Renal tubule and pericaeal microenvironment simulation system based on vein-imitating network chip and construction method and application of renal tubule and pericaeal microenvironment simulation system

Abstract

The invention relates to a simulated vein network chip-based renal tubule and peripheral microenvironment simulation system and a construction method and application thereof. The system comprises a simulated vein network micro-fluidic chip, immortalized human proximal renal tubule epithelial cells, human umbilical vein endothelial cells and a cell culture medium, the method comprises the following steps: respectively pouring cell suspensions of immortalized human near-end renal tubular epithelial cells and immortalized human near-end renal tubular epithelial cells and human umbilical vein endothelial cells into a chip, and continuously injecting a cell culture medium to simulate an in-vivo liquid flowing microenvironment after the cells are adhered to the wall, so as to obtain a corresponding simulation system; the system can effectively simulate close cell interaction and signal communication between a renal tubule and a peripheral capillary in a dynamic fluid environment, and provides a high-fidelity in-vitro research tool for renal tubule physiological mechanism research, chronic injury pathological change analysis and a hereditary nephropathy model; meanwhile, the method can also be used for evaluating the kidney clearance rate characteristic and the kidney toxicity risk of the medicine.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and organ-on-a-chip technology, specifically to a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip, its construction method, and its applications. Background Technology

[0002] Kidney disease is a major global health challenge. According to the 2023 International Society of Nephrology (ISN) Global Kidney Health Atlas, the median prevalence of chronic kidney disease worldwide is as high as 9.5%, with an associated mortality rate of 2.4%. Currently, the progression of kidney disease is considered irreversible, and patients with end-stage renal disease must rely on renal replacement therapy, while kidney transplantation is the only curative treatment. However, current treatment models face severe challenges: dialysis is expensive and accompanied by various complications, seriously affecting patients' quality of life; while kidney transplantation, the best option, has long been limited by a severe shortage of donor organs. Therefore, the global burden of kidney disease is heavy, and there is an urgent need to conduct in-depth research on the mechanisms of kidney damage in order to develop more effective treatments or innovative renal replacement therapies.

[0003] With the rapid development of tissue engineering and microfluidics, organ-on-a-chip technology has become an important platform for simulating the complex structure and physiological microenvironment of human organs in vitro, and is expected to revolutionize drug development and life science research. This technology simulates key physiological conditions in vivo by efficiently delivering nutrients, applying fluid shear stress stimulation, and constructing a three-dimensional culture environment within a microfluidic system. In the field of kidneys, the glomerular filtration function can be well simulated by various biomaterials, but effective methods for simulating complex functions such as renal tubular reabsorption still lack. Although organ-on-a-chip technology has made significant progress, most existing renal tubular chip models suffer from overly simple structures and insufficient biomimicry. Specifically, challenges include: precisely controlling the peritubular microenvironment (such as higher-order biochemical signal transduction and gradient distribution), inducing physiological-level fluid shear stress, increasing model complexity (such as integrating the vascular system, stromal cells, or immune cells), simulating multi-organ interactions, and reducing the model's own variability (such as batch-to-batch differences in renal tubular size, structure, and function). Therefore, developing novel biomimetic chips that can highly simulate renal physiological functions, especially the complex microenvironment of the renal tubules, is crucial for advancing kidney research and achieving in vitro functional replacement.

[0004] Constructing functional renal tubular microarrays is highly dependent on suitable cell sources. Currently, the most commonly used cells for such models include various immortalized renal tubular epithelial cell lines, such as MDCK cells from canine kidneys, LLC-PK1 cells from porcine kidneys, and human HK-2 cells. However, these cell lines cannot fully reproduce the phenotype of primary cells and often exhibit insufficient functional differentiation during culture, limiting the physiological relevance of the models. Human primary proximal tubular epithelial cells are generally considered the "gold standard" for in vitro modeling, as they better preserve in vivo function, but their application faces significant bottlenecks: the cell source exhibits significant donor variability, limited self-renewal and expansion capabilities, resulting in low experimental reproducibility and difficulty in obtaining sufficient cell quantities for high-throughput studies. Although the passage number can be extended through special treatments (such as using antisense nucleotides or RNA interference), the problems of donor variability and standardization have not been fundamentally solved.

[0005] In summary, although microfluidic technology and the organ-on-a-chip concept provide powerful tools for kidney research, and some studies have attempted to use biomimetic design to improve chip performance—for example, Gershlak et al. (Gershlak JR, Hernandez S, Fontana G, et al. Crossing kingdoms: Using decellularized plants asperfusable tissue engineering scaffolds. Biomaterials. 2017. 125: 13-22.) pioneered the use of decellularized plant leaf veins as perfusion-compatible tissue engineering scaffolds, demonstrating the potential of natural leaf vein networks in transporting fluids and supporting cell growth; and Professor He Jiankang's team in China successfully constructed a highly realistic "renal tubule-interstitial" chip using microfluidic chip tissue engineering technology—this field is still on the eve of a critical breakthrough. Even some more advanced renal tubular microarray models, such as those based on the high-throughput 3D microfluidic platform Nephrscreen (Naik S, Wood AR, Ongenaert M, et al. A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy. Int J Mol Sci. 2021. 22(10): 5361.), and models constructed using conditionally immortalized or primary cells (Vriend J, Nieskens T, Vormann MK, et al. Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip. AAPS J. 2018. 20(5): ), may not be fully realized. 87.), There are still fundamental deficiencies in the simulation of core physiological functions, mainly reflected in: difficulty in achieving full polarization of renal tubular epithelial cells and stable high expression of functional transport proteins in vitro; inability to effectively simulate the complex material exchange and interaction between renal tubular epithelium and vascular endothelium, as well as the real renal tubular peritubular microenvironment. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention aims to provide a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip, its construction method and application. This system can effectively simulate the close cell interaction and signal communication between the renal tubule and peritubular capillaries in a dynamic fluid environment, providing a high-fidelity in vitro research tool for the study of renal tubular physiological mechanisms, analysis of pathological changes in chronic injury and hereditary nephropathy models. It can also be used to evaluate the renal clearance characteristics and nephrotoxicity risk of drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A renal tubule and peritubular microenvironment simulation system based on a leaf vein-inspired network chip includes a biomimetic leaf vein network microfluidic chip, immortalized human proximal renal tubule epithelial cells, human umbilical vein endothelial cells, and cell culture medium. The microfluidic channel network of the biomimetic leaf vein network microfluidic chip is in the form of a renal tubule structure, and a cell layer is attached to the inner wall of the lumen of the microfluidic channel network. When the cell layer is immortalized human proximal renal tubule epithelial cells, a renal tubule simulation system is formed. When the cell layer is a cell suspension of immortalized human proximal renal tubule epithelial cells and human umbilical vein endothelial cells, a renal tubule peritubular microenvironment simulation system is formed. The cell activity in the renal tubule simulation system and the renal tubule peritubular microenvironment simulation system is maintained by continuously perfusing fresh cell culture medium into the biomimetic leaf vein network microfluidic chip and draining metabolically degraded cell culture medium. The ratio of immortalized human proximal renal tubule epithelial cells to human umbilical vein endothelial cells in the renal tubule peritubular microenvironment simulation system is 1:2.

[0008] A method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein-inspired network chip includes the following steps: Step S1: Preprocess the biomimetic leaf vein network microfluidic chip; Step S2: Assemble the pretreated biomimetic leaf vein network microfluidic chip on the fixing device, and connect one end of the inlet tube and one end of the outlet tube to the inlet and outlet of the microfluidic channel network of the biomimetic leaf vein network microfluidic chip, respectively. Connect the other end of the inlet tube to the syringe, which can be detachably clamped to the micro-injection pump. Connect the other end of the outlet tube to the recovery cup to complete the assembly of the entire bioreactor. Step S3: Place the assembled bioreactor under a UV lamp and rinse the biomimetic leaf vein network microfluidic chip with sterile PBS for sterilization. Step S4: After sterilization of the bioreactor, the target cells are injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip using a microinjection pump. The perfusion of target cells is complete when target cells flow out of the recovery cup. The bioreactor with perfused target cells is then transferred to a cell culture incubator and allowed to stand to allow the target cells to adhere. After the target cells adhere, fresh cell culture medium is continuously injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip at a preset flow rate using a microinjection pump. The old cell culture medium is then discharged through the outlet of the microfluidic channel network to achieve dynamic replacement of the cell culture medium, simulating the in vivo fluid flow microenvironment and maintaining the in vitro activity of the target cells. When the target cells are only immortalized human proximal renal tubular epithelial cells, the simulation system is a renal tubule simulation system. When the target cells are a cell suspension of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells in a ratio of 1:2, the simulation system is a renal tubular peritubular microenvironment simulation system.

