A microfluidic pig intestinal-vascular-liver multi-organ chip and a construction method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2026-03-20
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies lack liver and intestinal organ research models applicable to large animals such as pigs, and traditional organoid culture lacks fluid shear force and mechanical force stimulation, resulting in insufficient timeliness and accuracy of research.
A multi-organ chip consisting of pig intestine, blood vessels, and liver was constructed using microfluidic technology. Pig intestinal organoids, pig vascular endothelial cells, and pig liver organoids were co-cultured in three dimensions to simulate the 'intestinal absorption-blood circulation-liver metabolism' pathway. The chip was cultured using matrix gel and the culture medium was dynamically perfused via a microinjection pump.
It has achieved a high-fidelity simulation of the combined injury and repair model of pig intestine and liver, providing a species-specific physiological correlation research platform to support long-term research and drug evaluation, and improving the accuracy and reproducibility of the research.
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Abstract
Description
Technical Field
[0001] This invention discloses a microfluidic porcine intestinal-vascular-liver multi-organ chip, its construction method and application, which belongs to the interdisciplinary field of microfluidic chip and organoid technology. Background Technology
[0002] The intestine is a vital digestive organ in pigs, responsible for the digestion and absorption of most nutrients, and also serving as the first line of defense against external toxins and harmful substances. The intestine is a complex, functional microsystem with diverse cell types. It contains not only intestinal epithelial cells responsible for nutrient absorption, goblet cells that secrete mucus, and Paneth cells that produce antimicrobial peptides, but also endocrine cells, immune cells, and neurons, all working together to perform multiple physiological functions such as digestion, barrier defense, and signal regulation. The efficiency of nutrient absorption in the intestine directly affects the body's energy supply level and metabolic stability, thus determining the nutrient utilization and health status of all tissues and organs.
[0003] The liver is a vital metabolic and detoxification center in pigs, undertaking the core functions of nutrient transformation, storage, and distribution. It is also a key chemical plant for the degradation of endogenous metabolic waste and exogenous toxins. The liver is a complex and multifunctional biochemical processing system. Its core functional units—the hepatic lobules—contain not only hepatocytes responsible for metabolism, bile duct cells that secrete bile, and stellate cells that store vitamins, but also Kupffer cells that clear pathogens and hepatic progenitor cells with regenerative potential. Together, they collaboratively complete numerous life activities, including synthesis, decomposition, detoxification, immune regulation, and storage. The liver's metabolic regulation of nutrients directly affects the body's blood glucose stability, lipid balance, and protein synthesis, thereby determining the energy supply and homeostasis of all tissues and organs.
[0004] Organoids are collections of organ-specific cell types that develop from stem cells or organ progenitor cells. In vitro, they can spontaneously form functional cell types and spatial distributions similar to real tissue structures. Porcine intestinal organoids develop from stem cells in intestinal crypts and can differentiate into various cell types. Compared to isolated intestinal epithelial cells (IPECs), they more closely resemble the actual physiological state of animals and are more accurate and direct in studying the interaction between specific intestinal segments and reactants. Porcine liver organoids develop from progenitor cells or liver stem cells in liver tissue and can differentiate into complex structures containing various cell types such as hepatocyte-like cells and bile duct-like cells. Compared to traditional hepatocyte lines (such as HepG2), they better mimic the complex physiological structure and function of the liver, exhibiting higher physiological relevance and predictive value in studying liver-specific metabolism, pathogen infection, and drug toxicity responses. Furthermore, organoids have long culture periods, stably maintaining multicellular composition and functional characteristics, facilitating long-term research. Organoid chips, which integrate organoids into micro-engineered compartments, enable the co-culture of different types of organoids, simulating the body's physiological microenvironment for imaging, biochemical detection, and functional analysis of each part.
[0005] Microfluidics is a technology for precisely controlling and manipulating microscale fluids, enabling precise control of the flow rate, direction, mixing, and culture environment of liquids within organoid chips. Traditional organoid culture exists in a static environment, lacking physiological stimuli such as fluid shear forces and mechanical forces; microfluidics, on the other hand, provides fluid shear forces to organoids, continuously supplies nutrient-rich culture media, removes metabolic waste, and provides a dynamic yet stable environment for organoid growth.
[0006] Currently, research on the liver and intestine mainly relies on in vivo animal experiments and in vitro static cell culture, which have significant limitations in terms of timeliness and accuracy. Furthermore, most studies use human and mouse models, lacking effective models applicable to larger animals. Therefore, this invention aims to overcome the shortcomings of existing technologies by providing a microfluidic porcine intestinal-vascular-liver multi-organ on-chip system with strong species specificity, high physiological relevance, and standardized operation, along with its complete construction method and application scheme for establishing intestinal-liver injury repair models. Summary of the Invention
[0007] To overcome the shortcomings of the prior art, the present invention provides the following technical solution: The first aspect of the present invention provides a microfluidic porcine intestinal-vascular-liver multi-organ chip, including porcine intestinal organoids, porcine vascular endothelial cells and porcine liver organoids, which construct an in vitro porcine hepato-enterocirculatory microphysiological system containing the "intestinal absorption-blood circulation-liver metabolism" pathway through three-dimensional, dynamic, tandem co-culture.
