μm.

[0041] The afferent microartery channel 5 consists of 1-2 serpentine microchannels extending from the edge of the lower layer of the chip to the central region, with a channel width of 100 μm and a height of 50 μm. This channel simulates the blood flow characteristics of the afferent arterioles, which are thick, short, and have a large diameter but high resistance. The serpentine, meandering structure increases the total length of the flow channel, and combined with the narrow channel cross-section (width-to-height ratio 2:1), it generates significant fluid resistance within the limited chip area, creating a high-resistance environment similar to that of the afferent arterioles in vivo before the perfusion fluid enters the central organoid region. Arterial endothelial cells are seeded on the inner wall of the channel to form a dense monolayer under pulsatile flow conditions, and then perfused with VEGF-containing culture medium to stimulate angiogenesis within the organoid.

[0042] In other specific embodiments, the bifurcation structure of the efferent capillary network channel 6 includes: a primary main channel, 2 to 3 secondary channels formed by bifurcation of the primary main channel, and 2 to 4 tertiary channels formed by further bifurcation of each secondary channel; the bifurcation angle of the secondary and tertiary channels is 40° to 75°.

[0043] The efferent capillary network channels feature a biomimetic branching structure: 1-2 main channels emerge from the other side of the central area, immediately branching into 2-3 secondary channels with bifurcation angles ranging from 45° to 75°, mimicking the physiological bifurcation angle range of blood vessels in vivo (30-50 μm wide, 30 μm high). Each secondary channel further branches into 2-4 tertiary microchannels with branching angles of 40° to 60°. Some tertiary channels continue to branch into even finer terminal channels (30 μm wide, 30 μm high), forming a complete tree-like fractal network. These channels meander and coil on the other side of the chip, eventually converging at the outlet. This simulates the anatomical structure of efferent arterioles branching into a perivascular capillary network.

[0044] In some other specific embodiments, the renal tubular channel 7 is a serpentine channel located in the upper chip layer 1 in the region corresponding to the efferent capillary network channel 6. It is used to culture proximal / distal tubular epithelial cells and is another independent channel network that overlaps with and is closely adjacent to the capillary network. The width of the renal tubular channel 7 (150-200 μm wide and 100 μm high) is greater than the width of the efferent capillary network channel 6.

[0045] In other embodiments, the porous membrane 3 has a pore size of 0.4 to 10 μm, and the porous membrane 3 at least covers the opening area of ​​the central groove 4 and the area between the renal tubular channel 7 and the efferent capillary network channel 6.

[0046] In some other specific embodiments, the upper chip layer 1 has an opening at the location of the corresponding organoid accommodating area, and the opening is provided with an openable cover plate 9.

[0047] In specific implementation, the upper chip layer 1 is provided with an inlet 71 and an outlet 72 at both ends of the renal tubule channel 7, and the upper chip layer 1 is provided with a drainage hole above the collection tank 10, which is connected to an external drainage tube.

[0048] It should be noted that there is no porous membrane above the collection tank 10, but it is directly attached to the upper chip layer (the edge of the porous membrane only covers the area between the capillary network and the renal tubular channels, and does not contact the collection tank).

[0049] The collection tank 10 is an arc-shaped tank with two drainage holes located at the points that divide the collection tank into three equal parts. Flexible tubes can be added to the drainage holes to draw out and collect the fluid.

[0050] This invention also discloses a method of using a biomimetic capillary network coupled renal functional microfluidic chip, which employs the above-mentioned chip and includes: The kidney organoids were placed in the organoid housing area so that the kidney organoids came into contact with the porous membrane; Perfusion fluid is injected through the afferent microartery channel, allowing the fluid to pass through the fluid distribution area and exchange substances with the renal organoids via a porous membrane before flowing out through the efferent capillary network channel. Pulsating flow was applied to the ingressor microartery channel for perfusion culture.

[0051] In addition, the afferent microartery channel and the efferent capillary network channel can also be used to add two different types of vascular cells for culture, simulating the effects of arteries and veins.

[0052] In practice, the chip substrate can be made of biocompatible materials such as PDMS, PMMA, or COC, and prepared using soft lithography or injection molding processes. The porous membrane 3 can be made of polycarbonate or PET.

[0053] Fluid simulation This embodiment uses computational fluid dynamics (CFD) to numerically simulate the fluid flow behavior inside the chip, focusing on analyzing the velocity field, wall shear stress, and transmembrane flux distribution in the ingress channel, capillary network, and renal tubular channels.

[0054] Simulation conditions: A pulsating flow with an average flow rate of 0.8 μL / min and a frequency of 1–2 Hz was applied at the inlet of the inlet microartery channel 5.