[0009] Furthermore, the biomimetic leaf vein network microfluidic chip includes a PDMS leaf vein network underchip and a PDMS top cover chip that is laminated and bonded to the PDMS leaf vein network underchip.

[0010] Furthermore, the fabrication method of the chip under the PDMS leaf vein network specifically includes: Rinse fresh osmanthus leaves with deionized water, then soak the rinsed fresh osmanthus leaves in a 3.5-5 wt% NaOH solution, heat to boiling and maintain for 30-40 minutes. Remove the leaves and remove any remaining leaf tissue to obtain the leaf vein network skeleton. Air dry the leaf vein network skeleton to obtain a leaf vein network skeleton specimen. The leaf vein network skeleton specimen was photographed to obtain a high-resolution digital image of the leaf vein network skeleton. This high-resolution image was then imported into LEAF GUI software. The image binarization algorithm in LEAF GUI software was used to process the leaf vein network skeleton into black, and the non-flow channel areas outside the leaf vein network skeleton into white, resulting in a binarized image of the leaf vein network skeleton. The integrity of the leaf vein network skeleton specimen was observed. If the skeleton structure was intact, a 400-500nm chromium coating was sputtered onto the leaf vein network skeleton specimen to obtain chromium-plated leaf veins. If the skeleton structure was incomplete, a fresh osmanthus leaf was selected to prepare a new leaf vein network skeleton specimen. Hexamethyldisilane is spin-coated onto a silicon wafer and cured to form a 3-4 μm thick hexamethyldisilane coating. A photosensitive polymer is spin-coated onto the surface of the hexamethyldisilane coating and cured to form a 3-4 μm thick photosensitive polymer coating. The silicon wafer with the hexamethyldisilane coating and the photosensitive polymer coating is placed in an exposure station. A chromium-plated leaf vein is used as a photomask and aligned with the surface of the silicon wafer. The silicon wafer is irradiated with ultraviolet light. The ultraviolet-irradiated silicon wafer is then placed in a 0.5-1 wt% NaOH solution. The photosensitive polymer decomposes in the NaOH solution to obtain a silicon wafer with a leaf vein network negative polymer pattern. A chromium layer with a thickness of 150~200 nm is uniformly sputtered onto a silicon wafer with a leaf vein network negative polymer pattern. After the chromium layer is deposited, it is immersed in an acetone solution to dissolve the leaf vein network negative polymer pattern, thus obtaining a chromium-plated silicon wafer. The chromium-plated silicon wafer is then etched to obtain a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern. A first vacuum degassing treatment is performed on a mixed solution of polydimethylsiloxane prepolymer and curing agent with a mass ratio of (9~10):1. An alkylation treatment is then performed on a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern. The mixed solution of polydimethylsiloxane prepolymer and curing agent after the first vacuum degassing treatment is cast onto the alkylated silicon wafer leaf vein mold, and a second vacuum degassing treatment is performed. After the second vacuum degassing treatment, the mixed solution of polydimethylsiloxane prepolymer and curing agent in the silicon wafer leaf vein mold is dried and cured. After the mixed solution of polydimethylsiloxane prepolymer and curing agent has cured and formed, the silicon wafer leaf vein mold with the leaf vein network negative polymer pattern is peeled off to obtain a PDMS leaf vein network underchip with a leaf vein network negative polymer pattern.

[0011] Furthermore, the method for preparing the PDMS cover sheet specifically includes: A first vacuum degassing treatment was performed on a mixed solution of polydimethylsiloxane prepolymer and curing agent with a mass ratio of (9~10):1. The mixed solution of polydimethylsiloxane prepolymer and curing agent after the first vacuum degassing treatment was cast into a cell culture dish and subjected to a second vacuum degassing treatment. After the second vacuum degassing treatment, the mixed solution of polydimethylsiloxane prepolymer and curing agent in the cell culture dish was dried and cured. After the mixed solution of polydimethylsiloxane prepolymer and curing agent was cured and shaped, the cell culture dish was peeled off to obtain a PDMS cover sheet.

[0012] Furthermore, the method for preparing the immortalized human proximal renal tubular epithelial cells specifically includes: a) Primary human renal epithelial cells were resuscitated, resuspended in DMEM / F12 complete medium preheated to 37°C, and seeded into cell culture dishes at a cell density of 2-4*10^6. The cells were then cultured in a cell culture incubator at 37°C and 5% CO2. The DMEM / F12 complete medium consisted of 90% DMEM / F12 basal medium, 10% fetal bovine serum, and 0.5-1% penicillin-streptomycin antibiotics. b) When the primary human renal epithelial cells reach 70-80% confluence, discard the old DMEM / F12 complete medium and replace it with DMEM / F12 basal medium. Infect the primary human renal epithelial cells with lentivirus SV40T and lentivirus hTERT for 12-24 hours in a cell culture incubator at 37°C and 5% CO2. The MOI value of the lentivirus SV40T is 150, the MOI value of the lentivirus hTERT is 150, and the molar ratio of the lentivirus SV40T to the lentivirus hTERT is 1:1. c) After infection, discard the old DMEM / F12 basal medium and culture the cells in a 37°C, 5% CO2 cell culture incubator using DMEM / F12 complete medium containing 1 μg / mL Puromycin for 48-96 h. d) After the selection culture is completed, discard the old DMEM / F12 complete medium containing Puromycin and use the new DMEM / F12 complete medium for routine passage culture, passage once every 48~96 hours to obtain semi-immortalized human kidney epithelial cells. e) After passage 2-3 times, when the semi-immortalized human renal epithelial cells have grown to 80-90% confluence, identify the passaged semi-immortalized human renal epithelial cells by immunofluorescence staining; if the identified semi-immortalized human renal epithelial cells successfully express rabbit polyclonal antibody CK-18, immortalized human proximal renal tubular epithelial cells are obtained; if the identified semi-immortalized human renal epithelial cells do not successfully express rabbit polyclonal antibody CK-18, repeat step ae until rabbit polyclonal antibody CK-18 is successfully expressed.

[0013] Further, step S1 specifically includes: The biomimetic leaf vein network microfluidic chip was subjected to plasma hydrophilization treatment, sterilization treatment, and gelatin treatment in sequence.

[0014] The use of a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip as described above, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip constructed by any of the above methods, for simulating renal tubules and peritubular capillaries in vitro.

[0015] The application of a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip as described above, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip constructed by any of the above methods, in evaluating the renal clearance characteristics and nephrotoxicity risk of drugs, characterized in that: the drugs include, but are not limited to, antitumor drugs, antibiotics, and immunosuppressants.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention constructs a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip, which solves the problem that traditional in vitro two-dimensional static culture models cannot simulate the three-dimensional spatial configuration, material exchange interface and mechanical microenvironment of renal tubules and peritubular capillaries in vivo. It achieves the effect of supporting the formation of apical-basal polar functional monolayers of renal tubular epithelial cells and long-term stable expression of key transport proteins (such as OAT1 and OCT2) under dynamic fluid conditions. It has the advantages of the model being closer to physiology, maintaining cell function for a longer time, and expressing more stable proteins.

[0017] 2. This invention fabricates a microfluidic chip with a biomimetic leaf vein network structure, which solves the problem that existing organ-on-a-chip technology is unable to accurately mimic the complex branch structure of the peritubular capillary network in vivo at the micrometer scale. It achieves the effect of simulating the morphology of the capillary network, realizing controllable fluid perfusion, and forming a stable co-culture interface with the renal tubular lumen. It has the advantages of high structural biomimicry, uniform fluid distribution, good fabrication repeatability, and easy integration and expansion.

