Method for constructing carbon tetrachloride damage model based on three-dimensional liver chip and evaluating nutrition intervention

Abstract

The invention discloses a carbon tetrachloride damage model construction and nutrition intervention evaluation method based on a three-dimensional liver chip, and belongs to the technical field of microfluidics and biological medicines. According to the method, an in-vitro bionic liver tissue model is constructed by utilizing a three-dimensional liver chip, liver cell injury is induced by perfusing carbon tetrachloride, on the basis, substances such as algae-derived extracellular vesicles are added for repairing intervention, and meanwhile, contrastive analysis is performed with a two-dimensional plate culture system. Results show that the secretion level and the function maintenance time of albumin, urea and the like of liver cells in a liver chip model are superior to those of two-dimensional culture, and after nutrient intervention is carried out, liver injury indexes such as ALT, AST and the like in the chip model are remarkably reduced, and the cell activity and the metabolic function are obviously recovered. The model can be used for evaluating the curative effect of nutrient substances on chemical liver injury, overcomes animal experiment species difference and two-dimensional culture limitation, provides an efficient and sensitive in-vitro research platform for medicine and nutrition function screening, and has wide application prospects and industrialization potential.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and biomedicine, specifically relating to a method for constructing a carbon tetrachloride injury model based on a three-dimensional liver chip and evaluating nutritional intervention. Background Technology

[0002] The liver is a vital organ for metabolism and detoxification, and its dysfunction can lead to various metabolic diseases. Carbon tetrachloride (CCl4) is a commonly used chemical inducer of liver injury. It can trigger oxidative stress and lipid peroxidation through the generation of free radicals, leading to damage to the hepatocyte membrane structure and dysfunction. It is widely used in animal liver injury models (BasuS). Toxicology Letters , 2003, 145(3):265–272; Manibusan MK, Odin M, Eastmond DA. Critical Reviews in Toxicology , 2007, 37 (8): 701-715; Knockaert L, BersonA, Ribault C, et al. Laboratory Investigation , 2012; 92(3):396–410.). However, traditional animal models suffer from problems such as large species differences, long experimental cycles, ethical restrictions, and high costs, and their experimental results often deviate significantly from human physiological states (van der Worp HB, Howells DW, Sena ES, et al.). PLoS Medicine , 2010, 7 (3): e1000245; Seok J, Warren HS, Cuenca AG, et al. PNAS ,2013, 110 (9): 3507-3512; Van Norman G A. JACC: Basic to Translational Science , 2020, 5 (7): 830-841.).

[0003] To overcome the limitations of animal experiments, researchers have developed various in vitro liver injury models. For example, two-dimensional plate culture systems based on human hepatocytes or hepatocyte lines can be used for some metabolic studies, but the cells in this system exhibit flattened morphology, loss of polarization, and decreased metabolic enzyme activity, making it difficult to maintain liver-specific functions in the long term. In recent years, "liver-on-a-chip" based on microfluidic technology has provided a new research tool for in vitro biomimetic livers. Existing published research and patents mainly focus on drug toxicology or inflammatory stress. For example, patent CN115896224A (Soochow University) discloses a method for evaluating drug-induced liver injury under inflammatory stress based on microfluidic technology. It induces an inflammatory response through lipopolysaccharide (LPS) and evaluates drug toxicity, which can reflect the drug-related liver injury mechanism to a certain extent. However, this method mainly targets inflammatory stress drugs and does not involve chemical CCl4 damage or nutritional intervention applications. Patent CN118581188A (Southern Medical University) proposes a multi-organ microphysiological culture system based on microfluidic technology for detecting hepatotoxicity and nephrotoxicity of traditional Chinese medicine. Its design focuses on the joint analysis of multi-organ interactions and drug metabolism pathways, but it does not establish a specific model for single-organ chemical damage and repair. Patent CN118222397A reports a method for evaluating the anti-inflammatory effects of probiotics based on intestinal microarrays, demonstrating the potential of organ-on-a-chip in screening functional substances; however, its model belongs to the intestinal system and has limited correlation with liver metabolic function.

