Vascularized tissue culture chip as well as construction method and application thereof

Abstract

The invention discloses a vascularized tissue culture chip as well as a construction method and application thereof, and belongs to the technical field of biological tissue engineering and biological medicines. The vascularization tissue culture chip is formed by stacking five layers of sub-chips with different function requirements, a fluid channel and a culture chamber are formed, a highly integrated system is formed, and the design of a micro-column array further optimizes the fluid dynamic characteristics and reduces the dead angle effect of fluid. In-vivo microcirculation can be simulated, and a stable and controllable nutrition and metabolite exchange environment is provided for skin and liver tissue. The vascularized tissue culture chip provided by the invention provides a powerful research platform for a plurality of biomedical research fields such as personalized medical treatment, disease modeling, drug screening, toxicity testing, disease model research and the like.

Description

Technical Field

[0001] This invention relates to the fields of biological tissue engineering and biomedicine, and in particular to a vascularized tissue culture chip, its construction method, and its application. Background Art

[0002] Drug-induced hepatotoxicity and liver injury are major causes of acute liver failure and have become a significant health problem. Drug hepatotoxicity is also one of the main reasons for the failure of drug clinical trials. Currently, drug hepatotoxicity is usually assessed in animal models, but animal models often cannot accurately predict clinical responses due to species differences compared to humans. Therefore, developing an accurate and effective model for assessing drug hepatotoxicity is of great importance.

[0003] Adverse skin reactions caused by drugs are prevalent worldwide, often resulting from oral or intravenous administration of medications after they enter the systemic circulation. In hospitalized patients, adverse skin reactions affect up to 10% of cases, with 2%–6.7% of these reactions progressing to severe and potentially life-threatening conditions. The incidence varies depending on the population and the triggering drug. Given this situation, prospective drug screening and prediction of adverse skin reactions caused by systemic medications are of significant importance.

[0004] With the advancement of science and technology, especially the integration of microfluidics, microfabrication technology and tissue engineering, organ-on-a-chip technology has emerged and is gradually becoming one of the ideal tools for large-scale drug screening.

[0005] Organ-on-a-chip technology can highly simulate the function of human organs in vitro and provides a highly biomimetic and controllable microenvironment, significantly improving the accuracy of predicting drug responses. However, current organ-on-a-chip drug testing models still have limitations.

[0006] To achieve hepatotoxicity assessment of systemic and transdermal drug delivery on organ-on-a-chip, it is necessary to simultaneously construct highly biomimetic liver and skin tissues on a single chip to improve the systematicity and accuracy of drug testing. Current organ-on-a-chip drug testing models for hepatotoxicity still have limitations: First, few studies include skin systems in organ-on-a-chips used for drug testing, making it difficult to simultaneously assess hepatotoxicity of systemic and transdermal drug delivery; second, precise in vitro control of the liver and skin microenvironments remains a significant challenge; third, the in vitro construction of vascularized tissue is a prerequisite for accurate drug testing, and the construction of vascularized tissue has always been a research difficulty. Current on-chip tissues often lack vascular structures, while the liver is a highly vascularized tissue, and the dermis of the skin also has abundant capillaries.

[0007] Skin-on-a-chip containing the liver provides a new research tool for studying drug-induced skin adverse reactions, but it still has the following limitations: First, the potential mechanisms of systemic drug-mediated skin adverse reactions are much more complex than those of contact skin adverse reactions, and existing in vitro skin drug adverse reaction research models are difficult to reproduce in vivo skin adverse reactions under the influence of the liver and immunity. Organ-on-a-chip models that can include upstream cellular processes (e.g., liver metabolism) are needed. Second, in existing multi-organ-on-a-chip systems, the system design is more complex than that of single-organ systems, and the difficulty of reconstructing tissues on complex chips is greater, which greatly limits the biomimeticity of liver and skin tissues on the chip. Third, the precise control of the skin and liver microenvironment in vitro is an important challenge that needs to be solved, which has a significant impact on the accuracy of drug testing. It is necessary to reproduce the dynamic gas-liquid interface culture of skin tissue and the complex hemodynamic conditions of liver tissue on the chip.

[0008] The invention patent application with application number 202210937506.X discloses an organ-on-a-chip for multi-tissue co-culture and a method for detecting the regulatory effects of multi-tissue functions. However, the process of using this chip to detect the regulatory effects of multi-tissue functions is difficult to operate and has certain limitations. Specifically, it cannot simulate the effects of orally administered drugs on cells, tissues or organoids in the first culture chamber after digestion and absorption by the liver.