[0018] 3. Based on the construction of a biomimetic leaf vein network microfluidic chip, this invention integrates a two-cell renal tubule peritubular microenvironment simulation system with immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells as core functional components. This solves the problem that most existing models are single-cell cultures or simple mixed cultures, which cannot effectively simulate the close cell interaction and signal communication between renal tubules and peritubular capillaries in a three-dimensional spatial configuration and dynamic fluid environment. It achieves the effect of reproducing the cell-cell interaction in vivo and supporting the transport of substances across the barrier and paracrine signal transduction. It has the advantages of high physiological relevance of the model and the ability to more realistically study the key cell interaction mechanisms in the renal microenvironment homeostasis, damage and repair process.

[0019] 4. This invention prepares immortalized human proximal renal tubular epithelial cells using primary human renal epithelial cells, solving the problem that the expression levels of multiple key reabsorption transport proteins in in vitro culture of commonly used immortalized cell lines (such as HK-2) are significantly lower than in vivo levels, leading to insufficient model functionality. This invention provides a functionally enhanced renal tubular epithelial cell source that can be passaged for a long time. It can stably and highly express multiple key transport proteins in a biomimetic leaf vein network microfluidic chip system, and has the advantages of reliable cell source, functional characterization that is closer to the human physiological state, and significantly improved accuracy of in vitro renal tubular model function prediction.

[0020] 5. The renal tubule simulation system based on a leaf vein-like network chip of the present invention can effectively reproduce the active transport, reabsorption, and secretion functions of the renal tubule by simulating the renal tubule, providing a high-fidelity in vitro research tool for the study of renal tubular physiological mechanisms, analysis of pathological changes in chronic injury, and models of hereditary nephropathy. The renal tubule peritubular microenvironment simulation system based on a leaf vein-like network chip of the present invention can effectively reproduce the material exchange and mechanical stimulation between the renal tubule and capillary by simulating the peritubular capillaries of the renal tubule and applying physiologically relevant fluid shear forces, and can be used to study the renal tubular barrier function, acute injury response, and the interaction mechanism between renal tubular epithelial cells and vascular endothelial cells.

[0021] In summary, this invention constructs a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip. This system can effectively simulate the close cell interaction and signal communication between the renal tubule and peritubular capillaries in a dynamic fluid environment, providing a high-fidelity in vitro research tool for the study of renal tubular physiological mechanisms, analysis of pathological changes in chronic injury, and hereditary nephropathy models. It can also be used to evaluate the renal clearance characteristics and nephrotoxicity risk of drugs. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the construction method of the renal tubule and peritubular microenvironment simulation system based on a leaf vein-like network chip according to the present invention.

[0023] Figure 2 This is a binarized image of the leaf vein network skeleton of the present invention.

[0024] Figure 3 This is a schematic diagram illustrating the process of peeling off a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern according to the present invention; wherein, Figure 3 (a) in the figure is a schematic diagram of the bonding state of the silicon wafer leaf vein mold and the chip under the PDMS leaf vein network before peeling; Figure 3 (b) is a schematic diagram of the chip under the PDMS leaf vein network after stripping.

[0025] Figure 4 This is a schematic diagram of the chip under the PDMS leaf vein network of the present invention.

[0026] Figure 5 This is a schematic diagram of the assembly structure of the bioreactor of the present invention.

[0027] Figure 6 This is a schematic diagram illustrating the hydrodynamic analysis of the microfluidic channel network of the chip under the PDMS leaf vein network of the present invention; wherein, Figure 6 (a) in the figure represents the fluid shear force of the microfluidic channel network when the main flow velocity is 5 μL / min; Figure 6 (b) represents the fluid shear force of the microfluidic channel network when the main flow velocity is 15 μL / min; Figure 6 (c) represents the fluid shear force of the microfluidic channel network when the main flow velocity is 30 μL / min; Figure 6 In the figure, (d) represents the pressure of the microfluidic channel network when the main flow velocity is 5 μL / min; Figure 6 In the figure, (e) represents the pressure of the microfluidic channel network when the main flow velocity is 15 μL / min; Figure 6 In the figure, (f) represents the pressure of the microfluidic channel network when the main flow velocity is 30 μL / min; Figure 6 In this context, (g) represents the flow rate of the microfluidic channel network when the main channel flow rate is 5 μL / min; Figure 6 In this context, (h) represents the flow rate of the microfluidic channel network when the main channel flow rate is 15 μL / min; Figure 6 In the figure, (i) represents the flow rate of the microfluidic channel network when the main channel flow rate is 30 μL / min.

[0028] Figure 7 This image shows the adhesion status and activity detection results of immortalized human proximal renal tubular epithelial cells in the renal tubular simulation system of the present invention; wherein... Figure 7 (a) shows the adhesion status and cell morphology of immortalized human proximal renal tubular epithelial cells in a PDMS leaf vein network chip; Figure 7 (b) is an AOPI staining image of immortalized human proximal renal tubular epithelial cells in a PDMS leaf vein network chip.

[0029] Figure 8 These are images showing the adhesion state and immunofluorescence staining identification of immortalized human proximal renal tubule epithelial cells and human umbilical vein endothelial cells in the renal tubular peritubular microenvironment simulation system of the present invention; wherein, Figure 8 (a) shows the adhesion state of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells in a PDMS leaf vein network chip; Figure 8 (b) shows the immunofluorescence staining identification of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells in a PDMS leaf vein network chip.

[0030] Figure 9This is a comparison image of immunofluorescence staining identification of the renal tubule simulation system of the present invention and immortalized human proximal renal tubule epithelial cells in a conventional two-dimensional culture dish; wherein, Figure 9 Image (a) shows the immunofluorescence staining identification of immortalized human proximal renal tubular epithelial cells in a PDMS leaf vein network chip; Figure 9 Image (b) shows the immunofluorescence staining identification of immortalized human proximal renal tubular epithelial cells in a PDMS leaf vein network chip.

[0031] Figure 10 Immunofluorescence staining comparison of reabsorption characterization of the renal tubule simulation system of this invention and immortalized human proximal renal tubule epithelial cells in conventional two-dimensional culture dishes. Figure 1 ;in, Figure 10 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells within a renal tubular mimicry system. Figure 1 ; Figure 10 (b) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells in a conventional two-dimensional culture dish. Figure 1 .

[0032] Figure 11 Immunofluorescence staining comparison of reabsorption characterization of the renal tubule simulation system of this invention and immortalized human proximal renal tubule epithelial cells in conventional two-dimensional culture dishes. Figure 2 ;in, Figure 11 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells within a renal tubular mimicry system. Figure 2 ; Figure 11 (b) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells in a conventional two-dimensional culture dish. Figure 2 .

[0033] Figure 12 This invention provides a comparison of the reabsorption characterization immunofluorescence staining of the renal tubular peritubular microenvironment simulation system of the present invention with that of immortalized human proximal renal tubular epithelial cells in conventional two-dimensional culture dishes. Figure 1 ;in, Figure 12 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells within a renal tubular peritubular microenvironment simulation system. Figure 1 ; Figure 12 (b) shows the immunofluorescence staining results of immortalized human proximal renal tubular epithelial cells in a conventional two-dimensional culture dish. Figure 1 .

[0034] Figure 13This invention provides a comparison of the reabsorption characterization immunofluorescence staining of the renal tubular peritubular microenvironment simulation system of the present invention with that of immortalized human proximal renal tubular epithelial cells in conventional two-dimensional culture dishes. Figure 2 ;in, Figure 13 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells within a renal tubular peritubular microenvironment simulation system. Figure 2 ; Figure 13 (b) shows the immunofluorescence staining results of immortalized human proximal renal tubular epithelial cells in a conventional two-dimensional culture dish. Figure 2 .

[0035] Figure 14 These are schematic diagrams of three culture systems of the present invention; wherein, Figure 14 (a) in the text represents the standard culture system in 2D petri dishes; Figure 14 (b) in the figure represents the 2.5D transwell culture system; Figure 14 (c) in the figure represents the renal tubule simulation system based on a 3D leaf vein network chip.