[0004] In addition, some academic studies have explored the CCl4-induced liver injury and repair process using two-dimensional cell or three-dimensional organoid models. Although three-dimensional spherical culture improves the spatial structure and intercellular interaction to some extent, it lacks a similar fluid shear force and metabolic supply environment in vivo, making it difficult to achieve continuous perfusion and multi-cell synergistic response, resulting in a gradual decline in cell viability and enzyme activity during the culture period. Existing liver chip models generally have the following common shortcomings: (1) lack of controllable liver injury models induced by chemical toxins (such as CCl4), which cannot realistically simulate the in vivo oxidative stress and lipid peroxidation process; (2) most models rely on pump-driven or static culture, making it difficult to provide a stable and physiologically relevant low-shear fluid environment; (3) lack of evaluation system for the repair capacity of nutrient-active substances, resulting in the application scope being limited to drug toxicology; (4) some models use two-dimensional culture, and cell polarization, CYP activity and secretion function are prone to rapid decline, making it difficult to carry out long-term injury and repair research. Most microfluidic chips used rely on pump perfusion culture, and the complexity of the pipeline design and the limitations of operation restrict the model construction and evaluation of nutrient intervention. In summary, there is currently a lack of an integrated evaluation model for CCl4 chemical damage and nutritional intervention based on a three-dimensional microfluidic liver chip. Existing technologies cannot simultaneously simulate the dynamic metabolic, immune, and hemodynamic environments of the liver in vitro, nor can they achieve continuous control of damage induction and repair intervention within the same system. Therefore, it is difficult to systematically and quantitatively evaluate the effects of nutritional factors on liver function recovery. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to overcome the limitations of current in vitro hepatocyte models, such as their singular structure, lack of dynamic fluid environment, and inability to accurately reflect liver metabolism and repair processes. The invention provides a carbon tetrachloride (CCl4) injury model and nutritional intervention evaluation method based on a three-dimensional liver chip. This method reconstructs a three-dimensional liver tissue structure with shear stress and metabolic microenvironment by culturing human HepG2 hepatocytes within a three-dimensional liver chip. Under dynamic conditions, CCl4-induced hepatocyte injury is achieved, and specific nutritional factors are introduced for repair intervention. This method utilizes pump-free dynamic system-on-a-chip technology to reconstruct a biomimetic liver microenvironment in vitro, achieving an integrated and dynamic simulation of CCl4 chemical damage and nutritional intervention processes. This allows for efficient and repeatable evaluation of the liver function protection and repair effects of nutrients. Compared to traditional two-dimensional culture or organoid models, the microarray model established in this invention can more realistically simulate the in vivo physiological state of the liver, maintaining cell polarization and metabolic function in the long term. Its damage and intervention processes are highly controllable and reproducible. Detection indicators such as albumin, total bilirubin, urea synthesis, and γ-glutamyl transferase changes in hepatocytes within the liver microarray system more closely resemble in vivo responses. Simultaneously, this system enables parallel screening of multiple components and comparison of pharmacological effects, significantly improving experimental efficiency and data reliability. Therefore, this invention is the first to introduce a three-dimensional microfluidic liver microarray into a CCl4 liver injury-nutritional repair system, achieving precise simulation and quantification of intervention effects on liver function changes under dynamic in vitro conditions, demonstrating significant innovation and broad application value.

[0006] To achieve the above objectives, a three-dimensional liver microarray system was first constructed. The microarray consists of a top cover, a core, and a base. The core contains three sets of parallel cell culture chambers (small chamber pores) and sample inlet ports. A fluid channel connects the two ends of each set of sample inlet ports and small chamber pores. Insertable culture chambers are installed within the small chamber pores. By culturing human hepatocytes, a three-dimensional liver tissue model with metabolic function is formed under continuous fluid shear force.

[0007] In one embodiment, the three-dimensional liver chip comprises, from top to bottom, an upper cover, a core, a connecting membrane, and a base.

[0008] In one embodiment, the top cover and the bottom base are sealed together by a microporous membrane in the middle. The membrane material can be polycarbonate or polytetrafluoroethylene to ensure good air permeability and cell adhesion performance.

[0009] In one embodiment, the core contains multiple sets of parallel culture units, each culture unit including one or more small chambers and two sample inlets located on both sides of the small chambers; the sample inlets and the lower part of the small chambers are connected by a channel; the channel width is about 5 to 30 mm and the height is 0.1 to 0.6 mm, which can achieve stable microfluidic shear force.

