Ssepsis model construction method based on lung and kidney organ chip and application

Abstract

The invention provides a construction method and application of a sepsis model based on a lung and kidney organ chip. The method comprises the following steps: S1, carrying out ultraviolet sterilization on an organ chip device; s2, modifying a transwell plug-in unit; s3, inoculating human lung microvascular endothelial cells and human umbilical vein endothelial cells; S4, inoculating human alveolar epithelial cells and human renal tubular epithelial cells; s5, transwell inserts are arranged in two holes in the middle of the organ chip device respectively, a mixed culture medium is added into liquid adding holes in the two sides of the organ chip device, and the lung chip and the kidney chip are communicated through a lower channel of the organ chip device; s6, dynamically culturing the lung and kidney organ chip; and S7, sucking up the culture medium in the constructed lung and kidney organ chip, adding the LPS-containing mixed culture medium into a lower channel, and respectively adding the LPS-containing mixed culture medium into the human alveolar epithelial cell side and the human renal tubular epithelial cell side of the lung and kidney organ chip and the transwell plug-in hole for culturing for 2 days. According to the invention, the lung and kidney organ chip is applied to the sepsis model for the first time, and the constructed sepsis model is more suitable for basic research and drug development of sepsis.

Description

Technical Field

[0001] This invention relates to the field of disease model construction, specifically to a method and application for constructing a sepsis model based on lung and kidney organ-on-a-chip. Background Technology

[0002] Animal experiments are a traditional research method in disease model construction, but they have the following drawbacks: 1) Species differences. Animals and humans inevitably differ in physiological structure, organ function, and life maintenance, and animal models cannot accurately simulate the physiological and pathological environment of humans. [1,3] 2) Because animal growth takes time, model creation often requires a longer period of time; 3) Animal models are subject to ethical controversies. [1,3] While cell culture is a commonly used research method, it has some drawbacks: 1) Cell culture is a static culture, lacking the influence of in vivo hemodynamics. Studies have shown that hemodynamics affects cell function, including drug absorption. This explains why some drugs are effective in cell experiments but ineffective in humans. [1,4] ;2) Cell culture models usually contain only one type of cell, lacking intercellular interactions and unable to maintain the differentiation and expression of tissue-specific functions. The human body is a complex organism, and the occurrence and development of diseases are often the result of the combined action of multiple cells or even multiple organs. Studying only one type of cell, lacking the influence of other cells or tissues, cannot reflect the true situation of the human body.

[0003] Therefore, current technology cannot meet the needs of basic research and drug development for sepsis in clinical practice, and a new sepsis model is urgently needed.

[0004] Sepsis is a life-threatening multiple organ dysfunction syndrome (MODS) caused by a dysregulation of the host's response to infection.

[12] Globally, there are approximately 31.5 million cases of sepsis and 19.4 million severe cases each year, resulting in about 5.3 million deaths.

[13] This condition places immense pressure on patients' families and society. The lungs and kidneys are the organs most commonly affected by sepsis, frequently leading to acute lung injury (ALI) and acute kidney injury (AKI). The lungs are the primary site of gas exchange, providing essential oxygen to all parts of the body by inhaling oxygen and expelling carbon dioxide. The kidneys, on the other hand, regulate fluid and electrolyte balance, filtering waste products and excess water from the blood to produce urine and excrete it, thus maintaining blood cleanliness and normal circulation. Both organs are interconnected and influence each other through physiological mechanisms such as blood circulation, the neuroendocrine system, and the regulation of water-salt and acid-base balance. Currently, there are cell models of sepsis-related lung injury. [6]and cell models of sepsis-related kidney injury. [7] Animal models of sepsis [5] However, traditional two-dimensional cells lack the influence of hemodynamics and the in vivo microenvironment, making it impossible to co-culture multiple organs. Animal experiments, on the other hand, cannot meet the needs of clinical research due to species and ethical issues. Therefore, there is an urgent need for a sepsis model that can assess lung and kidney involvement in vitro.