[0036] Figure 15 The figures show the results of glucose quantitative reabsorption rate determination in immortalized human proximal renal tubular epithelial cells in conventional 2D culture dish culture system, 2.5D transwell chamber culture system, and 3D leaf vein network chip-based renal tubular simulation system, respectively. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments: The core reagents involved in this invention include: PDMS dimethylsilane (Dow Corning / China), hexamethyldisilane (Aladdin / China), DMEM / / F12 basal medium (Cellmax / China), paraformaldehyde (Saiwell / China), Triton X-100 (Beyotime / China), fetal bovine serum (Fetal Bovine Serum, Sigma / USA), penicillin-streptomycin (GIBCO / China), 0.25% Trypsin (1X), 0.25% Trypsin (1X), Phenol Red, with EDTA (Synthetium / China), rabbit polyclonal antibody SGLT2 (Proteintech / China), rabbit polyclonal antibody OAT3 (Aibotek / China), mouse polyclonal antibody GLUT2 (Proteintech / China), mouse polyclonal antibody AQP1 (Proteintech / China), rabbit polyclonal antibody CK-18 (Proteintech / China), CoraLite488-conjugated Goat Anti-Mouse IgG (Proteintech / China), CoraLite594-conjugated Goat Anti-Rabbit IgG (Proteintech / China), PBS (Saiwell / China).

[0038] A renal tubule and peritubular microenvironment simulation system based on a leaf vein-inspired network chip includes a biomimetic leaf vein network microfluidic chip, immortalized human proximal renal tubule epithelial cells, human umbilical vein endothelial cells, and cell culture medium. The microfluidic channel network of the biomimetic leaf vein network microfluidic chip has a renal tubule structure, and the inner wall of the microfluidic channel network's lumen is attached with a cell layer. When the cell layer is immortalized human proximal renal tubule epithelial cells, a renal tubule simulation system is formed; when the cell layer is immortalized human proximal renal tubule epithelial cells... When the cell suspension of human umbilical vein endothelial cells is mixed with the renal tubule peritubular microenvironment simulation system, a renal tubule peritubular microenvironment simulation system is formed. Then, by continuously perfusing fresh cell culture medium into the biomimetic leaf vein network microfluidic chip and discharging the old cell culture medium after metabolism, the microenvironment of fluid flow in the body is simulated, thereby maintaining the cell activity in the renal tubule simulation system and the renal tubule peritubular microenvironment simulation system. The ratio of immortalized human proximal renal tubule epithelial cells to human umbilical vein endothelial cells in the renal tubule peritubular microenvironment simulation system is 1:2.

[0039] The human umbilical vein endothelial cells in this embodiment were obtained from Suzhou Haixing Biotechnology Co., Ltd. The introduced cell culture medium was DMEM / F12 complete medium, which consists of 90% DMEM / F12 basal medium, 10% fetal bovine serum and 0.5~1% penicillin-streptomycin double antibiotic.

[0040] See Figure 1 A method for constructing a simulation system of renal tubules and peritubular microenvironment based on a leaf vein-inspired network chip, specifically including the following: Step S1: Preprocess the biomimetic leaf vein network microfluidic chip; The fabrication method of the biomimetic leaf vein network microfluidic chip specifically includes: Preparation of leaf veins: Rinse fresh osmanthus leaves with deionized water, then soak the rinsed fresh osmanthus leaves in a 3.5-5 wt% NaOH solution, heat to boiling and maintain for 30-40 minutes. Remove the leaves and brush them with a soft brush to remove any remaining leaf tissue, thus obtaining the leaf vein network skeleton. Place the leaf vein network skeleton in a fume hood at 20-25℃ and air dry for 12-14 hours to obtain the leaf vein network skeleton specimen. Leaf vein network skeleton screening: Leaf vein network skeleton specimens were photographed using a single-lens reflex camera to obtain high-resolution digital images of the leaf vein network skeleton. These images were then imported into LEAF GUI (Leaf Extraction and Analysis Framework Graphical User Interface) software. The image binarization algorithm in LEAF GUI software was used to process the leaf vein network skeleton into black, while the non-flow channel areas outside the leaf vein network skeleton were processed into white, resulting in the following image: Figure 2 The binarized image of the leaf vein network skeleton with distinct boundaries is shown. The integrity of the leaf vein network skeleton specimen is observed. If the skeleton structure is complete, a 400-500 nm chromium coating is sputtered onto the leaf vein network skeleton specimen using an Explorer14 magnetron sputtering system (Denton Vacuum, America) to obtain chromium-plated leaf veins. If the skeleton structure is incomplete, fresh osmanthus leaves need to be selected again to prepare a new leaf vein network skeleton specimen. Furthermore, the binarized image of the leaf vein network skeleton in this embodiment is also used as an analysis template for subsequent leaf vein flow channel prediction, thereby obtaining the evaluation values ​​of the pressure field, shear stress field, and flow velocity of the leaf vein network skeleton through fluid dynamics analysis software. Preparation of leaf veins on silicon wafers: Hexamethyldisilane (HMDS) is spin-coated onto a silicon wafer (i.e., silicon slab) at a speed of 900~1000 r / min, and cured at 95~100℃ for 15~20 min to form a hexamethyldisilane coating with a thickness of 3~4 μm; a photosensitive polymer is then applied at 700... Spin-coating a photosensitive polymer coating with a viscosity of ~800 r / min onto a viscous hexamethyldisilane coating surface and curing it at 95~100℃ for 15~20 min to form a photosensitive polymer coating with a thickness of 3~4 μm; placing the silicon wafer with the hexamethyldisilane coating and photosensitive polymer coating onto an exposure station, using chromium-plated leaf veins as a photomask and aligning it with the surface of the silicon wafer, irradiating the silicon wafer with ultraviolet light for 12~20 seconds, and then placing the ultraviolet-irradiated silicon wafer into a 0.5~1 wt% NaOH solution, allowing the photosensitive polymer to decompose in the NaOH solution, to obtain a silicon wafer with a leaf vein network negative polymer pattern; Preparation of silicon wafer leaf vein mold: A chromium layer with a thickness of 150~200 nm is uniformly sputtered onto a silicon wafer with a leaf vein network negative polymer pattern. After the chromium layer is deposited, it is immersed in an acetone solution to dissolve the leaf vein network negative polymer pattern, thus obtaining a chromium-plated silicon wafer. The chromium-plated silicon wafer is then dry-etched in an inductively coupled plasma (ICP) etching machine for 100~120 min to obtain a microfluidic channel network with a depth of 150 μm, thereby obtaining a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern. In this embodiment, the process gas pressure set during sputtering is 0.5~1.0 Pa, and the deposition time after sputtering is 10~15 minutes. Fabrication of PDMS Leaf Vein Network Chip: A polydimethylsiloxane (PDMS) prepolymer solution and curing agent are uniformly mixed at a mass ratio of (9~10):1. The mixture is then subjected to a first vacuum degassing treatment to remove air bubbles. An alkylation treatment is performed on a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern at 20~25℃ for 20-30 minutes. The PDMS prepolymer and curing agent mixture after the first vacuum degassing treatment is cast onto the alkylated silicon wafer leaf vein mold, and a second vacuum degassing treatment is performed in a vacuum chamber. After the second vacuum degassing treatment, the mixture is dried and cured in an oven at 50~60℃. Once the PDMS prepolymer and curing agent mixture has cured and formed, as shown... Figure 3 (a) and Figure 3 As shown in (b), the silicon wafer leaf vein mold with leaf vein network negative polymer pattern is peeled off to obtain a PDMS leaf vein network under chip with leaf vein network negative polymer pattern; the alkylating agent in this embodiment is perfluorosilane with a mass fraction of 1~2%, and the thickness of the PDMS leaf vein network under chip is 2~3mm. from Figure 2 and Figure 4 As can be seen, the leaf vein network negative polymer pattern in this embodiment is a microfluidic channel network of a biomimetic leaf vein network microfluidic chip, which is modeled after the structure of a human renal tubule. This renal tubule structure includes a main channel with openings at both ends, and multiple secondary channels that are directly and indirectly connected to the main channel. The openings at both ends of the main channel are an inlet and an outlet, respectively. The inner diameter of the main channel is larger than the inner diameter of the secondary channels. In this embodiment, the inner diameter of the main channel is 100~150μm, and the smallest inner diameter of the secondary channels is 10~20μm. The diameters of the main channel and the secondary channels are close to the diameter range of physiological renal tubules.