[0010] In one embodiment, a pluggable culture chamber is provided inside the chamber hole; the bottom of the pluggable culture chamber is provided with a porous bottom membrane, the edge of which is attached to the connecting membrane and suspended above the channel.

[0011] In one embodiment, the entire chip is treated with plasma or ultraviolet light to enhance interlayer bonding and hydrophilicity, and then assembled and sealed under aseptic conditions. The resulting device has high transparency and good sealing, enabling real-time observation of cell morphology and culture status under microscopic conditions, providing a stable microenvironment for biomimetic liver tissue culture.

[0012] During cell culture, cells are seeded in pluggable culture chambers, which are then placed into the chamber wells. Culture medium is injected into the channels through the injection wells. After the cells adhere, the three-dimensional liver chip is placed on a rocking culture platform, and cell culture is performed using a pump-free reciprocating rocking method, ensuring continuous flow of the culture medium within the channels and its contact with the cells. The specific method is as follows: The sterilized chip was placed under aseptic conditions, and sterile culture medium was added to the channels and culture chambers respectively. The mixture was incubated at 36-38℃ and 4-6% CO2 for 12-24 hours to fully wet the channels and membrane surfaces, after which residual liquid was discarded. Hepatocytes in good passaged condition (such as human hepatocyte lines HepG2, LO2, or primary human hepatocytes) were prepared to a cell density of 1×10⁻⁶. 5 ~5×10 5 Cells were prepared by adding 50–150 μL of the cell suspension to the microarray culture chambers to ensure cell adhesion. Then, an appropriate amount of complete culture medium was added to each chamber, and the cells were pre-cultured on a dynamic culture platform at 36–38°C and 4–6% CO2 for 12–24 hours. After the cells had adhered stably, the tilt angle of the dynamic culture platform was set to 2°–15°, and the rocking frequency to 0.5–5 Hz, for continuous culture for 24–168 hours.

[0013] When establishing a chemical liver injury model, chemical agents are injected into a three-dimensional liver chip to induce hepatocyte damage. The specific method is as follows: After obtaining a stable liver microarray model, pre-prepared CCl4-containing culture medium was slowly injected into the main fluid channel to create continuous flow. CCl4 is soluble in complete culture medium containing 0.1% dimethyl sulfoxide (DMSO) to improve its stability and solubility; the final concentration was controlled between 0.2 and 20 mmol / L. The microarray was connected to a microfluidic perfusion system or a low-speed circulating pump, and continuous perfusion was maintained at 37 °C and 5% CO2 for 24–72 hours. Cell morphology changes were observed in real-time using an optical microscope or fluorescent probes during culture. After induction, the CCl4-containing culture medium was discarded, and fresh culture medium was used for another 24 hours to obtain a three-dimensional chemically induced liver injury model with typical hepatocyte injury characteristics. This model stably reflects the in vivo liver injury process induced by CCl4 in terms of cell morphology, metabolic activity, and inflammatory response.

[0014] Furthermore, after establishing the carbon tetrachloride liver injury microarray model, interventions were conducted using selected nutrients. The specific method was as follows: The nutrients to be evaluated are dissolved or dispersed in a complete culture medium to prepare a working solution of the required concentration. These nutrients include, but are not limited to, marine polysaccharides, proteins, polyphenolic compounds, and / or algal extracellular vesicles. The prepared nutrient solution is injected into the chip channels or culture chambers at a volume of 100–300 μL to maintain a final concentration of 10–100 μmol / L (depending on the type and potency of the substance). An equal volume of blank culture medium is added to the control group. The treated chip is placed in a constant-temperature perfusion culture device or a dynamic rocking platform and dynamically cultured at 37 ℃ and 5% CO2 for 24–72 hours, with a rocking angle of 2°–15° and a rocking frequency of 0.5–5 Hz to maintain stable microfluidic shear forces. During the culture period, the culture medium containing the drug or nutrients is replaced every 24 hours for continuous dynamic intervention.