[0005] Organ-on-a-chip, a new technology reported in 2004, is a cell culture device implemented through a microfluidic system. It contains continuously perfused chambers filled with live cells simulating the physiological state of tissues or organs. The lung-on-a-chip, for example, utilizes a microfluidic system comprising two tightly packed microchannels separated by a specific polydimethylsiloxane (PDMS) membrane. A collagen-coated interphase membrane is used, and human alveolar epithelial cells and human pulmonary microvascular endothelial cells are cultured on opposite sides of this membrane. Figure 1 As shown, once the cells grow to the point of convergence, air is introduced into the upper chamber to form an air-liquid interface, more accurately simulating the alveolar cavity. [1] .like Figure 2 As shown, the kidney chip is also implemented through a microfluidic system containing two tightly packed microchannels separated by a specific polydimethylsiloxane (PDMS) membrane. The middle membrane is coated with laminin, and human induced pluripotent stem cell (hiPSC)-derived podocytes are cultured on the top of the membrane. Primary human glomerular endothelial cells are cultured on the other side of the same membrane, simulating the urinary zone and capillary chamber of the glomerulus, respectively. Simultaneously, the circulatory aspiration of the two hollow chambers on both sides simulates the dynamic mechanical action of the glomerulus due to the periodic pulsation of renal blood flow. [2] .

[0006] Multi-organ-on-a-chip (MIB) connects multiple organs via microfluidic channels. Through the interaction of culture medium flow, it can simulate organ-organ crosstalk, such as multicellular structures, tissue-tissue interfaces, physicochemical microenvironments, and vascular perfusion. MIB produces tissue and organ functional levels that are impossible to achieve with traditional 2D or 3D culture systems. [9] In recent years, reports on multi-organ-on-a-chip (MIB) technology have been gradually increasing, such as multi-organ-on-a-chip models based on human kidney and liver organoids, which study the therapeutic effects and biodistribution of extracellular vesicles derived from mesenchymal matrix cells.

[10] Shafagh et al. developed a hepatopancreatic organ-on-a-chip device that provides new insights into the complex metabolic interactions involved in prediabetic hyperglycemia.

[11] However, there are currently no applications of organ-on-a-chip technology for the lungs and kidneys, nor its application in sepsis. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for constructing and applying a sepsis model based on lung and kidney organ-on-a-chip, so as to solve the problem that the existing technology cannot meet the needs of basic research and drug development for sepsis in clinical practice.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: According to one aspect of the present invention, a method for constructing a sepsis model based on lung and kidney organ-on-a-chip is provided, comprising the following steps: S1. subjecting the organ-on-a-chip device to ultraviolet sterilization; S2. modifying the transwell insert with type I collagen (colI); S3. seeding human pulmonary microvascular endothelial cells and human umbilical vein endothelial cells: in the preparation of both the lung and kidney chips, 50 μL of 1 × 10⁻⁶ cells is seeded. 6 / ml of cell suspension was placed at the bottom center of the wells of a 24-well plate. The Transwell insert was placed in the well of the 24-well plate. The 24-well plate with the Transwell insert was inverted and placed in a 37°C 5% CO2 incubator for 2 hours, then upright. S4. Seeding of human alveolar epithelial cells and human renal tubular epithelial cells: In the preparation of lung and kidney microarrays, 100ul of 1×10 5 S5. Seed the transwell insert with a 1 / ml cell suspension and incubate overnight at 37°C with 5% CO2. S6. Remove the transwell inserts used for the lung and kidney microarrays and place them in the two middle wells of the organ-on-a-chip device. Aspirate the liquid from the transwell insert of the lung microarray. Simultaneously, add 2ml of mixed culture medium to the liquid addition wells on both sides of the organ-on-a-chip device. Connect the lung and kidney microarrays through the lower channel of the organ-on-a-chip device. S7. Place the prepared lung and kidney organ-on-a-chip on a shaker and culture dynamically for 5 days, changing the culture medium every 1-2 days to complete the construction of the lung and kidney organ-on-a-chip. S8. Aspirate the culture medium from the constructed lung and kidney organ-on-a-chip. Add 2mL of mixed culture medium containing 10μg / mL lipopolysaccharide to the lower channel. Add 100ul of mixed culture medium containing 10μg / mL LPS to the wells of the transwell inserts on the human alveolar epithelial cell and human renal tubular epithelial cell sides of the lung and kidney organ-on-a-chip, respectively. Change the culture medium daily and incubate on a shaker at 37°C with 5% CO2 for 2 days.

[0009] Optionally, in the above method for constructing a sepsis model based on lung and kidney organ-on-a-chip, in step S2, type I collagen coll is diluted in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution. 100 μL of the modification solution is added to the Transwell plug-in, and 0.5 ml of the modification solution is added to the well of the plate and left overnight.