[0041] Preparation of PDMS cover sheet: A polydimethylsiloxane prepolymer solution and a curing agent were uniformly mixed at a mass ratio of (9-10):1. The mixture was then subjected to a first vacuum degassing treatment to remove air bubbles. The degassed solution was then poured into a cell culture dish with an inner diameter of 9 cm and a thickness of 3-4 mm, and subjected to a second vacuum degassing treatment in a vacuum chamber. After the second degassing treatment, the mixture was dried and cured in an oven at 50-60°C. Once the mixture had solidified, the cell culture dish was peeled off to obtain PDMS. Top cover sheet; In this embodiment, the mass ratio of polydimethylsiloxane (PDMS) prepolymer solution to curing agent is 1:10, and Dow Corning DC184 kit is used. The core components of this kit are polydimethylsiloxane (PDMS) prepolymer solution and curing agent. The chip under the PDMS leaf vein network is cut to center the negative polymer pattern of the leaf vein network under the chip; then the PDMS cover is cut to the same size as the chip under the PDMS leaf vein network; in this embodiment, the cutting size is 8cm long and 4cm wide. The PDMS leaf vein network underchip and PDMS cover sheet are integrated together by lamination bonding to obtain a biomimetic leaf vein network microfluidic chip. During the lamination bonding process, the bottom surface of the PDMS cover sheet is tightly attached to and completely covers the leaf vein network negative polymer pattern of the PDMS leaf vein network underchip, so that a flow channel with open ends and closed sides is formed inside the biomimetic leaf vein network microfluidic chip.

[0042] Step S1 specifically includes: The biomimetic leaf vein network microfluidic chip was subjected to plasma hydrophilization treatment, sterilization treatment, and gelatin treatment in sequence.

[0043] During the plasma hydrophilization treatment, a Harrick plasma cleaner is used. The specific operation is as follows: place the chip under the PDMS leaf vein network in the Harrick plasma cleaner, close the chamber door, turn on the vacuum pump to create a vacuum environment in the cleaning chamber, and then turn on the "POWER" button of the plasma cleaner. A glow will appear in the chamber, which means that the plasma cleaning has started. After the glow lasts for 3 to 5 minutes, turn off the "POWER" button, turn off the vacuum pump, open the air inlet, and wait for the air pressure inside and outside the chamber to equalize. Then open the chamber door, take out the chip under the PDMS leaf vein network, and the plasma hydrophilization treatment is completed. The specific sterilization process is as follows: Place the PDMS leaf vein network chip after plasma hydrophilization treatment into a pressure sterilizer, close the valve, set the sterilization temperature to 100℃ and the time to 30 minutes, and then click the start sterilization button; The specific procedure for gelatin treatment is as follows: Place the sterilized PDMS leaf vein network chip in a sterile cell culture dish, and place the cell culture dish together in a sterile cell culture worktable. Use 3-5 ml of 0.1% gelatin solution to drop onto the PDMS leaf vein network chip, ensuring complete coverage. Place it in a 4°C refrigerator for 12-16 hours, then remove it and discard the excess gelatin solution. The PDMS leaf vein network chip should then be placed back in a sterile cell culture dish for later use.

[0044] Step S2: Assemble the pretreated biomimetic leaf vein network microfluidic chip on the fixing device, and connect one end of the inlet tube and one end of the outlet tube to the inlet and outlet of the microfluidic channel network of the biomimetic leaf vein network microfluidic chip, respectively. Connect the other end of the inlet tube to the syringe, which can be detachably clamped to the micro-injection pump. Connect the other end of the outlet tube to the recovery cup to complete the assembly of the entire bioreactor. See Figure 5 This invention provides an assembly diagram of a bioreactor: The bioreactor includes a biomimetic leaf vein network microfluidic core, a fixing device, an inlet pipe 3, an outlet pipe 4, a micro-injection pump, and a recovery cup; the biomimetic leaf vein network microfluidic core includes a PDMS leaf vein network lower chip 12 and a PDMS upper cover 11 laminated and bonded to the PDMS leaf vein network lower chip; the fixing device includes two opposing upper clamping plates 21, a lower clamping plate 22, multiple bolts 23, and nuts 24; the PDMS Two liquid flow holes 110 are provided through the upper cover plate 11, which are coaxially opposite to the liquid inlet and liquid outlet of the microfluidic channel network 120 of the PDMS leaf vein network chip 12, respectively; a biomimetic leaf vein network microfluidic chip 1 is provided between the upper clamping plate 21 and the lower clamping plate 22. The upper clamping plate is provided with multiple first fixing holes 210, and the lower clamping plate is provided with multiple second fixing holes 220. The first fixing holes 210 and the second fixing holes 220 are coaxially opposite and evenly distributed around the periphery of the biomimetic leaf vein network microfluidic chip; the bolts 23 are inserted vertically in sequence. The first fixing hole 210 and the second fixing hole 220 are connected to the threaded end of the bolt 23, which is connected to a nut 24. By tightening the nut 24, the upper clamping plate 21 and the lower clamping plate 22 are clamped and fixed to the biomimetic leaf vein network microfluidic core 1. The upper clamping plate 21 is provided with two liquid guiding holes 211 that are coaxially opposite to the liquid flow hole 110. The two liquid guiding holes 211 are located between the multiple fixing holes 210. The liquid inlet pipe 3 and the liquid outlet pipe 4 pass through the liquid guiding holes 211 and the liquid flow hole 110 in sequence, and are connected to the liquid inlet and liquid outlet of the microfluidic channel network 120. The bioreactor of this embodiment has been disclosed in the utility model patent application with patent application number CN202422586233.6, and will not be described in detail in this invention.

[0045] Step S3: Place the assembled bioreactor under a UV lamp and rinse the biomimetic leaf vein network microfluidic chip with sterile PBS (sterile phosphate buffer) for sterilization. Step S4: After sterilization of the bioreactor, the target cells are injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip using a microinjection pump. The infusion of target cells is complete when target cells flow out of the recovery cup. The bioreactor, now infused with target cells, is then transferred to a 37°C, 5% concentration. In a CO2 cell culture incubator, the target cells are allowed to adhere to the culture vessel wall for 4-6 hours. After adhesion, fresh cell culture medium is continuously injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip at a preset flow rate using a microinjection pump. The old cell culture medium after metabolism is discharged through the outlet of the microfluidic channel network to achieve dynamic replacement of the cell culture medium, simulating the in vivo fluid flow microenvironment and maintaining the in vitro activity of the target cells, thereby obtaining the corresponding simulation system. When the target cells are only immortalized human proximal renal tubular epithelial cells, the simulation system is a renal tubule simulation system. When the target cells are a cell suspension of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells in a ratio of 1:2, the simulation system is a renal tubular peritubular microenvironment simulation system.

[0046] The microinjection pump in this embodiment is a Lande dual-channel injection pump, which is equipped with two 50ml syringes. When injecting target cells, the Lande dual-channel injection pump adopts a fast-push mode with a flow rate of 500ml / h. When injecting cell culture medium, in order to simulate the microenvironment of in vivo fluid flow, it is necessary to consider the pressure field and shear stress field of the microfluidic channel network (i.e., leaf vein network skeleton) of the biomimetic leaf vein network microfluidic chip. Therefore, it is necessary to replace the syringes mounted on the Lande dual-channel injection pump in batches and set an appropriate flow rate to continuously infuse cell culture medium into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip to simulate the microenvironment of in vivo fluid flow and maintain the stable in vitro culture state of cells. The recovery cup is used to receive the waste cell culture medium. The method for confirming the preset flow rate of fresh cell culture medium injected into the biomimetic leaf vein network microfluidic chip in step S4 is as follows: The binarized image of the leaf vein network skeleton was input into the fluid dynamics analysis software (version FLOW-3D) to establish a three-dimensional fluid analysis model of the leaf vein microchannel. The flow velocity of the main channel of the leaf vein microchannel was set, and three typical flow velocities of 5 μL / min, 15 μL / min and 30 μL / min were selected. The pressure field and shear stress field distribution inside the leaf vein network skeleton were evaluated by the fluid dynamics analysis software. The influence of flow velocity change on the pressure gradient of the microfluidic channel inside the leaf vein network skeleton was revealed, and the difference in the distribution of shear stress at the bottom and middle of the channel was clarified, so as to select a suitable inlet flow velocity for the cell culture medium.

[0047] Figure 6(ai) is a fluid analysis diagram showing the fluid shear force, pressure, and velocity in the chip under the leaf vein network at different inlet flow velocities. The legend in the lower right corner of each diagram shows the specific range of fluid shear force, pressure, and velocity in the chip under the leaf vein network at different inlet flow velocities. According to the physiological shear force, pressure, and velocity experienced by renal tubular epithelial cells in vivo under physiological conditions, they are 0.01~0.1 Pa, 1.33~2.67 kPa, and 10 kPa, respectively. -3 ~10 -4 The inlet flow rate is mm / s. Therefore, the results show that choosing an inlet flow rate of 15 uL / min is more in line with the physiological flow rate, shear force and pressure in the renal tubules.