[0008] Furthermore, the porcine intestinal organoids are cultured on a microarray using a 10-30% Matrigel concentration, the porcine vascular endothelial cells are cultured on a microarray using a 5-15% Matrigel concentration, and the porcine liver organoids are cultured on a microarray using a 10-30% Matrigel concentration; preferably, the intestinal and liver organoids are cultured on a microarray using a 20% Matrigel concentration, and the vascular endothelial cells are cultured on a microarray using a 10% Matrigel concentration.
[0009] Furthermore, the porcine vascular endothelial cells are porcine hip artery vascular endothelial cells, at a concentration of 0.5~1.5×10⁻⁶. 6 Inoculate at a seeding density of 1 × 10⁻⁶ cells / mL; preferably, 1 × 10⁻⁶ cells / mL. 6 Inoculate at a density of cells / mL.
[0010] Furthermore, the chip is fabricated using a dual-layer soft lithography technique, comprising a first layer of interconnected grooves and a second layer of three main channels, with channel thicknesses of 15~25μm and 150~250μm, respectively; preferably, the channel thicknesses are 20μm and 200μm, respectively.
[0011] Furthermore, the chip is dynamically cultured by continuously perfusing culture medium at a flow rate of 2~5μL / h using a micro-injection pump; preferably, the flow rate is 3μL / h.
[0012] The second aspect of the present invention provides a method for constructing a microfluidic porcine intestinal-vascular-liver multi-organ chip according to claim 1, comprising the following steps: (1) extraction and culture of porcine intestinal organoids; (2) extraction and culture of porcine liver organoids; (3) preparation of chip carrier; and (4) multi-organ chip inoculation and culture of porcine intestinal organoids, porcine vascular endothelial cells and porcine liver organoids.
[0013] Furthermore, the extraction and culture of the porcine intestinal organoids includes: taking fresh piglet intestinal tissue, washing to remove contents, scraping off intestinal mucosa and villi, cutting into small pieces, incubating with EDTA-2Na using shaking, filtering to collect crypts, resuspending in a mixture of organoid culture medium and matrix gel for inoculation and culture, and passaged periodically.
[0014] Furthermore, the extraction and culture of the pig liver organoids includes: taking fresh piglet liver tissue, washing and cutting it into small pieces, adding digestive fluid for water bath digestion, filtering to stop digestion, resuspending it in diluted matrix gel for inoculation and culture, and passaged periodically.
[0015] Furthermore, the fabrication of the chip carrier includes: designing and simulating the chip using AutoCAD and COMSOL, preparing a mold by spin coating with negative photoresist, pouring in a polydimethylsiloxane mixture for curing, drilling holes and bonding it with a glass slide, and coating the channels with poly-D-lysine after ultraviolet irradiation.
[0016] Furthermore, the multi-organ microarray seeding culture includes: seeding porcine intestinal organoids at a density of 3-8 per μL, preferably 5-7 per μL, with 20% Matrigel inoculated into the left channel; and seeding porcine vascular endothelial cells at a density of 0.5-1.5 × 10⁻⁶. 6 For porcine liver organoids, add 10% Matrigel at a density of 6-10 cells / mL and inoculate the middle channel. For porcine liver organoids, add 20% Matrigel at a density of 8 cells / mL and inoculate the right channel. Place the channel on a 30° inclined support and perfuse the medium at a flow rate of 3 μL / h for 7 days.
[0017] A third aspect of the present invention provides an application of the microfluidic porcine intestinal-vascular-liver multi-organ chip according to the first aspect, wherein the application is for establishing a model of combined intestinal-liver injury and repair.
[0018] Furthermore, the construction of the intestinal-liver combined injury and repair model includes: a normal culture period, an injury period (with the addition of LPS 100 µg / mL and APAP 20 mM) and a repair period (with the addition of 5 mM sodium butyrate and 100 ng / mL HGF).
[0019] Furthermore, it is used for research on pig nutritional metabolism, including simulating intestinal absorption and liver metabolic processes.
[0020] Furthermore, it is used for drug evaluation, including assessing the toxicity or protective effect of candidate drugs on the gut-hepatic axis.
[0021] Furthermore, it is used for the construction of pig disease models, including simulating temporal changes in intestinal-liver damage and assessing the expression of key proteins.
[0022] The beneficial effects of this invention are mainly reflected in the following aspects: (1) Highly simulates the physiological microenvironment: Through microfluidic technology, three-dimensional dynamic tandem co-culture of porcine intestinal organoids, vascular endothelial cells and liver organoids is realized, constructing a complete "intestinal absorption-blood circulation-liver metabolism" pathway. Compared with traditional static culture, it more realistically reproduces the dynamic physiological and pathological processes in vivo, improving the specificity and accuracy of the model.