[0015] After culture, supernatant and / or cell samples from the microarray channels were collected. Liver function-related indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), albumin, urea, glutathione (GSH), and cytochrome P450 activity, were detected using routine biochemical or immunoassay methods. Simultaneously, inflammatory factors (such as TNF-α and IL-6) and oxidative stress levels were measured to comprehensively assess the effect of nutrients on liver damage repair. The above operating conditions and parameter ranges can be appropriately adjusted according to different cell types, nutrient properties, or microarray specifications to ensure system stability and reproducibility of experimental results.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention establishes a CCl4 chemical liver injury model in a three-dimensional microfluidic dynamic environment for the first time. It achieves uniform exposure through continuous perfusion, avoiding the problems of uneven diffusion and unstable reaction of CCl4 in the traditional two-dimensional model.

[0017] (2) Using the optimized low-shear swing fluid drive mode (5°, 2 Hz), the high level of hepatocyte metabolic function can be maintained for a long time without the need for an external pump. The secretion of albumin and urea is maintained at 1.4–1.6 times that of the two-dimensional group, which is significantly better than the existing static model.

[0018] (3) The “damage-intervention-repair” coherent system constructed in this invention can monitor changes in hepatocyte function in real time. CCl4-induced increases in ALT / AST, MDA accumulation, and α-SMA upregulation are all stably reproduced in the model.

[0019] (4) Algal extracellular vesicle intervention can significantly reduce γ-GGT and MDA levels and increase albumin and urea secretion, proving that the present invention has significant effects in the field of screening of nutritional active substances and provides a new technical platform for the intersection of food and medicine. Attached Figure Description

[0020] Figure 1 This is a schematic diagram and exploded view of the pump-free three-dimensional liver chip system of the present invention.

[0021] Figure 2 This invention compares the secretion factors of hepatocytes cultured in two-dimensional static culture and dynamic culture on a liver microarray under different culture cycles, including changes in (A) albumin, (B) urea, (C) γ-glutamyltransferase (γ-GGT) and (D) total bilirubin.

[0022] Figure 3 The liver chip model of carbon tetrachloride liver injury in this invention was established, and the liver chip function was compared after intervention treatment using algal extracellular vesicles. The results included changes in (A) albumin, (B) total bilirubin, (C) gamma-glutamyl transferase (γ-GGT) and (D) urea.

[0023] Figure 4 This invention assesses the intervention effect of model nutrients in a carbon tetrachloride liver microarray model by detecting α-SMA index. (A) Three-dimensional liver microarray α-SMA immunofluorescence staining images of different treatment groups after intervention and (B) relative fluorescence quantitative histogram.

[0024] Figure 5 The liver function was evaluated for different treatment groups, specifically the intracellular alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0025] Figure 6To compare the oxidative stress levels of different treatment groups, i.e., the intracellular malondialdehyde (MDA) content. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited to these examples.

[0027] Example 1: Cell culture method for liver microarray The pump-free three-dimensional liver chip used in this embodiment consists of a top cover, a core, a connecting membrane, and a base. The core contains three sets of parallel sample inlet holes and chamber holes. A fluid channel, approximately 12 mm wide, 0.4 mm high, and 40 mm long, runs through each set of sample inlet holes and chamber holes. The chamber holes are circular with a diameter of 6.5 mm. The sample inlet holes and chamber holes are perforated, and their walls are sequentially connected to the connecting membrane and the base to form closed culture chambers. Insertable culture chambers are installed within the chamber holes. The bottom of each culture chamber has a porous bottom membrane structure with a pore size of 3 μm, made of polycarbonate (PC), polytetrafluoroethylene (PTFE), or polyester (PET) microporous membrane. The edge of the porous bottom membrane adheres to the connecting membrane and is suspended above the fluid channels, allowing material diffusion between the culture chamber and the fluid channels without direct communication. The chip top cover and base are sealed together by a microporous polycarbonate membrane to form an independent culture space. The chip employs a tilting, oscillating platform to drive the periodic reciprocating flow of liquid, achieving stable microfluidic shear force without an external pump. The system structure is as follows: Figure 1 As shown. The pluggable culture chamber was placed in the chamber opening, and the top cover, core, connecting membrane, and base were assembled from top to bottom. After assembly, it was vacuum degassed for 10 min to remove air bubbles, and then sterilized by UV irradiation for 60 min. The sterilized liver chip was placed in a laminar flow hood, and sterile complete culture medium (DMEM high glucose + 10% FBS + 1% P / S) was added to the channels and culture chambers. After incubation at 37 ℃ and 5% CO2 for 12 h, the residual medium was discarded. Human hepatocyte HepG2 cell suspension (density 3×10⁻⁶) was taken. 5 Approximately 100 μL of culture medium was added to the culture chamber of the chip, and 1.5 mL of complete culture medium was added to the chip channel through the sample injection well. The culture was then allowed to adhere under static conditions for 6 h. Subsequently, a rocking culture platform was connected, and dynamic culture was performed at a tilt angle of 6° and a frequency of 3 Hz. Figure 1 The culture medium was changed every 24 hours during the culture process, and the culture was carried out continuously for 120 hours.