[0009] Therefore, in order to comprehensively evaluate the hepatotoxicity of drugs under different routes of administration or the adverse skin reactions of systemic administration on the organ-on-a-chip platform, it is necessary to construct an on-a-chip that simultaneously contains highly biomimetic liver and skin tissue to enhance the systematicness and predictive accuracy of drug testing. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides a vascularized tissue culture chip containing both vascularized skin and vascularized liver, along with a precisely controllable fluid microenvironment, its construction method, and its applications.

[0011] The vascularized tissue culture chip includes a first sub-chip 1 and a fifth sub-chip 5 stacked from top to bottom; The first sub-chip 1 is provided with a liquid inlet 101 and a liquid outlet 103, and two culture chambers 102 are provided between the liquid inlet 101 and the liquid outlet 103. A porous membrane 3 is also provided between the first sub-chip 1 and the fifth sub-chip 5, and the porous membrane 3 covers the lower end of each culture chamber 102. The fifth sub-chip 5 has a connecting channel 501 on its upper surface. The liquid inlet 101 and the liquid outlet 103 are connected by the connecting channel 501 to form a U-shaped channel. The connecting channel 501 has an array of top pillars 502. The top pillars 502 have a flow groove 503 at their upper ends. The upper ends of the top pillars 502 abut against the lower end of the porous membrane 3.

[0012] The porous membrane 3 is provided with a first sealing gasket 2 and a second sealing gasket 4 on its upper and lower sides, respectively. The upper plate 1 and the first sealing gasket 2 are closed and clamped together. The first sealing gasket 2 and the second sealing gasket 4 clamp the porous membrane 3. The first sealing gasket 2 is provided with a second through hole 202 aligned with each culture chamber 102. The second sealing gasket 4 is provided with a through groove 401. The first sealing gasket 2 and the second sealing gasket 4 are also provided with a first through hole 201 and a third through hole 203. The first through hole 201 and the third through hole 203 are aligned with the liquid inlet 101 and the liquid outlet 103, respectively.

[0013] Preferably, the porous membrane 3 has a pore size of 0.4~1 μm and a porosity of 5%~20%.

[0014] Cells cannot pass through porous membranes, which can physically support tissue culture. In addition, the porous nature of the membrane can ensure that nutrients from the culture medium are transported to the tissue for long-term culture, and metabolic waste from the tissue can also diffuse into the culture medium through the porous membrane.

[0015] Preferably, the porous membrane is a polycarbonate (PC) porous membrane, a polytetrafluoroethylene (PTFE) porous membrane, or a polyethylene terephthalate (PET) porous membrane.

[0016] Preferably, a sink 402 is provided at the through waist groove 401, and the porous membrane 3 is placed in the sink 402.

[0017] The area of ​​the through groove 401 is slightly smaller than that of the porous membrane 3. The sink groove 402 serves to position the porous membrane 3, preventing misalignment during installation. It also presses down on the outer edge of the porous membrane 3 to prevent horizontal movement.

[0018] Preferably, the connecting channel 501 has a plurality of rotatable adjusting columns 6 arranged in a straight line in the center. Each adjusting column 6 has a strip-shaped baffle 601. One end of the adjusting column 6 has a rotating shaft 602. The rotating shaft 602 is rotatably connected to and passes through the fifth sub-chip 5. The end of the rotating shaft 602 has an annular groove 603 and a plurality of disc springs 7. Each disc spring 7 has a U-shaped slot 702. The bottom end of the U-shaped slot 702 has a locking part 701. The locking part 701 locks into the annular groove 603. The outer edge of the disc spring 7 abuts against the bottom end of the fifth sub-chip 5.

[0019] The top column 502 has a smooth surface without any sharp edges. The top column design, which eliminates sharp edges, prevents air bubbles from getting trapped during fluid flow.

[0020] The materials used for the first chip layer 1 and the fifth chip layer 5 include polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or polylactic acid (PLA). Non-toxic and non-irritating materials are selected to fabricate organ-on-a-chip systems, minimizing adverse effects on cell growth.

[0021] The processing methods for the first and fifth sub-chip layers include 3D printing, laser etching, CNC milling, or hot pressing.

[0022] The vascularized tissue culture chip is assembled from sub-chips using ultrasonic welding, plasma sealing, fixture assembly, threaded assembly, or adhesive bonding assembly methods.