[0023] (2) High species specificity: Using porcine-derived cells and organoids, designed for large livestock animals (such as pigs), it fills the gap in existing models (such as human or mouse) in the nutritional metabolism, drug evaluation and disease research of large animals, and provides more relevant physiological simulation and predictive value.
[0024] (3) Standardized in vitro platform: The chip construction method is standardized, including organoid extraction, chip preparation and dynamic perfusion culture, which facilitates operation and reproducible experiments, and provides a reliable tool for nutritional metabolism research (such as intestinal absorption efficiency and liver metabolic regulation).
[0025] (4) Construction of damage and repair model: A time-controlled intestinal-liver combined damage and repair model can be established. By adding damage agents (such as LPS and APAP) and repair factors (such as sodium butyrate and HGF), the dynamic changes of intestinal barrier, vascular network and liver function can be evaluated, supporting detection such as immunofluorescence staining to verify the repair effect.
[0026] (5) High application scalability: It is suitable for evaluating the combined toxicity or protective effects of candidate drugs, toxins, prebiotics, etc. on the gut-liver axis. It has unique value, especially in simulating the first-pass effect, chronic toxicity and repair mechanism, and improves the efficiency of livestock health management and drug development. Attached Figure Description
[0027] Figure 1 Growth diagram of pig intestinal organoids; Figure 2 Growth diagram of pig liver organoids; Figure 3 Growth diagram of pig liver organoids; Figure 4 Immunofluorescence staining of porcine liver organoid marker proteins; Figure 5 Growth of porcine intestinal organoids at different matrix gel concentrations; Figure 6 Growth of porcine liver organoids at different matrix gel concentrations Figure 7 Growth of porcine hip artery endothelial cells at different matrix gel concentrations Figure 8 Growth of porcine hip artery endothelial cells under different inoculation densities; Figure 9 Multi-organ microarray growth diagram; Figure 10 Multi-organ microarray immunofluorescence staining; Figure 11 A multi-organ-on-a-chip model of intestinal-liver injury and repair; Figure 12 Temporal changes of intestinal organoids in a multi-organ-on-a-chip model of intestinal-liver injury and repair; Figure 13 Expression of key proteins in intestinal organoids in a multi-organ-on-a-chip model of intestinal-liver injury and repair; Figure 14 Temporal changes of liver organoids in a multi-organ-on-a-chip model of intestinal-liver injury and repair; Figure 15 Expression of key liver organoid proteins in a multi-organ-on-a-chip model of intestinal-liver injury and repair; Figure 16Differential expression of key proteins in intestinal and liver organoids in a multi-organ-chip model of intestinal-liver injury and repair. Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0029] Example 1: Preparation of Intestinal-Blood Vessel-Liver Organoids 1. Extraction and culture of porcine intestinal and liver organoids (1) Extraction and culture of porcine intestinal organoids Step 1: Take about 10 cm of fresh piglet intestinal tissue and place it in a culture dish. Cut the intestinal segment longitudinally with the intestinal lumen facing upward. Use a pipette to draw cold DPBS and rinse repeatedly to remove the contents. Use a coverslip to gently scrape away the intestinal mucosa and villi. After rinsing again, cut the intestinal segment into two small pieces of 2 mm and place them in a 50 mL conical tube. Use cold DPBS to blow and wash several times until the supernatant is clear. Step 2: Place the tissue block in ice-cold DPBS containing 2.5 mM disodium ethylenediaminetetraacetate (EDTA-2Na) and incubate at 80 rpm for 30 minutes. After incubation, wait for the tissue block to settle, then aspirate the supernatant and resuspend it in cold DPBS containing 0.1% BSA. Use a pipette to blow the supernatant up and down and filter it through a 70 μm cell sieve. Collect the fraction and place it on ice. Step 3: Take a fraction and observe the number of crypts under a microscope. Adjust the inoculation density to 500 crypts per well. Transfer the required volume of fraction to a 15 mL centrifuge tube, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, resuspend in cold DMEM / F12 complete medium, centrifuge at 300 g for 5 min, and discard the supernatant again. Step 4: Gently pipette and resuspend the crypts in 25 times the volume of organoid culture medium and Matrigel mixture (culture medium: Matrigel = 1:3), take 25 μL and inoculate it into a 24-well plate, let it stand at 37°C for 10 minutes to allow Matrigel to solidify completely, then add 750 μL of culture medium and culture, changing the medium every two days. Step 5: After the organoids have grown for 5 days, passage them, add PBS buffer and gently pipette them into a 15 mL centrifuge tube, incubate at 4°C for 20 min to dissolve the matrix gel, centrifuge at 300 g for 5 min and discard the supernatant, add 2 mL PBS, mechanically pipette and centrifuge again, add organoid culture medium and matrix gel mixture according to the amount of precipitate, pipette and resuspend, and spot culture.