[0028] Comparative Example 1: Dynamic Culture Time-Gradient Liver Microarray Model To compare the effects of different culture cycles on liver microarray performance, liver microarrays were prepared using the same seeding method as in Example 1, followed by time-gradient dynamic culture. The seeded microarrays were placed on a temperature-controlled rocking culture device with an inclination angle of approximately 6° and a rocking frequency of 3 Hz, and cultured at 37 ℃ and 5% CO2. Periodic rocking drove the culture medium to flow back and forth along the microchannels, creating a stable low-shear fluid environment to maintain cell viability. Culture times were set at 1, 3, 5, and 7 days (i.e., 24, 72, 120, and 168 hours), with the culture medium replaced every 24 hours. After culture, the supernatant from the microarray culture medium at different time points was collected to assess the functional secretion levels of hepatocytes under long-term dynamic conditions, thus obtaining dynamic liver microarray models with different culture cycles.

[0029] Comparative Example 2: Static Culture Time-Gradient Liver Microarray Model To compare the effects of dynamic culture on hepatocyte function maintenance, the same chip preparation and cell seeding methods as in Example 1 were used, but without rocking or fluid-driven treatment. The chips were placed horizontally in an incubator at a 0° tilt angle, without mechanical vibration, and statically cultured at 37 °C and 5% CO2. To ensure nutrient supply, the culture medium was replaced with fresh medium every 24 hours. Culture times were set to 1, 3, 5, and 7 days (i.e., 24–168 hours), and liver function secretion indicators were periodically measured to obtain liver chip models with different culture periods under static conditions. This comparative example was used to compare the differences in cell metabolic activity and function maintenance with the dynamic culture model.

[0030] Example 2: Analysis of Cell Culture Results from Liver Microarray Effects of different culture times and culture modes on hepatocyte function: The experiments were conducted according to the methods of Example 1, Comparative Example 1, and Comparative Example 2, comparing the effects of culture times of 1, 3, 5, and 7 days, and the effects of dynamic and static culture modes on the results. All cultured cells had their complete culture medium replaced every 24 hours, and the supernatant was collected on days 1, 3, 5, and 7 for functional index detection. The detection indicators included albumin, urea, total bilirubin, and gamma-glutamyl transferase (γ-GGT). Commercial kits from Nanjing Jiancheng were used for the measurements. Albumin was measured using the bromocresol green method, urea using the urease-indophenol method, total bilirubin using the diazo coupling method, and γ-glutamyl transferase using the p-nitroaniline release method.

[0031] like Figure 2As shown, these indicators (albumin, urea, total bilirubin, γ-GGT) reflect the metabolic and functional maintenance capabilities of hepatocytes under two-dimensional static and planar rocking dynamic culture conditions. The blue bars in the figure represent static culture liver microarray models without fluid drive (Comparative Example 2); the purple bars represent dynamic culture liver microarray models, where a low-shear fluid environment is created through rocking (Example 1). The horizontal axis represents the number of culture days (1, 3, 5, 7 days), and the vertical axis represents the corresponding indicator values. Under dynamic culture conditions, albumin and urea secretion levels continuously increased with increasing culture time, indicating that hepatocyte anabolic metabolic functions were well maintained; while in the static culture group, these indicators decreased significantly from day 3. Compared to the static culture group, the levels of total bilirubin and γ-GGT in the dynamic group continuously increased and remained stable until day 5. The dynamic pump-free microfluidic liver microarray can maintain the activity and function of hepatocytes in vitro for a long time, significantly better than traditional static culture, providing a reliable technical basis for subsequent chemical injury models and nutritional intervention studies. Furthermore, the liver microarray remained basically stable on day 5, and subsequent experiments used day 5 as the modeling starting point.