[0023] The method for constructing the vascularized tissue culture chip includes the following steps: S1. After assembling and sterilizing the sub-chips and accessories of the vascularized tissue culture chip, a vascularized tissue culture chip is obtained. S2, Vascular tissue construction: The umbilical vein endothelial cell suspension is seeded into the first and second culture chambers of the vascularized tissue culture chip in step S1, and placed in an incubator for a period of time to allow the umbilical vein endothelial cell suspension to solidify and form a preliminary vascular tissue structure. S3, Skin tissue construction: Fibroblast suspension is seeded into the first culture chamber of the vascularized tissue culture chip in step S2, covering the formed vascular tissue. It is placed in an incubator and left to stand for a period of time to allow the fibroblast suspension to solidify. The dermal tissue of the skin is directly located above the vascular tissue. Then, keratinocyte suspension is seeded on the surface of the solidified fibroblast suspension and left to stand for a period of time to allow the keratinocytes to adhere. S4. Dynamic culture: Culture medium I is injected into the vascularized tissue culture chip in step S3 through the liquid inlet, and culture medium II is added from the top of the first culture chamber to carry out dynamic culture. S5. Inducing epidermal differentiation: After the initial formation of skin tissue, culture medium II is aspirated from the first culture chamber, and culture medium III is injected through the inlet to replace the culture medium in the U-shaped channel. This constructs a gas-liquid interface culture microenvironment for skin tissue, and dynamically cultivates and forms vascularized skin tissue with a stratum corneum. S6. Liver tissue construction: The liver tissue cell suspension is seeded into the second culture chamber of the vascularized tissue culture chip in step S5, with the liver tissue cell suspension covering the vascularized tissue. The cell suspension is placed in an incubator and left to stand for a period of time to allow it to solidify. Culture medium IV is added to the second culture chamber, and the cell suspension is dynamically cultured to form functionally mature vascularized liver tissue.

[0024] The method for constructing the vascularized tissue culture chip is performed entirely using aseptic techniques.

[0025] In step S1, before assembly, except for the porous membrane, the other sub-chips and sealing gaskets are cleaned with water and then soaked in 75% alcohol for a certain period of time. The sub-chips are then taken out and placed in a clean bench to air dry naturally. All sub-chips, sealing gaskets and porous membranes are then placed in a biosafety cabinet for ultraviolet irradiation sterilization. All accessories are sterilized by high-pressure steam sterilization at 120 °C.

[0026] Thoroughly clean the chip with water to remove surface dust, dirt, and organic residue; ensure gentle handling during cleaning to avoid physical damage to the chip; soaking in 75% alcohol helps to further remove any remaining organic matter or oil stains and reduces the risk of biological contamination; ultraviolet light has a strong bactericidal ability and can further eliminate biological contaminants.

[0027] Preferably, in step S1, the sterilization includes ultraviolet irradiation sterilization or ethylene oxide sterilization.

[0028] Preferably, in step S2, the concentration of the umbilical vein endothelial cell suspension is 1×10⁻⁶. 6 ~5×10 6 per mL.

[0029] Preferably, the suspensions of the umbilical vein endothelial cells, fibroblasts, and liver tissue cells include at least one of the following: type I rat tail collagen solution, fibrin gel solution, hyaluronic acid solution, laminin solution, or a mixed solution of type I rat tail collagen and bovine fibrin.

[0030] Preferably, the concentration of the type I rat tail collagen solution is 2.5 mg / mL.

[0031] Preferably, the concentration of the bovine fibrin solution is 2.5 mg / mL.

[0032] Preferably, when the suspension is a type I rat tail collagen solution, the pH of the suspension is adjusted to 7-7.4 under ice bath conditions before inoculation.

[0033] Preferably, when bovine fibrin solution is selected as the suspension, thrombin is added under ice bath conditions before inoculation, and the concentration of thrombin in the suspension is 3 U / mL, and the mixture is quickly mixed evenly.

[0034] In a further preferred embodiment, when the suspensions of the umbilical vein endothelial cells, fibroblasts, and liver tissue cells are a mixture of type I rat tail collagen solution and bovine fibrin solution, the volume ratio of type I rat tail collagen solution to bovine fibrin solution is 0.5 to 2:1.

[0035] Preferably, the suspension of the keratinocyte suspension includes a keratinocyte culture medium.

[0036] Preferably, the concentration of the keratinocyte suspension is 1×10⁻⁶. 6 ~2×10 6 per mL.

[0037] Preferably, the concentration of the fibroblast suspension is 0.5 × 10⁻⁶. 6 ~3×10 6 per mL.

[0038] Preferably, the culture medium I uses high-glucose DMEM culture medium as the base medium, and adds 0.1% antibiotics, 30~50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum.