[0010] Optionally, in the above method for constructing a sepsis model based on lung and kidney organ-on-a-chip, the mixed culture medium used in steps S5 to S7 is prepared by mixing RPMI 1640, HULEC-5a, DMEM / F12 and ECM ScienCell 1001 culture media in a volume ratio of 1:1:1:1.

[0011] Optionally, in the above method for constructing a sepsis model based on lung and kidney organ-on-a-chip, in steps S6 and S7, the rotation speed of the shaker is 5 rpm and the oscillation angle is 10°.

[0012] According to another aspect of the present invention, the application of the lung and kidney organ-on-a-chip-based sepsis model constructed by the above method in basic research on sepsis and in the development of drugs for the treatment of sepsis is provided.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to apply a lung-kidney organ-on-a-chip (LOA) to a sepsis model, which is far superior to traditional methods. The cells used in this invention are all human-derived, closely resembling the real human condition compared to single-cell culture. The LOA simulates both the air-fluid interface structure of the lungs and the structure of the renal tubules, and connects the special structures of the two different organs through channels, ensuring the interconnection between the lungs and kidneys. Then, it is placed on a shaker to simulate the characteristics of human blood flow, forming the LOA. The sepsis model is created using LPS, which is something that traditional cell and animal models cannot fully achieve. This invention provides a shorter construction time for the LOA-kidney chip, better stability, and overcomes various problems associated with animal research, such as racial differences, animal ethics, time constraints, and stability issues. Therefore, the LOA-kidney organ-on-a-chip sepsis model of this invention is more suitable for basic research on sepsis and drug development, and has greater clinical value. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0015] Figure 1 This is a schematic diagram of the structure of an existing lung-on-a-chip device; Figure 2 This is a schematic diagram of an existing kidney-on-a-chip device; Figure 3 This is a schematic diagram of the lung and kidney organ chip of the present invention; Figure 4 These are three-dimensional imaging images of the lung and kidney organ chip of this invention; Figure 5 This is a plan view of each layer in the lung and kidney organ chip of the present invention; Figure 6 This is the result of the penetration of lung and kidney organ-on-a-chip and blank chip; Figure 7 This refers to the resistance value detection results of the lung and kidney organ chips; Figure 8 This shows the cell growth of lung and kidney organs at different culture times on an on-chip. Figure 9 These are the results of a multi-organ microarray permeability study of LPS-induced sepsis. Figure 10 Detection results of resistance values ​​on a multi-organ microarray chip for sepsis. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0017] This invention provides a method for constructing and applying a sepsis model based on lung and kidney organ-on-a-chip, solving the problems associated with current animal experiments, cell experiments, and single organ-on-a-chip methods. Specifically, in the lung and kidney organ-on-a-chip model, this invention uses four types of human cells: human alveolar epithelial cells, human pulmonary microvascular endothelial cells, human renal tubular epithelial cells, and human umbilical vein endothelial cells. This provides a richer variety of cell types and more closely resembles the real human condition compared to culturing a single cell type. Furthermore, it employs new organ-on-a-chip technology to construct the lung-kidney chip. The lung chip simulates the air-blood barrier structure of the lungs and displays the crucial functional alveolar-capillary interface, while the kidney chip simulates the microvascular lumen and tubular structure of the renal tubules. This invention provides a shorter construction time for the lung-kidney chip, better stability, and overcomes various problems associated with animal research, such as racial differences, animal ethics, time constraints, and stability issues.

[0018] The following provides a detailed description of the construction method and application of the sepsis model based on lung and kidney organ-on-a-chip according to the present invention.

[0019] 1. Experimental Materials Preparation of FITC-Dextran solution: Weigh an appropriate amount of FITC-Dextran powder and dissolve it in physiological saline to prepare a 1 mg / mL solution. Prepare fresh before use and store in the dark.

[0020] Human type II alveolar epithelial cells and human pulmonary microvascular endothelial cells were kindly donated by the Dalian Institute of Chemical Physics, while human renal tubular epithelial cells and human umbilical vein endothelial cells were kindly donated by the Nephrology Institute of the First Medical Center of the General Hospital of the Chinese People's Liberation Army. The required reagents and instruments are listed in Tables 1-3.