[0048] The specific method for preparing the immortalized human proximal renal tubular epithelial cells is as follows: a) Primary human renal epithelial cells were resuscitated, resuspended in DMEM / F12 (Dupuy's modified Eagle medium) complete medium pre-warmed to 37°C, and seeded into cell culture dishes at a cell density of 2-4*10^6. The cells were then cultured in a 37°C, 5% CO2 cell culture incubator. The DMEM / F12 complete medium consisted of 90% DMEM / F12 basal medium, 10% fetal bovine serum, and 0.5-1% penicillin-streptomycin antibiotics. In this embodiment, the inner diameter of the cell culture dish was 6 cm. The primary human renal epithelial cells used in this embodiment were derived from human urinary renal tubular epithelial cells. The resuscitation process of the primary human renal epithelial cells was a routine procedure, with the core steps being rapid thawing, centrifugation to remove cryopreservation solution, resuscitation in the culture medium, and seeding and culture. These steps will not be described in detail in this embodiment. b) When the primary human renal epithelial cells reach 70-80% confluence, discard the old DMEM / F12 complete medium and replace it with DMEM / F12 basal medium. Infect the primary human renal epithelial cells with lentivirus SV40T and lentivirus hTERT for 12-24 hours in a cell culture incubator at 37°C and 5% CO2. The MOI (multiple of infection) of the lentivirus SV40T is 150, the MOI of the lentivirus hTERT is 150, and the molar ratio of the lentivirus SV40T to the lentivirus hTERT is 1:1. c) After infection, discard the old DMEM / F12 basal medium and culture the cells in a 37°C, 5% CO2 cell culture incubator using DMEM / F12 complete medium containing 1 μg / mL Puromycin for 48–96 h. d) After the selection culture was completed, the old DMEM / F12 complete medium containing puromycin was discarded, and new DMEM / F12 complete medium was used for routine passage culture, passaged every 48-96 hours to obtain semi-immortalized human kidney epithelial cells. When performing cell passage culture, the use of 0.25% Trypsin (1X), Phenol Red, with EDTA can significantly improve the cell viability, adhesion rate and stability after passage. e) After passage 2-3 times, when the semi-immortalized human renal epithelial cells have grown to 80-90% confluence, identify the passaged semi-immortalized human renal epithelial cells by immunofluorescence staining; if the identified semi-immortalized human renal epithelial cells successfully express rabbit polyclonal antibody CK-18 (cytokeratin 18), immortalized human proximal renal tubular epithelial cells are obtained for later use; if the identified semi-immortalized human renal epithelial cells do not successfully express rabbit polyclonal antibody CK-18, repeat step ae until rabbit polyclonal antibody CK-18 is successfully expressed.

[0049] The identification of passaged, semi-immortalized human kidney epithelial cells by immunofluorescence staining specifically includes: When the semi-immortalized human renal epithelial cells reached 80-90% confluence, the old DMEM / F12 complete culture medium was discarded, and the semi-immortalized human renal epithelial cells were washed twice with 3-5 ml PBS, each time for 5-10 min. Then, the semi-immortalized human renal epithelial cells were fixed with 3-5 ml of 4% paraformaldehyde at 20-25℃ for 15-30 min. After fixation, the semi-immortalized human renal epithelial cells adhered firmly to the cell culture dish. Discard the 4% paraformaldehyde on the cell culture dish, wash the semi-immortalized human kidney epithelial cells twice with 3-5 ml PBS, each wash lasting 5-10 min, and then soak the semi-immortalized human kidney epithelial cells on the cell culture dish in 3-5 ml of 0.1% Triton X-100 permeabilized cell culture for 15-20 min at 4°C. Discard the 0.1% Triton X-100 on the cell culture dish, wash the semi-immortalized human kidney epithelial cells twice with 3-5 ml PBS, each wash lasting 5-10 min, and then block the semi-immortalized human kidney epithelial cells on the cell culture dish with 3-5 ml of 10% donkey serum at 20-25℃ for 30-60 min; Discard the 10% donkey serum on the cell culture dish, and add 3-5 ml of rabbit polyclonal antibody diluted 1:100 to the semi-immortalized human kidney epithelial cells on the cell culture dish. Then place the dish in a refrigerator at 4°C for 12-18 hours. Discard the rabbit polyclonal antibody CK-18 on the cell culture dish, wash the semi-immortalized human kidney epithelial cells three times with 3-5 ml PBS, each wash lasting 5-10 min; after washing, add FITC (fluorescein isothiocyanate) labeled secondary antibody diluted 1:100 (v / v) to the semi-immortalized human kidney epithelial cells on the cell culture dish, and then place them at 20-25℃ for 1-2 h; Discard the FITC secondary antibody on the cell culture dish, and wash the semi-immortalized human renal epithelial cells three times with 3-5 ml of PBS, each wash lasting 5-10 min. After washing, add 3-5 ml of DAPI (4',6-diamidinyl-2-phenylindole) to the semi-immortalized human renal epithelial cells on the cell culture dish and incubate at 20-25°C for 5-10 min. After incubation, observe the semi-immortalized human renal epithelial cells on the cell culture dish using a fluorescence microscope. If the cells show 80-90% green fluorescence, it indicates successful expression of rabbit polyclonal antibody CK-18, thus obtaining immortalized human proximal renal tubular epithelial cells.

[0050] The use of a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip as described above, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip constructed by any of the above methods, for simulating renal tubules and peritubular capillaries in vitro.

[0051] The application of a renal tubule and peritubular microenvironment simulation system based on a leaf vein-like network chip, as described above, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein-like network chip constructed by any of the above methods, in evaluating the renal clearance characteristics and nephrotoxicity risk of drugs, characterized in that: the drugs include, but are not limited to, antitumor drugs (such as cisplatin, methotrexate), antibiotics (such as aminoglycosides, vancomycin), and immunosuppressants (such as cyclosporine, tacrolimus). Through the enhanced prediction and detection capabilities of the dynamic fluid microenvironment, it can sensitively reflect early cell damage, barrier function changes, and transport protein function inhibition caused by drugs, providing earlier response signals and higher detection sensitivity than conventional static culture, and can be used for preclinical drug screening, dosage optimization, and toxicity mechanism studies.

[0052] The application effects of this invention will be described in detail below with reference to simulation experiments.

[0053] Figure 7 (a) is a diagram of immortalized human proximal renal tubular epithelial cells seeded on a PDMS leaf vein network chip and observed under a microscope in white light. It includes the main flow channels and secondary flow channels in the microfluidic channel network. The cells are well adhered to the chip. Figure 7 (b) is an AOPI staining image of immortalized human proximal renal tubular epithelial cells seeded in a chip under the PDMS leaf vein network. The image shows that the cells are in good condition.

[0054] Figure 8 (a) is a microscopic image observed under white light after immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells were seeded together on a PDMS leaf vein network chip. The cells are dense and well adhered to the chip. Figure 8 (b) shows the characteristic immunofluorescence staining of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells seeded in the PDMS leaf vein network chip. The characteristic protein of immortalized human proximal renal tubular epithelial cells is LTL, which appears green, and the characteristic protein of human umbilical vein endothelial cells is CD31, which appears red. The cell nuclei are dyed blue with DAPI, indicating that the characteristic proteins of the two types of cells are well expressed.

[0055] Figure 9 (a) shows the characterization staining of immortalized human proximal renal tubular epithelial cells seeded in a PDMS leaf vein network chip. As can be seen from the figure, the immortalized human proximal renal tubular epithelial cells present a reticular structure, and the characteristic protein LTL appears green and is strongly expressed. Figure 9 (b) shows the characterization and staining of immortalized human proximal renal tubular epithelial cells seeded in a two-dimensional culture dish. The cells are laid out flat, and although the characteristic protein LTL appears green, its expression is less than that of the PDMS leaf vein network chip.