[0055] Simulation results show that the afferent serpentine microartery channel (100 μm wide and 50 μm high) generates a pressure drop of approximately 85 Pa at an average flow rate of 0.8 μL / min. After the fluid enters the efferent capillary network channel 6 through the central organoid region, the average flow velocity drops sharply from 0.75 mm / s in the main channel to 0.02–0.5 mm / s in the terminal channels due to multi-level branching (36 microchannels in total). The transmembrane flux distribution is uniform on both sides of the porous membrane 3. The flow velocity in the renal tubular channel 7 is approximately 0.15 mm / s, which is consistent with the physiological flow conditions of the proximal tubule.

[0056] In addition, the amplitude of the arterial pulsation decreases by about 40% after passing through the afferent channel, and further decreases to less than 10% of the inlet amplitude after entering the capillary network. The flow pattern changes from pulsation to a nearly constant steady laminar flow, which accurately reproduces the physiological phenomenon that blood flow tends to be stable after the branching of the efferent arteriole in the body.

[0057] In summary, simulations have verified that the chip highly replicates the hemodynamic microenvironment of the nephron in key indicators such as fluid resistance, shear force distribution, and pulsation attenuation.

[0058] Cell seeding and functional validation (a) Cell inoculation Phase 1: Microarray preparation and cell seeding (Days -3 to 0) Day -3 (first three days): After the chip is sterilized (ultraviolet / plasma), extracellular matrix proteins (such as collagen IV and laminin) are perfused into each channel through the inlet of the afferent microartery channel 5, the capillary network channel pore 61 and the inlet 71 of the renal tubular channel 7, coating all channel walls and the surface of the porous membrane 3 to simulate the basement membrane environment.

[0059] Day -2 (First two days): Digest renal tubular epithelial cells and seed them into renal tubular channel 7 through inlet 71; invert the chip for 2-4 hours to allow cells to adhere to the upper sidewall of the channel (simulating the polarity of the lumen in vivo); after 4 hours, seed renal tubular cells again into renal tubular channel 7 through inlet 71 and culture upright. Subsequently, digest arterial endothelial cells and venous / capillary endothelial cells separately. Seed arterial endothelial cells into afferent arteriole channel 5 through inlet 5, allowing them to form a dense monolayer on the inner wall of the channel; seed venous / capillary endothelial cells into efferent capillary network channel 6 through efferent capillary network channel pore 61. Then close the efferent capillary network inlet, and culture medium is used for static or very low flow rate culture through inlet 5 of afferent arteriole channel, while renal tubular channel culture medium is perfused through inlet 71 of renal tubular channel 7.

[0060] Phase Two: Core Integration and Vascular Sprouting (Days 0-7) Day 0: The pre-cultured, healthy kidney organoids are gently implanted into the organoid receiving area of ​​the central groove 4 through the opening of the upper chip layer 1. The cover plate 9 is then closed to ensure good contact between the organoid and the bottom porous membrane 3. At this time, the organoid already has a primitive endothelial network (CD31+).

[0061] Days 1-7: Initiate perfusion of the shared culture medium at static or very low flow rates. Slow perfusion begins in the afferent microartery channel 5, the efferent capillary network channel 6, and the renal tubule channel 7. The aim is to allow the organoid to adapt to the microarray microenvironment and to enable the three types of cells (afferent artery endothelial cells, capillary network endothelial cells, and organoid internal cells) to begin "sensing" each other's presence, inducing angiogenesis through the secretion of factors such as VEGF.

[0062] Phase 3: Functional Induction and Pulsating Flow Activation (After Day 7) From day 7 onwards: Physiological pulsatile flow is initiated. An arterial-like pulsatile flow (frequency 1–2 Hz, simulating real arterial waveforms) is applied to the afferent microartery channel 5 to drive endothelial cell maturation and induce vascular directional invasion into the glomeruli within the organoid. The flow pattern in the efferent capillary network channel 6 transitions to near-continuous laminar flow (low shear force), simulating real peritubular capillary blood flow. A continuous, slow laminar flow is applied to the renal tubule channel 7, simulating the flow of primary urine in the renal tubules (flow rate 0.5–1.0 nL / min).

[0063] (II) Functional Verification 1. Filtration barrier verification: Fluorescently labeled molecules of different sizes (e.g., 3 kDa and 70 kDa dextran) were perfused into the afferent microartery channel 5. Small-molecule dextran was found in the collection fluid at the outlet 72 of the renal tubular channel 7, while large molecules were effectively blocked, demonstrating the formation of a functional filtration barrier.

[0064] 2. Verification of reabsorption function: Physiological concentrations of glucose or urea nitrogen were added to the culture medium in the afferent microartery channel 5, and the concentration changes of these substances at the outlet 72 of the renal tubular channel 7 were detected. If the concentration was significantly lower than that at the inlet, it indicated that reabsorption had occurred.