[0021] Table 1 Primary antibodies used in the experiment Table 2. Secondary antibodies used in the experiment Table 3. Reagents and equipment used in the experiment 2. Construction of a sepsis model based on lung and kidney organ-on-a-chip 2.1. The steps for constructing a sepsis model based on lung and kidney organ-on-a-chip include: S1. Sterilize the organ-on-a-chip device with ultraviolet light: Expose the organ-on-a-chip device to ultraviolet light overnight.

[0022] S2. Modify the Transwell insert with type I collagen (coll): Dilute type I collagen (coll) in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution. Add 100 μL of the modification solution to the Transwell insert and 0.5 ml of the modification solution to the wells of a 24-well plate and incubate overnight.

[0023] S3. Seeding of human pulmonary microvascular endothelial cells and human umbilical vein endothelial cells: In the preparation of lung and kidney microarrays, 50 μL of 1 × 10⁻⁶ cells were seeded. 6 Place a 1 / ml cell suspension at the bottom center of each well in a 24-well plate. Place the Transwell insert into each well of the 24-well plate. The cell suspension will spread rapidly after contact with the Transwell insert. Invert the 24-well plate containing the Transwell insert and place it in a 37°C 5% CO2 incubator for 2 hours. Then invert the plate.

[0024] S4. Seeding of human alveolar epithelial cells and human renal tubular epithelial cells: In the preparation of lung and kidney microarrays, 100 μL of 1 × 10⁻⁶ cells was seeded. 5 / ml cell suspension was seeded onto the surface of the Transwell insert and incubated overnight at 37°C in a 5% CO2 incubator.

[0025] S5. Remove the transwell inserts for the lung and kidney microarrays and place them into the two middle wells of the organ-on-a-chip device. For the lung microarray, aspirate the liquid from the transwell insert to create an air-liquid interface. Simultaneously, add 2 ml of mixed culture medium (RPMI 1640: HULEC-5a: DMEM / F12: ECM ScienCell1001 = 1:1:1:1, volume ratio) to the two liquid addition wells on both sides of the organ-on-a-chip device. The lung and kidney microarrays are connected through the lower channel of the organ-on-a-chip device. Note: Remove any air bubbles from under the transwell insert.

[0026] S6. Place the prepared lung and kidney organ-on-a-chip on a shaker for dynamic culture for 5 days, such as Figure 3 As shown, A is an image of the prepared lung and kidney organ microarray dynamically cultured on a shaker in a carbon dioxide incubator; B is a working unit of the lung and kidney organ microarray; C is a schematic diagram of the side structure of the lung and kidney organ microarray. The shaker speed is 5 rpm, the shaking angle is 10°, and the mixed culture medium is changed every 1-2 days to complete the construction of the lung and kidney organ microarray. The mixed culture medium used in step S6 is the same as that in step S5.

[0027] S7. Establishment of the sepsis model: The culture medium in the constructed lung and kidney organ-on-a-chip was aspirated, and 2 mL of mixed culture medium containing 10 μg / mL LPS was added to the lower channel. 100 μL of mixed culture medium containing 10 μg / mL LPS was added to the wells of the Transwell plug on the side of the human alveolar epithelial cells and human renal tubular epithelial cells of the lung and kidney organ-on-a-chip. The culture medium was changed daily. The cells were cultured for 2 days in a 37°C, 5% CO2 incubator on a shaker at a speed of 5 rpm and a shaking angle of 10°.

[0028] 2.2. Immunofluorescence staining on lung and kidney organ microarrays The immunofluorescence staining steps for lung and kidney organ microarrays include: a. Both the upper and lower channels of the lung chip were rinsed with PBS, 3 min × 2 times (the chip was placed on a shaker during rinsing). b. Fix with 4% paraformaldehyde at room temperature for 20 minutes; c. Rinse with PBS for 5 min × 3 times (place on a shaker during rinsing); d. HC / IF specific blocking solution for 10 minutes; e. Incubate primary antibody (diluted 1:100 with blocking buffer): overnight at 4°C, protected from light; f. Rinse with PBS for 5 min x 3 times (on a shaker); g. Incubate the secondary antibody (diluted 1:1000 with blocking buffer): at room temperature for 45 minutes; h. Under light-protected conditions, rinse with PBS for 5 min × 3 times (place on a shaker during rinsing); i. Double staining of nuclei: Add DAPI and incubate in the dark for 5-10 minutes; j. Under light-protected conditions, rinse with PBS for 3 min × 3 times to remove excess DAPI, and observe under fluorescence.