[0032] Example 3: Effects of algal extracellular vesicles on hepatocyte damage 1. The effect of algal extracellular vesicles as nutrients on liver function in the intervention of carbon tetrachloride-induced liver injury. Algal extracellular vesicles were prepared according to the method described in patent CN120536337A: Fresh Ulva lactuca was purchased and placed in seawater, washed twice with pre-cooled physiological saline to remove impurities, and then homogenized at 4 °C for 90 s. The volume ratio of Ulva lactuca to physiological saline was 1:5. After homogenization, the Ulva lactuca physiological saline solution was filtered three times through 300-mesh gauze. The obtained filtrate was purified using a tangential flow system: tangential flow rate 2 m / s, transmembrane pressure 0.3 MPa, 500 g of raw material yielded 120 mL of algal extracellular vesicle solution with a concentration of 12 mg / mL. To ensure the bioactivity of ULEVs, the tangential flow purified sample was stored at -80 °C.

[0033] After obtaining stable three-dimensional cultured liver microarrays, carbon tetrachloride was dispersed in complete culture medium under aseptic conditions (if necessary, DMSO was used as a stock solution before dilution, with a final concentration of DMSO ≤ 0.1%). Working solution was prepared at a final concentration of 8 mM and slowly injected into the main channel of the microarray. The modeling group was continuously cultured at 37 ℃ and 5% CO2 for 24 h. The intervention group, while ensuring the same carbon tetrachloride damage, was co-cultured with algal extracellular vesicles (100 μM protein final concentration) for 24 h. The normal group was cultured with an equal volume of culture medium for 24 h. The algal extracellular vesicle (EVs) group was co-cultured with only algal extracellular vesicles (100 μM protein final concentration). All groups were cultured at a rocking angle of 6° and a frequency of 3 Hz. After modeling, the culture supernatant was collected for albumin, urea, total bilirubin, and γ-GGT detection.

[0034] Figure 3 This is a comparative graph showing liver function secretion indicators in different treatment groups after the establishment of the carbon tetrachloride (CCl4) liver injury chip model in this invention. The experiment was divided into four groups: normal group, algal extracellular vesicle (EV) group alone, CCl4 injury group, and CCl4+EV intervention group. The results showed that the secretion of all factors was significantly increased in the CCl4 injury group, indicating that the synthetic function and metabolic activity of hepatocytes were impaired. The indicators of the EV group alone were similar to those of the normal group, indicating that algal EVs themselves have no cytotoxicity. In the CCl4+EV intervention group, albumin and urea levels were significantly restored, while total bilirubin and γ-GGT were significantly reduced, indicating that algal extracellular vesicles can effectively alleviate CCl4-induced chemical liver injury and promote liver function repair.

[0035] 2. The effect of algal extracellular vesicles as nutrients on carbon tetrachloride-induced liver injury and its influence on liver fibrosis. Following the experimental grouping and cell culture methods described in step 1, α-SMA immunofluorescence staining was performed to detect fibrosis in the cells. First, the culture medium in the chip was discarded, and the chip was slowly washed three times with PBS buffer to remove residual culture medium. Then, 4% paraformaldehyde solution was added, and the chip was fixed at room temperature for 15–20 min. After fixation, the chip was washed three more times with PBS, 5 min each time. To increase antibody permeability into cells, 0.1% Triton X-100 solution was added, and the chip was treated at room temperature for 10 min, followed by washing with PBS. 5% bovine serum albumin (BSA) blocking solution was added, and the chip was blocked at room temperature for 30 min to reduce non-specific binding. After blocking, α-SMA primary antibody (e.g., Abcam, ab5694, dilution 1:200) was added, and the chip was incubated overnight at 4 °C. The next day, the chip was removed, washed three times with PBS (5 min each time), and then fluorescently labeled secondary antibody (Alexa Fluor 488, dilution 1:500) was added, and the chip was incubated at room temperature in the dark for 1 h. After washing, the cell nuclei were counterstained with DAPI staining solution (staining for 5 min), and finally rinsed with PBS. The chip was observed and images were captured under an inverted fluorescence microscope or a confocal microscope. After image acquisition, the fluorescence intensity of α-SMA in different groups was quantitatively analyzed using ImageJ software, and the mean fluorescence intensity (MFI) was calculated. The results were used to assess the activation degree of hepatocytes or hepatic stellate cells.