[0030] The results are as follows Figure 1 As shown, the morphology of intestinal organoids changes with prolonged culture time: from early regular small round hollow structures, they gradually develop into dense structures with irregular shapes, complex internal folds, and multi-layered cell structures. This indicates that the organoids are growing and developing well, differentiating smoothly, and their tissue structure is becoming more mature and complex.
[0031] (2) Extraction and culture of porcine liver organoids Step 1: Take fresh piglet liver tissue and soak and wash it in a culture dish containing cold DPBS. Peel off the surface mucosa, take a small portion and put it in a 1.5 mL centrifuge tube with a small amount of buffer and cut it into small pieces. Step 2: Transfer the fully minced tissue into a 15mL centrifuge tube, add 5mL of tissue digestion solution, and digest in a 37℃ water bath for 30 minutes. Take a small amount of digestion solution every 10 minutes and observe it under a microscope. Stop digestion when you observe a large number of cell clusters or single cell groups smaller than 70 μm. Step 3: Filter using a 100 μm cell sieve and a 50 mL centrifuge tube, and wash the cell sieve with 3 times the volume of buffer to stop digestion. Aliquot the filtrate into 15 mL centrifuge tubes, centrifuge for 5 min at 300 g, and discard the supernatant (if the precipitate is red, it indicates the presence of red blood cells; add 1-2 mL of red blood cell lysis buffer to lyse for 2 min, add buffer to 10 min, centrifuge again for 5 min at 300 g, and discard the supernatant). If there are few or no red blood cells in the precipitate, add 1:3 diluted matrix gel according to the amount of precipitate, seed into 24-well cell culture plates, add about 25 μL of sample to each well, place the culture dish in a 37℃ incubator for 15 min to solidify, and then add 750 μL of culture medium to each well. Step 4: After the organoids have grown for 6 days, passage them, add PBS buffer and gently pipette them into a 15 mL centrifuge tube, incubate at 4°C for 20 min to dissolve the matrix gel, centrifuge at 300 g for 5 min and discard the supernatant, add 1 mL of digestion solution, pipette them into the tube and centrifuge again, add organoid culture medium and matrix gel mixture according to the amount of precipitate, pipette them into the resuspending state, and spot culture them into a plate.
[0032] The results are as follows Figure 2 As shown, with the extension of culture time, liver organoids gradually develop from small, dense cell clusters in the early stage to spherical bodies with a central cavity and uniform transparency inside. During this process, the organoid volume gradually increases, the outer boundary gradually thickens, the outline becomes clear, and the overall shape tends to be round and full.
[0033] 2. Immunofluorescence staining of porcine intestinal and liver organoids In porcine intestinal organoids, stem cells express transcription factor SOX2, microvilli on the brush border of intestinal epithelial cells express villiin, goblet cells express mucin (MUC2), endocrine cells express chromogranin (ChgA), and the intestinal cell tight junction proteins ZO-1, Occludin, and Claudin-1 can be detected by immunofluorescence staining.
[0034] In liver organoids, hepatocytes express ALB protein, bile duct cells express keratin CK19, hepatic stellate cells express desmin DES, and hepatic sinusoidal endothelial cells express CD31.
[0035] The results are as follows Figure 3-4 As shown: ChgA in intestinal organoids is mainly concentrated on the inner side of the lumen, indicating that the organoids have potential endocrine functions; VILLIN and SOX9 protein signals are uniformly distributed in a ring pattern inside and outside the lumen, forming a continuous strong signal band, indicating that the organoids have formed a complete and polarized epithelial monolayer structure and have good proliferative and regenerative potential; MUC2 protein signal is clearly located in a specific region on the outer side of the lumen, demonstrating the formation of the mucosal barrier function of the organoids. ALB protein in liver organoids is clearly and continuously located on the outer edge of the lumen, forming a ring-shaped strong signal band, indicating the presence of hepatocytes and that the organoids have liver metabolic functions; keratin CK19 forms a bright and continuous ring signal band on the periphery of the organoid and extends into the interior of the lumen, with clearly discernible patterns, consistent with the developmental characteristics of bile duct cells; desmin DES is distributed in a bright aggregate pattern on the periphery of the lumen, consistent with the distribution characteristics of stellate cells; CD31 is located in a specific region of the intima with clear signal, indicating that the endothelial cells have initially formed a vascular network-like structure.