[0065] 3. Disease Simulation: Using iPSC-induced kidney organoids from patients with polycystic kidney disease, pulsatile flow was applied to this chip. Studies have shown that pulsatile flow alone is sufficient to induce cyst formation without the need for drug induction, providing a powerful tool for studying the role of mechanical forces in disease development.

[0066] Industrial applicability The biomimetic capillary network-coupled renal function microfluidic chip and its usage method provided by this invention can construct a highly biomimetic nephron functional model in vitro. By integrating glomerular filtration and tubular reabsorption processes on the same platform, dynamic simulation of key physiological functions of the kidney can be achieved. It can be widely applied in drug screening and nephrotoxicity evaluation, research on the mechanisms of kidney-related diseases (such as acute kidney injury and chronic kidney disease), research on organoid development and functional regulation, and personalized precision medicine assessment based on patient-derived cells. Simultaneously, this chip can also serve as a key module in multi-organ-on-a-chip systems for constructing in vitro "human-on-a-chip" models, possessing significant industrial practical value in fields such as new drug development, regenerative medicine, and biomedical engineering.

[0067] The apparatus and methods disclosed in the embodiments are described in a relatively simple manner since they correspond to the methods disclosed in the embodiments. For relevant details, please refer to the description in the method section.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A biomimetic capillary network coupled renal function microfluidic chip, characterized in that, include: A superimposed upper chip layer (1) and a lower chip layer (2), and a porous membrane (3) disposed between the upper chip layer (1) and the lower chip layer (2); The lower chip layer (2) is provided with a central groove (4), an afferent microartery channel (5), and an efferent capillary network channel (6). The top opening of the central groove (4) extends to the upper chip layer (1). The central groove (4) is divided by the porous membrane (3) into an organoid receiving area above the porous membrane (3) and a fluid distribution area below the porous membrane (3). The afferent microartery channel (5) and the efferent capillary network channel (6) are respectively connected to the fluid distribution area. The efferent capillary network channel (6) includes at least two levels of bifurcated structures. The lower chip layer (2) is provided with a collection groove (10) downstream of the efferent capillary network channel (6). The upper chip layer (1) is provided with a renal tubular channel (7), which is arranged vertically opposite to the efferent capillary network channel (6) and separated by the porous membrane (3).

2. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The bottom of the central groove (4) of the lower chip layer (2) is provided with a micropillar array (8), and the porous membrane (3) is disposed above the micropillar array (8) and supported by the micropillar array (8).

3. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The ingress microartery channel (5) is a serpentine microchannel with a width of 80-120 μm and a height of 40-60 μm.

4. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The bifurcation structure of the efferent capillary network channel (6) includes at least a primary channel, a secondary channel and a tertiary channel. The secondary channel is formed by bifurcation of the primary channel, and the tertiary channel is formed by bifurcation of the secondary channel. The bifurcation angle of the secondary channel and the tertiary channel is 40° to 75°.

5. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The renal tubular channel (7) is a serpentine channel, located in the upper chip layer (1) in the region corresponding to the efferent capillary network channel (6). The width of the renal tubular channel (7) is greater than the width of the efferent capillary network channel (6).

6. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The porous membrane (3) has a pore size of 0.4 to 10 μm and at least covers the opening area of ​​the central groove (4) and the area between the renal tubular channel (7) and the efferent capillary network channel (6).

7. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The upper chip layer (1) has an opening at the position corresponding to the organoid accommodating area, and the opening is provided with an openable cover plate (9).

8. The renal function microfluidic chip coupled with a biomimetic capillary network according to claim 1, characterized in that, The upper chip layer (1) is provided with an inlet (71) and an outlet (72) at both ends of the renal tubule channel (7). The upper chip layer (1) is provided with a drainage hole that extends to the collection tank (10) and the drainage hole is connected to an external drainage tube.

9. A renal function microfluidic chip coupled with a biomimetic capillary network according to claim 8, characterized in that, The liquid collection tank (10) is an arc-shaped tank, and the drainage holes are multiple and arranged at equal arc length intervals along the arc path.

10. A method of using a biomimetic capillary network coupled renal function microfluidic chip, employing the chip described in any one of claims 1-9, characterized in that, include: The kidney organoid is placed in the organoid receiving area, so that the kidney organoid comes into contact with the porous membrane; Perfusion fluid is injected through the afferent microartery channel, allowing the perfusion fluid to pass through the porous membrane in the fluid distribution area and exchange substances with the renal organoids, before flowing out from the collection trough downstream of the efferent capillary network channel; Pulsating flow was applied to the ingressor microartery channel for perfusion culture.

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