[0036] Figure 4 This image shows the results of evaluating the repair effect of algal extracellular vesicles on a carbon tetrachloride liver injury chip model by detecting α-SMA (α-smooth muscle actin) expression levels. In the image, blue represents cell nuclei (DAPI staining), and green represents α-SMA labeling signals. The results show that the α-SMA fluorescence signal in hepatocytes of the normal group and the algal extracellular vesicle-only group was weak, indicating no significant activation. The α-SMA expression in the CCl4-injured group was significantly enhanced (p < 0.001), suggesting that chemical damage caused activation of hepatocytes or hepatic stellate cells, leading to a fibrotic trend. After intervention with algal extracellular vesicles, the α-SMA fluorescence signal was significantly weakened (p < 0.01), and quantitative analysis also showed a significant decrease in expression levels, indicating that algal extracellular vesicles can effectively inhibit CCl4-induced hepatocyte activation and alleviate inflammation and early fibrotic reactions. In summary, the α-SMA immunofluorescence results further validate the anti-inflammatory and anti-fibrotic effects of algal extracellular vesicles in the CCl4-induced liver injury model, providing strong evidence for their application as a natural nutritional repair factor.

[0037] 3. Effects of algal extracellular vesicles as nutrients on ALT and AST in the intervention of carbon tetrachloride-induced liver injury. Following the establishment of the carbon tetrachloride liver injury microarray model and the intervention culture of algal extracellular vesicles in step 1, the supernatant from each microarray was collected for detecting liver function-related enzyme indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Experimental groups included: normal group, EVs alone group, CCl4 injury group, and CCl4+ EVs intervention group. The Nanjing Jiancheng Bioengineering Institute kit was used for detection; all reagents and samples were equilibrated to room temperature before operation. The microarray supernatant was diluted according to the instructions and added to a 96-well microplate, with blank wells, standard wells, and sample wells for each group. The corresponding chromogenic substrate was added, gently mixed, and incubated at 37 ℃ for 15 min. The reaction was then terminated by adding the stop solution. The absorbance (A value) was measured at 510 nm (ALT) and 520 nm (AST), and the enzyme activity (U / L) in the samples was calculated based on the standard curve.

[0038] like Figure 5 As shown, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected in samples from each group in the carbon tetrachloride (CCl4)-induced liver injury microarray model. The results showed that ALT and AST were significantly elevated in the CCl4-induced group (p < 0.001), indicating that chemical injury leads to increased hepatocyte membrane permeability, leakage of intracellular enzymes, and mitochondrial damage and energy metabolism disorders. There was no significant difference between the normal group and the algal-derived extracellular vesicle group, suggesting that algal-derived extracellular vesicles themselves have no significant hepatotoxicity. The ALT level in the intervention group was significantly lower than that in the CCl4 group (p < 0.01), approaching that of the normal group, suggesting that EVs can effectively alleviate CCl4-induced cell membrane damage and have a significant protective effect. Comprehensive analysis indicates that the CCl4-induced liver microarray model can stably reproduce the typical biochemical characteristics of chemically induced hepatocyte injury, while algal-derived extracellular vesicle intervention can simultaneously reduce ALT and AST levels, alleviate membrane damage and mitochondrial damage, and promote liver function recovery. This further validates the application potential of the system of this invention in in vitro pharmacological repair and screening of functional nutrients.

[0039] 4. The effect of oxidative stress on the intervention of algal extracellular vesicles as nutrients in carbon tetrachloride-induced liver injury. Following the establishment of the carbon tetrachloride liver injury microarray model and the intervention culture of algal extracellular vesicles in step 1, the supernatant from each microarray was collected for detecting the content of the oxidative indicator malondialdehyde (MDA). Figure 6As shown, the intracellular malondialdehyde (MDA) content in each group was measured to evaluate the level of carbon tetrachloride (CCl4)-induced oxidative stress and the antioxidant effect of algal extracellular vesicle (EV) intervention. The results showed that the MDA content in the normal group and the algal extracellular vesicle-only group was low and the difference was not significant, indicating that algal extracellular vesicles themselves do not induce lipid peroxidation. The MDA level in the CCl4-damaged group was significantly increased (p < 0.001), indicating that chemical damage triggered a significant oxidative stress response, leading to enhanced cell membrane lipid peroxidation. In contrast, the MDA content in the intervention group was significantly decreased (p < 0.01), suggesting that algal extracellular vesicles can effectively scavenge free radicals and inhibit lipid peroxidation, thereby alleviating CCl4-induced oxidative damage.