[0036] 3. Construction of multi-organ-on-a-chip (1) Chip carrier fabrication Step 1: Design the chip using AutoCAD software, simulate it using COMSOL Multiphysics software, and fabricate the chip mold using a two-layer soft lithography technique. The first layer of the chip consists of interconnected grooves, which are uniformly spin-coated onto the silicon wafer with a 20µm thick negative photoresist (SU-2050) and baked under ultraviolet light. The second layer consists of three main channels with a thickness of 200µm, which are formed by spin-coating, exposure, and development using a high-resolution transparent mask to create the final microstructure on the silicon wafer. Step 2: Mix polydimethylsiloxane and curing agent thoroughly at a ratio of 10:1, pour into chip mold, vacuum for 30 minutes to remove air bubbles, and cure in an oven at 80°C for 3 hours. Then, drill holes at both ends of the chip channel to penetrate the upper and lower layers as entry and exit holes. Use a bonding machine to firmly bond the lower surface of the chip to the glass slide, and cure in an oven at 80°C for 3 hours. Step 3: Irradiate the prepared chip under a UV lamp for 30 min, coat the chip channels with poly-D-lysine at 37°C for 15 min, and then rinse the chip channels three times with purified water for later use. Rinse three times with sterile water to complete pretreatment. Next, prepare PIEC cells for inoculation by aspirating the culture dish supernatant, washing twice with room temperature PBS, adding 1.5 mL of trypsin and digesting at 37°C for 5 minutes, then adding 3 mL of ECM medium to stop digestion. Collect the cell suspension by pipetting, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend the cell pellet in a mixture of ECM and Matrigel, and adjust the concentration to 2 × 10⁻⁶. 6 Organoids were prepared in mL for later use. Intestinal and liver organoids were prepared separately. Approximately 300 organoids were collected by gently pipetting the organoid domes in a 24-well plate with pre-cooled PBS and transferred to centrifuge tubes. After standing at 4°C for 20 minutes, the tubes were centrifuged at 300g for 5 minutes, the supernatant was discarded, and the pellets were resuspended in 40 μL of dedicated culture medium. The pellets were then mixed with Matrigel. All organoid manipulations were performed on ice. Subsequently, microarray seeding was performed. PIEC cell suspension, intestinal organoid mixture, and liver organoid mixture were injected into the middle, left, and right channels of the microarray, respectively. The microarray was then placed in a 30°C, 5% CO2 incubator for 20 minutes to allow the Matrigel to harden, followed by 4 hours of static culture to promote attachment. When establishing the culture system, a 1 mL syringe connected to a capillary tube (0.46 mm inner diameter, 0.76 mm outer diameter) and a microfluidic injection pump were used to continuously perfuse the corresponding dedicated culture medium into the three channels at a flow rate of 3 μL / h. Finally, the chip was placed on a custom-made device with a 30° tilt angle and transferred into an incubator to ensure that the liver organoids remained stably positioned on the underside of the channel during culture.
[0037] (2) Optimal matrix gel concentration for porcine intestinal organoid microarray culture The growth status of porcine intestinal organoids was observed under a microscope at different matrix gel concentrations (5%, 10%, 20%, 30%, 40%), and the growth rate of diameter and area was statistically analyzed using ImageJ software.
[0038] The results are as follows Figure 5 As shown, under different concentrations of Matrigel, the growth of intestinal organoids exhibited typical developmental patterns: gradually increasing in size, evolving from initially dense small cell clusters or structures with small cavities into translucent spherical bodies with a prominent central cavity. The expansion and maturation process of organoids was particularly pronounced at a Matrigel concentration of 20%. Quantitative analysis using ImageJ software revealed that the growth rates of organoid diameter and area in this group were significantly higher than those in other concentration groups, indicating that 20% Matrigel most effectively supports the growth and development of intestinal organoids in the microarray, achieving optimal culture results.
[0039] (3) Optimal matrix gel concentration for porcine liver organoid microarray culture The growth status of porcine liver organoids was observed under a microscope at different matrix gel concentrations (10%, 20%, 30%), and the area growth rate was statistically analyzed using ImageJ software.
[0040] The results are as follows Figure 6 As shown, under three substrate gel concentrations (10%, 20%, and 30%), the morphological development of liver organoids on the microarray followed a relatively regular trend, with a gradual increase in volume. The structure evolved from an initial dense cell cluster to a more defined cavities, eventually developing into a well-formed structure with translucent edges. Among these, the liver organoids at a 20% substrate gel concentration showed the fastest area growth, reaching approximately 2.4% by day 6, significantly higher than the 10% and 30% groups. This group exhibited a consistently high growth rate in the later stages of culture (days 4-6), indicating that a 20% substrate gel concentration most effectively supports the structural development and morphological maturation of liver organoids, providing suitable conditions for their culture on the microarray.
[0041] (4) Exploration of conditions for implantation of porcine hip artery endothelial cells (PIEC) The formation of PIEC cell vascular networks at different matrix gel concentrations (0%, 10%, 20%, 30%) was observed under a microscope; the total branch length, intersections, mesh number, and node number were counted using ImageJ software.