[0029] 2.3. Identification of the integrity of lung and kidney organ-on-a-chip The integrity of lung-kidney organ-on-a-chip was assessed using cell junction proteins. The morphology of different cells and the expression of VE-cadherin, E-cadherin, and ZO-1 on the chip were observed using laser scanning confocal microscopy. The results showed that cells in both the lung and kidney chip systems formed tightly connected cell layers, and the expression of VE-cadherin, E-cadherin, and ZO-1 was clearly visible. Three-dimensional reconstruction results showed the formation of a continuous three-dimensional cell layer at the matrix gel interface. Figure 4 and Figure 5 ,in Figure 4 In the diagram, A and C are 3D images of the kidney chip in the lung-kidney organ-on-a-chip, with ZO1 marked in green, VE-cadherin marked in red, and DAPI in blue; B and D are 3D images of the lung chip in the lung-kidney organ-on-a-chip, with E-cadherin marked in green, VE-cadherin marked in red, and DAPI in blue. Figure 5 In the given information, A represents human alveolar epithelial cells (E-cadherin), B represents human pulmonary microvascular endothelial cells (VE-cadherin), C represents human renal tubular epithelial cells (ZO1), and D represents human umbilical vein endothelial cells (VE-cadherin).

[0030] 2.4. Identification of the permeability of lung and kidney organ-on-a-chip Alveolar-capillary barrier permeability was assessed by detecting the diffusion rate of FITC-glucan from the lower vascular layer to the upper alveolar channels. After co-culturing the lung-kidney organarray for 3 days, culture medium containing 1 mg / mL FITC-glucan was injected into the bottom channel of the chip. Two hours later, the culture medium was collected from the upper channel, and the fluorescence intensity was measured using a fluorescence microplate reader. The results showed that the permeability of the lung-kidney organarray was worse than that of the blank chip (P < 0.001 for both). Figure 6 The results (A and B in the image) indicate that the cell layers on the lung and kidney organ chip are intact, and the chip construction was successful.

[0031] 2.5. Identification of organ-on-a-chip resistance in the lungs and kidneys The successfully cultured lung-kidney microarrays were removed and placed into 24-well plates. 200 µL of PBS was added to the top of the microarray, and 700 µL of PBS was added to the bottom. Electrical resistance was measured. The blank wells without cells served as the control group. The results showed that the electrical resistance of the lung-kidney microarray was higher than that of the blank microarray, with P values ​​all < 0.001 (see [link to study].) Figure 7 The results indicate that the cell layers on the lung and kidney organ chip are intact, and the chip construction was successful.

[0032] 2.6. Timeframe for the development of the sepsis model based on lung and kidney organ-on-a-chip To explore the time required for the formation of a sepsis model on a lung and kidney organ-microarray, 2 mL of mixed culture medium containing 10 μg / mL LPS was added to the lower channel of the microarray, and 100 μL of mixed culture medium containing 10 μg / mL LPS was added to the upper side of the microarray. Cell growth on the microarray was assessed on days 1, 2, and 3 after the addition of the culture medium. Figure 8 The results showed that on the first day, the cell layer on the organ-on-a-chip was basically intact; on the second day, the cell layer was significantly damaged and the cell connections were not tight; on the third day, the cell layer was almost completely destroyed, with only a few cells remaining on the chip, indicating that the sepsis model based on the lung and kidney organ-on-a-chip was successfully constructed after two days of treatment with a mixed culture medium containing 10 μg / mL LPS.

[0033] 2.7. Changes in osmotic pressure in Lung and Kidney Organ-on-a-Chip Models of LPS-Induced Sepsis In a lung and kidney organ microarray, medium containing 10 µg / mL LPS was added and cultured for 2 days. All medium was then aspirated, and medium containing 1 mg / mL FITC-glucan was injected into the bottom channel of the microarray. Medium without FITC-glucan was added to the top layer. Two hours later, the medium was collected from the top channel, and the fluorescence intensity was measured using a fluorescence microplate reader. The results showed that the permeability of the lung and kidney organ microarray significantly increased after LPS treatment. Figure 9 A and B in the model also indicate that the blood-air barrier in the lung and kidney organ-on-a-chip is damaged, and kidney function is impaired, thus successfully constructing a sepsis model.