[0040] The combined results indicate that algal extracellular vesicles exhibit significant antioxidant activity and membrane protection in the CCl4-induced liver injury model, reducing MDA production and maintaining cell membrane stability and metabolic balance. This further demonstrates that the three-dimensional liver chip system of this invention can accurately reflect the chemical liver injury and antioxidant repair process, providing a reliable model and data support for in vitro studies of nutrients and natural functional factors.

[0041] The above embodiments are preferred embodiments of the present invention, but are not limited to the specific conditions and details in the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A three-dimensional liver chip system, characterized in that, The three-dimensional liver chip, from top to bottom, includes an upper cover, a core, a connecting membrane, and a base. The core contains multiple sets of parallel culture units, each culture unit including one or more small chambers and two sample inlet ports located on both sides of the small chambers; the sample inlet ports and the small chambers are connected below by a channel; The small chamber is provided with a pluggable culture chamber; the bottom of the pluggable culture chamber is provided with a porous bottom membrane, the edge of which is attached to the connecting membrane and suspended above the channel.

2. The three-dimensional liver chip system according to claim 1, characterized in that, The connecting membrane is a polycarbonate membrane; the porous bottom membrane is a polycarbonate microporous membrane, a polytetrafluoroethylene microporous membrane, or a polyester microporous membrane.

3. The three-dimensional liver chip system according to claim 1 or 2, characterized in that, The method for constructing the liver chip is as follows: (1) Chip pretreatment: Place the sterilized chip in a sterile environment, add sterile culture medium to the channel and culture chamber respectively, and let it stand for 12-24 hours at 36-38℃ and 4-6%CO2 to fully wet the channel and membrane surface, and then discard the residual liquid. (2) Cell seeding: 3×10 5 ~10 8 Hepatocytes at a density of cells / mL were seeded into the culture chamber, ensuring even distribution of cells on the porous bottom membrane surface, and then complete culture medium was added. (3) Dynamic culture: The chip is placed on an adjustable tilting culture platform with a tilt angle of 2° to 15° and a tilting frequency of 0.5 to 5 Hz, and dynamic culture is carried out at 36-38℃ and 4-6% CO2. (4) Medium replacement and harvesting: Fresh complete culture medium was replaced every 22-26 hours, and the culture was carried out continuously for 24-168 hours to obtain a dynamic three-dimensional liver chip model with complete structure and stable function.

4. The three-dimensional liver chip system according to claim 3, characterized in that, The hepatocytes include human hepatocyte lines HepG2, LO2, or primary human hepatocytes.

5. A method for assessing nutritional intervention in liver injury based on the three-dimensional liver chip system described in claim 3 or 4, characterized in that, Includes the following steps: (1) Establishing a chemical liver injury model: Carbon tetrachloride was injected into the three-dimensional liver chip and dynamically cultured for a period of time; the dynamic culture refers to placing the three-dimensional liver chip on a rocking culture platform with an inclination angle of 2° to 15° and a rocking frequency of 0.5 to 5 Hz for culture. (2) Nutritional intervention evaluation: Nutrients were added to the three-dimensional liver chip after step (1) for a period of time, and the changes in indicators in the chip culture medium and / or liver cells were detected.

6. The method according to claim 5, characterized in that, In step (1), the final concentration of added carbon tetrachloride is 0.2–20 mmol / L; the dynamic culture time is 24–72 hours.

7. The method according to claim 5, characterized in that, In step (2), the nutrients include marine polysaccharides, proteins, polyphenolic compounds and / or algal extracellular vesicles.

8. The method according to claim 5, characterized in that, In step (2), the intervention lasts for 24 to 72 hours.

9. The method according to claim 5, characterized in that, In step (2), the indicators include the levels of albumin, total bilirubin, gamma-glutamyl transferase and urea.

10. The application of the three-dimensional liver chip system according to any one of claims 1 to 4 in the evaluation of the safety and repair effects of nutrients, health foods or drugs.

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