[0042] The results are as follows Figure 7 As shown: Total branch length refers to the total length of all vascular branches, reflecting the development of vascular endothelial cells; nodes and intersections represent the connection points formed by the convergence of two or more vascular tubules; mesh count refers to the number of closed network structures. All three reflect the degree to which vascular endothelial cells form a vascular network. PIEC cells exhibit varying degrees of vascular development under different concentrations of Mastol. Quantitative analysis using ImageJ software showed that at a 10% Mastol concentration, the number of meshes, total branch length, nodes, and intersections formed by PIEC vascular endothelial cells were significantly higher than at other concentrations, indicating a superior degree of vascular network development.
[0043] The results are as follows Figure 8 As shown: at a cell density of 1×10 5 Under the cell / mL condition, the number of PIEC cells on the chip is sparse and it is difficult to form a vascular network; at 1×10 6 Under conditions of cells / mL, a dense network can be formed with clear nodes and branches, and good cell viability; at 1×10⁻⁶ cells / mL, it can form a dense network with clear nodes and branches, and the cells have good viability; 7 At a cell / mL level, cells begin to aggregate and shrink, and cell activity within the vascular network is poor. Therefore, 1×10 6cells / mL is most effective for PIEC vascular endothelial cells to form vascular networks on the chip.
[0044] (5) Seeding and culture of multi-organ microarrays Step 1: Porcine intestinal organoids were collected and resuspended at a density of 6 organs / μL, supplemented with 20% Matrigel, and seeded into the left channel of the microarray. Porcine hip artery endothelial cells (PIEC) were seeded at a density of 1×10⁻⁶ cells / μL. 6 Collect and resuspend porcine liver organoids at a density of 8 cells / μL, add 10% Matrigel, and seed them in the middle channel of the chip. Place the multi-organ chip on a 30° inclined support, ensuring that the liver organoids are on the bottom side. Step 2: Connect a 1mL syringe to a capillary tube with an inner and outer diameter of 0.46 × 0.76 mm, and use a microinfusion pump to continuously perfuse culture medium into the channel containing intestinal organoids, liver organoids and PIEC cells at a constant rate of 3 μL / h for 7 days.
[0045] The results are as follows Figure 9-10 As shown, under continuous flow culture for seven days, intestinal organoids gradually grew from initially small cell clusters, with a significant increase in volume. The morphology of vascular endothelial cells underwent regular changes, forming a gradually denser network structure with an increase in the number of meshes and branches. Liver organoids developed from a relatively loose cellular state into larger and denser structures with clearer boundaries, maintaining the integrity of their three-dimensional structure. This indicates that in the microfluidic organoid chip, the low-shear fluid environment generated by the peristaltic pump provides a stable nutrient exchange environment for the organoids, ensuring timely removal of metabolic waste, maintaining their morphological stability and metabolic activity, and driving vascular cells to form physiologically relevant barrier structures. After completing seven days of continuous flow culture, immunofluorescence staining of intestinal organoids, vascular endothelial cells, and liver organoids within the chip confirmed that this culture modality can completely maintain the complex cellular composition, specific differentiation state, and typical morphology of vascular endothelial cells in intestinal and liver organoids. In intestinal organoids, VILLIN, MUC2, CHGA, and ZO-1 were all expressed, indicating the establishment of a complex epithelial structure with polarity and barrier function. In liver organoids, the expression of ALB, CYP3A4, DESM, and CK19 demonstrated that different cells could coexist and that liver tissue possessed heterogeneity and core functions. In vascular endothelial cells, CD31 and VEGFA were stably expressed in a continuous monolayer network structure, serving as markers of their functional identity and intact intercellular connections. Example 2: Study on intestinal-liver injury and repair based on microfluidic multi-organ chip (1) Construction process of damage and repair model like Figure 11 As shown: Group A (Normal Culture): After being seeded and stabilized for 7 days, the multi-organ microarray was maintained for another 4 days using normal organoid complete culture medium under a dynamic perfusion system (flow rate 3 μL / h). This group was used to establish and confirm the stable state of the intestinal barrier, vascular network, and liver anabolic function on the microarray under continuous fluid shear stress.
[0046] B-cell liver repair group: This group aims to verify the effectiveness of the synergistic repair strategy and mainly includes the following three phases.
[0047] Culture period: 1 day of normal perfusion culture.
[0048] Injury phase: Two damaging agents were simultaneously added to the perfusion medium: lipopolysaccharide (LPS) at a final concentration of 100 µg / mL to specifically induce intestinal epithelial inflammation and tight junction disruption; and acetaminophen (APAP) at a final concentration of 20 mM to induce typical cellular metabolic stress and toxic damage in liver organoids. The injury intervention lasted for 2 days.