[0034] 2.8. Changes in electrical resistance in a lung and kidney organ-on-a-chip model of LPS-induced sepsis. In a lung and kidney organ microarray, culture medium containing 10 µg / mL LPS was added and incubated for 2 days. All culture medium was then aspirated, and the microarray was placed in a 24-well plate. 200 µL of PBS was added to the upper side of the microarray, and 700 µL of PBS was added to the lower side. Electrical resistance was then measured. The results showed that LPS treatment significantly reduced the electrical resistance of the lung and kidney organ microarray. Figure 10 A and B in the model also indicate that the blood-air barrier in the lung and kidney organ-on-a-chip is damaged, and kidney function is impaired, thus successfully constructing a sepsis model.

[0035] This invention is the first to apply lung-kidney organ-on-a-chip (LOA) technology to a sepsis model, which is far superior to traditional methods. The cells used in this invention are all human-derived. The LOA simulates both the air-fluid interface structure of the lungs and the structure of the renal tubules, and connects the special structures of the two different organs through channels, ensuring the interconnection between the lungs and kidneys. Then, it is placed on a shaker and shaken to simulate the characteristics of human blood flow, thus forming the LOA model. The sepsis model is created using LPS technology, which is something that traditional cell and animal models cannot fully achieve. Therefore, the LOA-kidney organ-on-a-chip sepsis model of this invention is more suitable for basic research on sepsis and the development of drugs for sepsis, and has greater clinical value.

[0036] The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or improve the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in the present invention; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0037] References 1. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, IngberDE. Reconstituting organ-level lung functions on a chip. Science. 2010 Jun25;328(5986):1662-8. 2. Ashammakhi N, Wesseling-Perry K, Hasan A, Elkhammas E, Zhang YS. Kidney-on-a-chip: untapped opportunities. Kidney Int. 2018 Dec;94(6):1073-1086. 3. Zhu L, Zhang J, Guo Q, Kuang J, Li D, Wu M, Mo Y, Zhang T, Gao X,Tan J. Advanced lung organoids and lung-on-a-chip for cancer research anddrug evaluation: a review. Front Bioeng Biotechnol. 2023 Nov 7;11:1299033. 4. Huang J, Yin Q, Wang Y, Zhou X, Guo Y, Tang Y, Cheng R, Yu X,Zhang J, Huang C, Huang Z, Zhang J, Guo Z, Huo X, Sun Y, Li Y, Wang H, YangJ, Xue L. EZH2 Inhibition Enhances PD-L1 Protein Stability in USP2-Mediated Colonization Cancer. Adv Sci (Weinh). 2024 Jun;11(23):e2308045. 5. Zhan Z, Lian Z, Bai H. Dexamethasone inhibited angiotensin II andits receptors to reduce sepsis-induced lung and kidney injury in rats. PLoSOne. 2024 Aug 23;19(8):e0308557. 6. Lu Z, Fang P, Li S, Xia D, Zhang J, Wu X, Pan J, Cai H, Fu L, SunG, You Q. Lactylation of Histone H3k18 and Egr1 Promotes EndothelialGlycocalyx Degradation in Sepsis-Induced Acute Lung Injury. Adv Sci (Weinh).2025 Feb;12(7):e2407064. 7. Wang HW, Wu MM, Zhu MM, Qin YY, Wang KQ, Wu CY, Zhang RR, Wang Y,Zhou C, Luo S, Lu CS, Pan JY. Growth differentiation factor 11 attenuatessepsis-associated acute kidney injury by reducing inflammation andcoagulation via PGC-1α / Nrf2 activation. Cell Mol Biol Lett. 2025 Aug 28;30(1):102. 8. Viravaidya K, Sin A, Shuler ML. Development of a microscale cellculture analog to probe naphthalene toxicity. Biotechnol Prog. 2004 Jan-Feb;20(1):316-23. 9. Schimek K, Frentzel S, Luettich K, Bovard D, Rütschle I, Boden L,Rambo F, Erfurth H, Dehne EM, Winter A, Marx U, Hoeng J. Human multi-organchip co-culture of bronchial lung culture and liver spheroids for substanceexposure studies. Sci Rep. 2020 May 12;10(1):7865. 10. Nguyen VVT, Ye S, Gkouzioti V, van Wolferen ME, Yengej FY,Melkert D, Siti S, de Jong B, Besseling PJ, Spee B, van der Laan LJW, HorlandR, Verhaar MC, van Balkom BWM. A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution ofmesenchymal stromal cell-derived extracellular vesicles. J ExtracellVesicles. 2022 Nov;11(11):e12280. 11. Zandi Shafagh R, Youhanna S, Keulen J, et al. Bioengineeredpancreas–liver crosstalk in a microfluidic coculture chip identifies humanmetabolic response signatures in prediabetic hyperglycemia. Adv Sci. 2022;9。