[0056] Figure 10 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubule epithelial cells within a renal tubule mimicry system. The results show that the cells express SGLT2 (red) more than GLUT2 (green). Figure 10 Image (b) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubule epithelial cells in a conventional two-dimensional culture dish. The results show that the cells express less SGLT2 (red) and more GLUT2 (green). By comparison, it can be seen that the reabsorption transport proteins expressed in immortalized human proximal renal tubule epithelial cells in the renal tubule simulation system and the two-dimensional culture dish are different. SGLT2 is mainly expressed in the renal tubule simulation system, while GLUT2 is mainly expressed in the two-dimensional culture dish.

[0057] Figure 11 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubule epithelial cells in the renal tubule simulation system. The results show that the cells express more AQP1 (green) and less OAT3 (red). Figure 11Image (b) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubule epithelial cells in a conventional two-dimensional culture dish. The results show that the cells express less AQP1 (green) and more OAT3 (red). By comparison, it can be seen that the reabsorption transport proteins expressed in immortalized human proximal renal tubule epithelial cells in the renal tubule simulation system and the two-dimensional culture dish are different. AQP1 is mainly expressed in the renal tubule simulation system, while OAT3 is mainly expressed in the two-dimensional culture dish.

[0058] Figure 12 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubule epithelial cells in the renal tubule peritubular microenvironment simulation system. The results show that the cells present a reticular structure, indicating that the cells have better tubular formation ability. The cells express more SGLT2 (red) and less GLUT2. Figure 12 (b) shows the immunofluorescence staining results of immortalized human proximal renal tubule epithelial cells in a conventional two-dimensional culture dish. The results show that the cells express less SGLT2 (red) and more GLUT2 (green). By comparison, it can be seen that the renal tubule peritubular microenvironment simulation system and the two-dimensional culture dish show different types of reabsorption transport proteins. SGLT2 is mainly expressed in the renal tubule peritubular microenvironment simulation system, while GLUT2 is mainly expressed in the two-dimensional culture dish. Moreover, the cells in the renal tubule peritubular microenvironment simulation system have better morphology and stronger tubule formation ability.

[0059] Figure 13 (a) shows the immunofluorescence staining results of two reabsorption transport proteins in immortalized human proximal renal tubular epithelial cells within the renal tubular peritubular microenvironment simulation system. The results show that the cells exhibit a reticular structure, indicating better tubular formation ability. The cells express more AQP1 (green) and less OAT3 (red). Figure 13 (b) shows the immunofluorescence staining results of immortalized human proximal renal tubule epithelial cells in a conventional two-dimensional culture dish. The results show that the cells express less AQP1 (green) and more OAT3 (red). By comparison, it can be seen that the renal tubule peritubular microenvironment simulation system and the two-dimensional culture dish show different types of reabsorption transport proteins. In the renal tubule peritubular microenvironment simulation system, AQP1 is mainly expressed, while in the two-dimensional culture dish, OAT3 is mainly expressed. Moreover, the cells in the renal tubule peritubular microenvironment simulation system have better morphology and stronger tubule formation ability.

[0060] The specific steps for immunofluorescence staining identification of two reabsorption transport proteins in the above-mentioned renal tubule simulation system, renal tubular peritubular microenvironment simulation system, and immortalized human proximal renal tubular epithelial cells in a two-dimensional culture dish are as follows: 1) Wash the immortalized human proximal renal tubular epithelial cells twice with 3-5 ml PBS, each time for 5-10 min. Then fix the immortalized human proximal renal tubular epithelial cells with 3-5 ml of 4% paraformaldehyde at 20-25℃ for 15-30 min. After fixation, the immortalized human proximal renal tubular epithelial cells adhere firmly to the cell culture dish. 2) Discard the 4% paraformaldehyde on the cell culture dish, rinse the immortalized human proximal renal tubular epithelial cells twice with 3-5 ml PBS, each rinse lasting 5-10 min, and then use 3-5 ml of 0.1% Triton X-100 permeabilized cell culture dish on the immortalized human proximal renal tubular epithelial cells for 15-20 min at 4℃. 3) Discard the 0.1% Triton X-100 on the cell culture dish, rinse the immortalized human proximal renal tubular epithelial cells twice with 3-5 ml PBS, each rinse lasting 5-10 min, and then block the immortalized human proximal renal tubular epithelial cells on the cell culture dish with 3-5 ml of 10% donkey serum at 20-25℃ for 30-60 min; 4) Discard the 10% donkey serum on the cell culture dish, and add 3-5 ml each of rabbit polyclonal antibody SGLT2 (sodium-glucose cotransporter 2) and mouse polyclonal antibody GLUT2 (glucose transporter 2) (or mouse polyclonal antibody AQP1 (aquaporin 1) and rabbit polyclonal antibody OAT3 (organic anion transporter 3)) diluted 1:200 to the immortalized human proximal renal tubular epithelial cells on the cell culture dish, and then place it in a refrigerator at 4°C for 12-18 hours; 5) Discard the rabbit polyclonal antibody SGLT2 and mouse polyclonal antibody GLUT2 (or mouse polyclonal antibody AQP1 and rabbit polyclonal antibody OAT3) solution on the cell culture dish, wash the semi-immortalized human proximal renal tubular epithelial cells three times with PBS, each wash lasting 5-10 min; after washing, add 3-5 ml of FITC-labeled secondary antibody (CoraLite594–conjugated Goat Anti-Rabbit IgG and CoraLite488-conjugated Goat Anti-Mouse IgG) diluted 1:100 to the immortalized human proximal renal tubular epithelial cells on the cell culture dish, and then place them at 20-25℃ for 1-2 h; 6) Discard the FITC secondary antibody on the cell culture dish, and wash the semi-immortalized human proximal renal tubular epithelial cells three times with 3-5 ml PBS, each time for 5-10 min; after washing, add DAPI to the immortalized human proximal renal tubular epithelial cells on the cell culture dish and incubate at 20-25℃ for 5-10 min; after incubation, observe the immortalized human proximal renal tubular epithelial cells on the cell culture dish using a fluorescence microscope. If the cells show red or green fluorescence, it indicates that the corresponding transporter protein is significantly expressed.

[0061] Figure 14 These are, respectively, a 2D culture dish conventional culture system, a 2.5D transwell chamber culture system, and a 3D leaf vein network chip-based renal tubule simulation system; Figure 14 In (a), immortalized human proximal renal tubular epithelial cells are spread evenly on the bottom surface of a 2D culture dish. Figure 14 (b) shows the three-dimensional growth of immortalized human proximal renal tubular epithelial cells on the pore membrane in a 2.5D transwell chamber. Figure 14 (c) shows that immortalized human proximal renal tubular epithelial cells in the leaf vein network can exhibit three-dimensional growth in the renal tubular simulation system.

[0062] Figure 15 The figures show the results of glucose reabsorption rate measurement in immortalized human proximal renal tubular epithelial cells in a conventional 2D culture dish, a 2.5D transwell chamber, and a renal tubular simulation system based on a 3D leaf vein network chip. The results indicate that the glucose reabsorption function of immortalized renal tubular epithelial cells is better in the renal tubular simulation system based on the 3D leaf vein network chip of this invention.

[0063] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions or improvements made by those skilled in the art within the spirit and principles of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A simulation system for renal tubules and peritubular microenvironment based on a leaf vein-inspired network chip, characterized in that: The system comprises a biomimetic leaf vein network microfluidic chip, immortalized human proximal renal tubule epithelial cells, human umbilical vein endothelial cells, and cell culture medium. The microfluidic channel network of the biomimetic leaf vein network microfluidic chip has a renal tubule structure, and the inner wall of the microfluidic channel network is attached with a cell layer. When the cell layer is immortalized human proximal renal tubule epithelial cells, a renal tubule simulation system is formed. When the cell layer is a cell suspension of immortalized human proximal renal tubule epithelial cells and human umbilical vein endothelial cells, a renal tubule peritubular microenvironment simulation system is formed. The cell activity in the renal tubule simulation system and the renal tubule peritubular microenvironment simulation system is maintained by continuously perfusing fresh cell culture medium into the biomimetic leaf vein network microfluidic chip and draining metabolically degraded cell culture medium. The ratio of immortalized human proximal renal tubule epithelial cells to human umbilical vein endothelial cells in the renal tubule peritubular microenvironment simulation system is 1:

2.