[0039] Preferably, when the keratinocytes are cell lines, the culture medium II uses high-glucose DMEM culture medium as the base medium, with the addition of 0.1% antibiotics and 10% fetal bovine serum; when the keratinocytes are primary extracted cells, the culture medium II is a commercially available keratinocyte culture medium.

[0040] Further preferably, the keratinocyte culture medium contains keratinocyte culture medium SFM.

[0041] Preferably, in steps S4 and S5, when the vascularized tissue culture chip is placed horizontally at rest, the liquid level of the culture medium in the U-shaped channel is not higher than the horizontal line of the upper surface of the skin tissue, and the liquid level in the second culture chamber is not higher than the horizontal line of the upper surface of the skin tissue.

[0042] Preferably, in step S6, the concentration of hepatocytes in the liver tissue cell suspension is 2 × 10⁻⁶. 6 ~4×10 6 The number of cells / mL was 0.35 × 10⁻⁶ / mL, and the density of hepatic sinusoidal endothelial cells was 0.35 × 10⁻⁶ / mL. 6 ~0.7×10 6 The concentration of hepatic stellate cells was 0.1~0.2×10⁻⁶ cells / mL. 6 The concentration of hepatic macrophages was 0.05 × 10⁶ cells / mL. 6 ~0.1×10 6 per mL.

[0043] Preferably, when the keratinocytes are cell lines, the culture medium III uses high-glucose DMEM culture medium as the base medium and adds 0.1% antibiotics, 1.8 mM calcium chloride, 20-50 ng / ml endothelial growth factor (EGF), and 10% fetal bovine serum; when the keratinocytes are primary extracted cells, the culture medium III uses DMEM culture medium as the base medium and adds 1.8 mM calcium chloride, 20-50 ng / ml endothelial growth factor (EGF), 0.1% antibiotics, 0.2% commercial keratinocyte growth factor additive (EDGS), and 10% fetal bovine serum.

[0044] High concentrations of calcium ions are crucial for inducing epidermal cell differentiation. In normal skin, calcium concentration is distributed in a gradient across the epidermal layers; as cells migrate towards the skin surface, calcium ion concentration increases, thereby promoting cell differentiation. In in vitro culture, this invention simulates this natural process by increasing calcium ion concentration, causing keratinocytes to stop dividing and begin differentiating, forming a structure similar to the skin surface. Endothelial growth factor (EGF) stimulates keratinocyte proliferation; fetal bovine serum (FBS) provides essential nutrients, promotes cell proliferation, and helps cells resist stress, improving cell survival. These components work synergistically to promote cell proliferation, maintain cell health, and prevent contamination, which is essential for successfully inducing epidermal differentiation. EGF is added to promote angiogenesis.

[0045] Preferably, the culture medium IV uses DMEM culture medium as the base medium and adds 0.1% antibiotics, 1 nM insulin, 2 mM glutamine, 50 μM hydrocortisone succinate and 10% fetal bovine serum.

[0046] DMEM, as a basic nutrient source, meets the high energy requirements of cells, supporting their rapid growth and metabolic activities. Antibiotics are primarily used to prevent bacterial and fungal contamination; low concentrations are sufficient to inhibit the growth of most microorganisms while minimizing potential toxicity to cells, ensuring a pure culture environment and healthy cell status. Insulin, as an important growth factor, regulates glucose uptake and protein synthesis, which is crucial for the normal physiological function of hepatocytes. In the in vitro culture environment, appropriate amounts of insulin help maintain cellular metabolic homeostasis and promote hepatocyte growth and functional expression. Glutamine, a commonly used non-essential amino acid in cell culture, serves not only as an energy source for cells but also... Glutamine is a source of nutrients and plays an important role in nucleic acid and protein synthesis. It also helps maintain the pH stability of the culture medium. Fetal bovine serum is rich in growth factors, hormones, vitamins, and lipids, providing comprehensive nutritional support for cells and promoting their healthy growth and differentiation. Hydrocortisone succinate is a glucocorticoid that can regulate various physiological processes in liver cell culture, including inflammatory responses, cell proliferation and differentiation. It can inhibit the activation of hepatic stellate cells, thereby reducing collagen production, helping to maintain the homeostasis of the culture system and preventing excessive fibrosis. It may also affect the phagocytic function of hepatic macrophages and the secretion of cytokines, thereby regulating the immune response. Under in vitro culture conditions, hydrocortisone succinate can help maintain the differentiation state and function of hepatocytes, such as bile acid secretion, drug metabolism, and protein synthesis. Hydrocortisone succinate can be used to simulate the in vivo hormonal environment in in vitro cell culture.