[0049] Repair phase: Remove the culture medium containing the damaging agent and replace it with a medium containing specific repair factors. Add 5 mM sodium butyrate (as a key energy source for intestinal epithelium and an inhibitor of histone deacetylase, designed to stabilize tight junctions, reduce inflammation, and promote intestinal barrier repair) and 100 ng / mL hepatocyte growth factor (HGF) (as a potent hepatocyte mitogen and anti-apoptotic factor, designed to drive hepatocyte regeneration and functional recovery) to the normal culture medium. Continue dynamic culture for 1 day.
[0050] The C intestinal-liver injury group served as a necessary control for group B, distinguishing between "active repair" and "natural recovery." Its treatment was identical to group B during the culture and injury periods. On day 4, the injury medium was replaced with normal medium, and culture continued for one more day. The results from this group were used to assess the organoid's own recovery potential after injury removal, serving as a key control to demonstrate the specificity of the repair factor.
[0051] (2) Evaluation methods for immunofluorescence staining At the experimental endpoint, the chip was fixed in situ and subjected to multiplex immunofluorescence staining using a standardized procedure to achieve simultaneous visual evaluation of its structure and function. The staining steps are as follows: Fixation: 4% paraformaldehyde was perfused at a flow rate of 12 μL / h using a micro-injection pump for 1 h. Washing: Use a microinfusion pump to perfuse PBS at a flow rate of 3 μL / min and wash for 30 min; Permeabilization: Perfuse 0.5% Triton X-100 PBS at a flow rate of 3 μL / min using a microinfusion pump for 30 min; Blocking: Use a microinfusion pump to perfuse 5% BSA-0.1% Triton X-100 PBS at a flow rate of 3 μL / min for 2 hours for blocking; Primary antibody: The primary antibody was diluted proportionally with 5% BSA-0.1% Triton X-100 PBS solution and incubated at 4°C for 12 h after perfusion. ZO1 and MUC2, CD31 and VEGFA, and CYP3A4 and ALB were injected into the intestinal organoid channel, vascular endothelial cell channel and liver organoid channel, respectively. Washing: Wash twice with PBS solution; Secondary antibodies: The secondary antibodies goat-anti-rabbit CoraLite® 594 and goat-anti-mouse CoraLite® 488 were prepared at a ratio of 1:200 using 5% BSA-0.1% Triton X-100 PBS solution, and incubated in the perfusion channel at room temperature in the dark for 1 hour. Washing: Wash twice with PBS solution; DAPI: Incubate DAPI for 5 minutes; Observation: Under a fluorescence microscope, ZO1, CD31, and CYP3A4 appear green, while MUC2, VEGFA, and ALB appear red.
[0052] Among them, ZO-1 (a tight junction core protein that directly quantifies intestinal barrier integrity); MUC2 (a mucin secreted by goblet cells that assesses mucus barrier function and goblet cell activity); CD31 (an endothelial cell marker used for three-dimensional reconstruction of vascular networks and calculation of branch length and node number); VEGFA (vascular endothelial growth factor that indicates endothelial cell activation, injury response, and paracrine function); ALB (albumin, the gold standard marker of hepatocyte-specific synthetic function); and CYP3A4 (a core metabolic enzyme that assesses hepatocyte metabolic capacity).
[0053] (3) Successful construction of damage and repair model Figure 12-15 The temporal changes and immunofluorescence staining of intestinal organoids and livers in the normal culture group, intestinal-liver repair group, and intestinal-liver injury group are presented sequentially.
[0054] In the normal culture group (Group A), both intestinal and liver organoids exhibited healthy, intact, and stable growth. DAPI staining showed dense and uniformly distributed cell nuclei. The MUC2 protein in the intestinal organoids formed a continuous, bright signal band at the luminal margin, indicating normal goblet cell function. The ZO-1 protein, serving as the core of tight junctions, should have formed a continuous and clear linear signal along the cell boundary in this group, collectively forming a robust intestinal barrier. Furthermore, the liver organoids showed strong positive fluorescence signals for both CYP3A4 and ALB, exhibiting widespread and uniform co-expression within the organoids, indicating that hepatocytes simultaneously maintain active metabolic and protein synthesis functions.
[0055] In the intestinal-hepatic injury group (Group C), both intestinal and liver organoids exhibited severe specific functional impairments. Specifically, the intestinal organoids showed irregular structural deformation, decreased nuclear density in some areas, and significantly weakened MUC2 signal intensity, which changed from a continuous band-like pattern to a diffuse and discontinuous distribution, indicating impaired goblet cell secretion. The ZO-1 signal in this group showed marked blurring and diffusion from the cell boundary into the cytoplasm, indicating disruption of tight junction structures and loss of physical barrier integrity. Furthermore, the overall structural integrity of the liver organoids was impaired, with blurred edges or loose structures in some areas. DAPI signaling showed sparse nuclear distribution, suggesting a reduction in cell number or viability. In addition, the fluorescence signal intensity of CYP3A4 and ALB was simultaneously and significantly weakened, with a reduced distribution range and diffuse and uneven signaling. This confirms that APAP injury not only disrupts organoid structure but also directly and severely inhibits the core metabolic and synthetic functions of hepatocytes.