2. A method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein-inspired network chip, characterized in that: Specifically, the steps include the following: Step S1: Preprocess the biomimetic leaf vein network microfluidic chip; Step S2: Assemble the pretreated biomimetic leaf vein network microfluidic chip on the fixing device, and connect one end of the inlet tube and one end of the outlet tube to the inlet and outlet of the microfluidic channel network of the biomimetic leaf vein network microfluidic chip, respectively. Connect the other end of the inlet tube to the syringe, which can be detachably clamped to the micro-injection pump. Connect the other end of the outlet tube to the recovery cup to complete the assembly of the entire bioreactor. Step S3: Place the assembled bioreactor under a UV lamp and rinse the biomimetic leaf vein network microfluidic chip with sterile PBS for sterilization. Step S4: After sterilization of the bioreactor, the target cells are injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip using a microinjection pump. The perfusion of target cells is complete when target cells flow out of the recovery cup. The bioreactor with perfused target cells is then transferred to a cell culture incubator and allowed to stand to allow the target cells to adhere. After the target cells adhere, fresh cell culture medium is continuously injected into the microfluidic channel network of the biomimetic leaf vein network microfluidic chip at a preset flow rate using a microinjection pump. The old cell culture medium is then discharged through the outlet of the microfluidic channel network to achieve dynamic replacement of the cell culture medium, simulating the in vivo fluid flow microenvironment and maintaining the in vitro activity of the target cells. When the target cells are only immortalized human proximal renal tubular epithelial cells, the simulation system is a renal tubule simulation system. When the target cells are a cell suspension of immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells in a ratio of 1:2, the simulation system is a renal tubular peritubular microenvironment simulation system.

3. The method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip according to claim 2, characterized in that: The biomimetic leaf vein network microfluidic chip includes a PDMS leaf vein network underchip and a PDMS top cover chip that is laminated and bonded to the PDMS leaf vein network underchip.

4. The method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip according to claim 3, characterized in that: The specific method for fabricating the chip under the PDMS leaf vein network includes: Rinse fresh osmanthus leaves with deionized water, then soak the rinsed fresh osmanthus leaves in a 3.5-5 wt% NaOH solution, heat to boiling and maintain for 30-40 minutes. Remove the leaves and remove any remaining leaf tissue to obtain the leaf vein network skeleton. Air dry the leaf vein network skeleton to obtain a leaf vein network skeleton specimen. The leaf vein network skeleton specimen was photographed to obtain a high-resolution digital image of the leaf vein network skeleton. This high-resolution image was then imported into LEAF GUI software. The image binarization algorithm in LEAF GUI software was used to process the leaf vein network skeleton into black, and the non-flow channel areas outside the leaf vein network skeleton into white, resulting in a binarized image of the leaf vein network skeleton. The integrity of the leaf vein network skeleton specimen was observed. If the skeleton structure was intact, a 400-500nm chromium coating was sputtered onto the leaf vein network skeleton specimen to obtain chromium-plated leaf veins. If the skeleton structure was incomplete, a fresh osmanthus leaf was selected to prepare a new leaf vein network skeleton specimen. Hexamethyldisilane is spin-coated onto a silicon wafer and cured to form a 3-4 μm thick hexamethyldisilane coating. A photosensitive polymer is spin-coated onto the surface of the hexamethyldisilane coating and cured to form a 3-4 μm thick photosensitive polymer coating. The silicon wafer with the hexamethyldisilane coating and the photosensitive polymer coating is placed in an exposure station. A chromium-plated leaf vein is used as a photomask and aligned with the surface of the silicon wafer. The silicon wafer is irradiated with ultraviolet light. The ultraviolet-irradiated silicon wafer is then placed in a 0.5-1 wt% NaOH solution. The photosensitive polymer decomposes in the NaOH solution to obtain a silicon wafer with a leaf vein network negative polymer pattern. A chromium layer with a thickness of 150~200 nm is uniformly sputtered onto a silicon wafer with a leaf vein network negative polymer pattern. After the chromium layer is deposited, it is immersed in an acetone solution to dissolve the leaf vein network negative polymer pattern, thus obtaining a chromium-plated silicon wafer. The chromium-plated silicon wafer is then etched to obtain a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern. A first vacuum degassing treatment is performed on a mixed solution of polydimethylsiloxane prepolymer and curing agent with a mass ratio of (9~10):

1. An alkylation treatment is then performed on a silicon wafer leaf vein mold with a leaf vein network negative polymer pattern. The mixed solution of polydimethylsiloxane prepolymer and curing agent after the first vacuum degassing treatment is cast onto the alkylated silicon wafer leaf vein mold, and a second vacuum degassing treatment is performed. After the second vacuum degassing treatment, the mixed solution of polydimethylsiloxane prepolymer and curing agent in the silicon wafer leaf vein mold is dried and cured. After the mixed solution of polydimethylsiloxane prepolymer and curing agent has cured and formed, the silicon wafer leaf vein mold with the leaf vein network negative polymer pattern is peeled off to obtain a PDMS leaf vein network underchip with a leaf vein network negative polymer pattern.

5. The method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip according to claim 3, characterized in that: The specific method for preparing the PDMS cover sheet includes: A first vacuum degassing treatment was performed on a mixed solution of polydimethylsiloxane prepolymer and curing agent with a mass ratio of (9~10):

1. The mixed solution of polydimethylsiloxane prepolymer and curing agent after the first vacuum degassing treatment was cast into a cell culture dish and subjected to a second vacuum degassing treatment. After the second vacuum degassing treatment, the mixed solution of polydimethylsiloxane prepolymer and curing agent in the cell culture dish was dried and cured. After the mixed solution of polydimethylsiloxane prepolymer and curing agent was cured and shaped, the cell culture dish was peeled off to obtain a PDMS cover sheet.

6. The method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip according to claim 2, characterized in that: The method for preparing immortalized human proximal renal tubular epithelial cells specifically includes: a) Primary human renal epithelial cells were resuscitated, resuspended in DMEM / F12 complete medium preheated to 37°C, and seeded into cell culture dishes at a cell density of 2-4*10^6. The cells were then cultured in a cell culture incubator at 37°C and 5% CO2. The DMEM / F12 complete medium consisted of 90% DMEM / F12 basal medium, 10% fetal bovine serum, and 0.5-1% penicillin-streptomycin antibiotics. b) When the primary human renal epithelial cells reach 70-80% confluence, discard the old DMEM / F12 complete medium and replace it with DMEM / F12 basal medium. Infect the primary human renal epithelial cells with lentivirus SV40T and lentivirus hTERT for 12-24 hours in a cell culture incubator at 37°C and 5% CO2. The MOI value of the lentivirus SV40T is 150, the MOI value of the lentivirus hTERT is 150, and the molar ratio of the lentivirus SV40T to the lentivirus hTERT is 1:

1. c) After infection, discard the old DMEM / F12 basal medium and culture the cells in a 37°C, 5% CO2 cell culture incubator using DMEM / F12 complete medium containing 1 μg / mL Puromycin for 48-96 h. d) After the selection culture is completed, discard the old DMEM / F12 complete medium containing Puromycin and use the new DMEM / F12 complete medium for routine passage culture, passage once every 48~96 hours to obtain semi-immortalized human kidney epithelial cells. e) After passage 2-3 times, when the semi-immortalized human renal epithelial cells have grown to 80-90% confluence, identify the passaged semi-immortalized human renal epithelial cells by immunofluorescence staining; if the identified semi-immortalized human renal epithelial cells successfully express rabbit polyclonal antibody CK-18, immortalized human proximal renal tubular epithelial cells are obtained; if the identified semi-immortalized human renal epithelial cells do not successfully express rabbit polyclonal antibody CK-18, repeat step ae until rabbit polyclonal antibody CK-18 is successfully expressed.

7. The method for constructing a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip according to claim 2, characterized in that: Step S1 specifically includes: sequentially performing plasma hydrophilization treatment, sterilization treatment, and gelatin treatment on the biomimetic leaf vein network microfluidic chip.

8. The use of a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip as described in claim 1, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein network chip constructed by any of claims 2-7, for simulating renal tubules and peritubular capillaries in vitro.

9. The application of a renal tubule and peritubular microenvironment simulation system based on a leaf vein-like network chip as described in claim 1, or a renal tubule and peritubular microenvironment simulation system based on a leaf vein-like network chip constructed by any of claims 2-7, in evaluating the renal clearance characteristics and nephrotoxicity risk of drugs, characterized in that: The drugs mentioned include, but are not limited to, anti-tumor drugs, antibiotics, and immunosuppressants.

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