[0038] 12. Hu D, Sheeja Prabhakaran H, Zhang YY, Luo G, He W, Liou YC.Mitochondrial dysfunction in sepsis: mechanisms and therapeutic perspectives.Crit Care. 2024 Sep 3;28(1):292. 13. Martin-Loeches I, Singer M, Leone M. Sepsis: key insights, futuredirections, and immediate goals. A review and expert opinion. Intensive CareMed. 2024 Dec;50(12):2043-2049.

Claims

1. A method for constructing a sepsis model based on lung and kidney organ-on-a-chip, characterized in that, Includes the following steps: S1. Perform ultraviolet sterilization on the organ-on-a-chip device; S2. Modify the Transwell plug with type I collagen (colI); S3. Seeding of human pulmonary microvascular endothelial cells and human umbilical vein endothelial cells: In the preparation of lung and kidney microarrays, 50 μL of 1 × 10⁻⁶ cells were seeded. 6 / ml of cell suspension was placed in the center of the bottom of the well of a 24-well plate. The transwell plug was placed in the well of the 24-well plate. The 24-well plate with the transwell plug was inverted and placed in a 37°C 5% CO2 incubator for 2 hours, then placed upright. S4. Seeding of human alveolar epithelial cells and human renal tubular epithelial cells: In the preparation of the lung chip and the kidney chip, 100 μL of 1 × 10⁻⁶ cells was seeded. 5 / ml cell suspension was seeded on the surface of the transwell plug and cultured overnight at 37°C with 5% CO2; S5. Take out the transwell plugs used for the lung chip and the kidney chip and place them in the two middle holes of the organ-on-a-chip device respectively. Absorb the liquid in the transwell plug of the lung chip. At the same time, add 2 ml of mixed culture medium to the liquid filling holes on both sides of the organ-on-a-chip device. The lung chip and the kidney chip are connected through the lower channel of the organ-on-a-chip device. S6. Place the prepared lung and kidney organ chip on a shaker for dynamic culture for 5 days, and change the culture medium every 1 to 2 days to complete the construction of the lung and kidney organ chip; S7. Aspirate the culture medium from the constructed lung and kidney organ chip. Add 2 mL of mixed culture medium containing 10 μg / mL lipopolysaccharide to the lower channel. Add 100 μL of mixed culture medium containing 10 μg / mL LPS to the wells of the transwell plug on the human alveolar epithelial cell and human renal tubular epithelial cell sides of the lung and kidney organ chip, respectively. Change the culture medium daily and culture on a shaker in a 37°C 5% CO2 incubator for 2 days.

2. The method for constructing a sepsis model based on lung and kidney organ-on-a-chip according to claim 1, characterized in that, In step S2, type I collagen coll was diluted in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution. 100 μL of the modification solution was added to the Transwell insert, and 0.5 ml of the modification solution was added to the wells of the plate. The mixture was left to stand overnight.

3. The method for constructing a sepsis model based on lung and kidney organ-on-a-chip according to claim 1, characterized in that, The mixed culture medium in steps S5 to S7 is prepared by mixing RPMI 1640, HULEC-5a, DMEM / F12 and ECM ScienCell 1001 culture medium in a volume ratio of 1:1:1:

1.

4. The method for constructing a sepsis model based on lung and kidney organ-on-a-chip according to claim 1, characterized in that, In S6 and S7, the rotational speed of the shaker is 5 rpm and the oscillation angle is 10°.

5. The application of the lung and kidney organ-on-a-chip sepsis model constructed by the method according to any one of claims 1 to 4 in basic research on sepsis.

6. The application of the lung and kidney organ-on-a-chip sepsis model constructed by the method according to any one of claims 1 to 4 in the development of drugs for the treatment of sepsis.

Images