[0056] In the intestinal-liver repair group (Group B), both intestinal and liver organoids showed clear signs of functional recovery. The intensity and continuity of MUC2 signal were improved compared to Group C, suggesting a certain degree of recovery in goblet cell function. Repair also promoted the reconstruction of ZO-1 signal continuity, with partial recovery of interrupted linear signals, indicating that tight junction structures were being repaired. Furthermore, the liver organoids exhibited more regular morphology and restored structural compactness. DAPI showed an increase in nuclear density. The most critical change was in the functional markers: the fluorescence signals of CYP3A4 and ALB both showed a significant increase in intensity, and their expression distribution within the liver organoids was more widespread and coherent. This indicates that the repair intervention of hepatocyte growth factors effectively promoted the synergistic recovery of damaged hepatocytes in terms of metabolic capacity and synthetic function.
[0057] like Figure 16As shown, the mean immunofluorescence intensities of key proteins MUC2, ZO1, ALB, and CYP3A4 in the entero-hepatic repair group (Group B) were significantly higher than those in the entero-hepatic injury group (Group C). For example, the mean fluorescence intensities of ZO1 in the normal culture group (Group A), entero-hepatic repair group (Group B), and entero-hepatic injury group (Group C) were 34.69 ± 1.41, 54.21 ± 2.42, and 22.49 ± 0.97, respectively. The model described in this invention can not only be used to assess the potency of the aforementioned nutrients (sodium butyrate) and growth factors (HGF), but its standardized procedures can also be directly transferred to evaluate the combined toxicity or protective effects of candidate drugs, toxins, prebiotics, and other substances on the entero-hepatic axis, especially in simulating first-pass effects, digestion and absorption, and chronic toxicity in vivo, where it has unique value.
Claims
1. A method for constructing a microfluidic porcine intestinal-vascular-liver multi-organ chip, characterized in that, Includes the following steps: (1) Extraction and culture of pig intestinal organoids; (2) Extraction and culture of pig liver organoids; (3) Preparation of chip carriers; (4) Multi-organ chip culture of pig intestinal organoids, pig vascular endothelial cells and pig liver organoids.
2. The construction method according to claim 1, characterized in that, The extraction and culture of the porcine intestinal organoids include: taking fresh piglet intestinal tissue, washing to remove contents, scraping off intestinal mucosa and villi, cutting into small pieces, incubating with EDTA-2Na with shaking, filtering to collect crypts, resuspending in a mixture of organoid culture medium and matrix gel for inoculation and culture, and passaged periodically.
3. The construction method according to claim 1, characterized in that, The extraction and culture of the pig liver organoids include: taking fresh piglet liver tissue, washing and cutting it into small pieces, adding digestive solution for water bath digestion, filtering to stop digestion, resuspending it in diluted matrix gel for inoculation and culture, and passaged periodically.
4. The construction method according to claim 1, characterized in that, The fabrication of the chip carrier includes: designing and simulating the chip using AutoCAD and COMSOL, preparing a mold by spin coating and exposure with negative photoresist, pouring in a polydimethylsiloxane mixture for curing, drilling holes and bonding it with a glass slide, and coating the channels with poly-D-lysine after ultraviolet irradiation.
5. The construction method according to claim 1, characterized in that, The multi-organ microarray seeding culture includes: seeding porcine intestinal organoids at a density of 3-8 organs / μL with 10-30% Matrigel in the left channel; and seeding porcine vascular endothelial cells at a density of 0.5-1.5 × 10⁻⁶ cells / μL. 6 Inoculate the middle channel with 5-15% Matrigel at a density of 6-10 cells / μL, and inoculate the right channel with 10-30% Matrigel. Place the channel on a 30° inclined support and perfuse the medium at a flow rate of 2-5 μL / h for 5-8 days.
6. A microfluidic porcine intestinal-vascular-liver multi-organ chip, characterized in that, The microfluidic porcine intestinal-vascular-liver multi-organ chip is prepared by the method described in any one of claims 1 to 5.
7. The application of the microfluidic porcine intestinal-vascular-liver multi-organ chip constructed by the method described in any one of claims 1 to 5, characterized in that, The application is for establishing a combined intestinal-liver injury and repair model, the construction of which includes: a normal culture period, an injury period, and a repair period.
8. The application according to claim 7, characterized in that, The application is for research on pig nutritional metabolism; preferably, the application is for research simulating intestinal absorption and liver metabolism.
9. The application according to claim 7, characterized in that, The application is for drug evaluation; preferably, the application is for evaluating the toxicity or protective effect of candidate drugs on the gut-hepatic axis.
10. The application according to claim 7, characterized in that, The application is for constructing pig disease models; preferably, the application is for simulating temporal changes in intestinal-liver injury and assessing the expression of key proteins.