[0047] While hydrocortisone can be used to mimic the in vivo hormone environment in in vitro cell culture, it is typically lipid-soluble, resulting in low solubility in aqueous media. For use in cell culture, it must first be dissolved in an organic solvent such as ethanol or DMSO and then diluted into the medium, limiting the maximum achievable concentration. Furthermore, excessively high concentrations of organic solvents can be toxic to cells. Hydrocortisone succinate, on the other hand, is more readily soluble in aqueous solutions and can therefore be added directly to cell culture media without the need for organic solvents. This improves its delivery efficiency and reduces potential solvent toxicity issues.

[0048] Preferably, the dynamic culture is carried out by placing the culture on a rocker arm or connecting it to a peristaltic pump system.

[0049] Further optimization involves placing the device on a rocker arm for dynamic cultivation, with a rocker arm amplitude of 15°, a rocking frequency of 15-50 rpm, and a motor power of 25 W.

[0050] In a further preferred embodiment, when the peristaltic pump system is connected for dynamic culture, the two ends of the peristaltic pump are connected to the inlet and outlet of the vascularized tissue culture chip to directly form a fluid circulation, or to form a circulation with the help of a culture flask, with a flow rate of 1~5 μL / min.

[0051] The application of the vascularized tissue culture chip in testing adverse skin reactions and / or hepatotoxicity of drugs or cosmetics includes the application of the constructed vascularized tissue culture chip in testing adverse skin reactions and / or hepatotoxicity of drugs or cosmetics.

[0052] The drug can be administered orally, by injection, or through the skin.

[0053] The application of the vascularized tissue culture chip in testing adverse skin reactions or liver toxicity of drugs or cosmetics includes the following steps: For injectable drugs, the fluid enters the fluid channel in the constructed vascularized tissue culture chip through the fluid inlet hole, and after dynamic culture for a certain period of time, the skin tissue and liver tissue are tested.

[0054] The application of the vascularized tissue culture chip in testing adverse skin reactions and / or hepatotoxicity of drugs or cosmetics includes the following steps: For orally administered drugs, the drug is added from the culture chamber of liver tissue culture to simulate the first-pass metabolism of the drug in the liver, and the skin and liver tissues are tested after dynamic culture for a certain period of time.

[0055] The application of the vascularized tissue culture chip in testing adverse skin reactions and / or hepatotoxicity of drugs or cosmetics includes the following steps: For transdermal drugs, the drug is added from above the culture chamber of the skin tissue culture, and then it penetrates into the blood circulation system to reach the liver. After dynamic culture for a certain period of time, the skin tissue and liver tissue are tested.

[0056] The application of the vascularized tissue culture chip in testing adverse skin reactions and / or hepatotoxicity of drugs or cosmetics specifically includes the following steps: Drugs are metabolized by the liver and circulate through the blood vessels, causing adverse skin reactions. For example, antitumor drugs such as methotrexate, antigout drugs such as allopurinol, antiepileptic drugs such as carbamazepine, and antipyretic analgesics such as acetaminophen are often accompanied by adverse skin reactions. The vascularized tissue culture chip constructed in this invention can mimic the liver metabolism process of drugs, thereby better understanding the metabolism and mechanism of action of drugs in the human body. Simultaneously, it can also be used to study the effects of drugs on different tissues and to assess the safety and efficacy of drugs. Furthermore, the vascularized tissue culture chip constructed in this invention can construct bioactive microorganism models in vitro for studying the effects of cosmetic ingredients on the skin and their effects on the liver after absorption through the skin.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Traditionally, drug testing and toxicity assessment often rely on single cell lines or animal models. The vascularized tissue culture chip provided by this invention includes both vascularized skin and vascularized liver models, which can not only more comprehensively reflect the mechanism of action of drugs in vivo, but also simulate the entire process of drug absorption from the skin (such as transdermal administration) to liver metabolism, as well as the adverse reactions of drugs on skin tissue after first-pass metabolism in the liver, thereby providing more accurate predictions of drug effects and toxicity.

[0058] 2. The vascularized tissue culture chip provided by this invention provides the necessary gas-liquid exchange interface and blood flow simulation for skin tissue and the necessary blood flow simulation for liver tissue to maintain the physiological state and function of the tissue; through microfluidic technology, it realizes the precise control of the fluid microenvironment, which helps to reproduce the real physiological conditions in the human body and improve the reliability and reproducibility of experimental results.

[0059] 3. Vascularization is crucial for achieving tissue function and presents a significant technical challenge in tissue engineering. This invention successfully cultured vascular tissue beneath the skin and liver to form an endothelial barrier, solving this problem and providing a more realistic model for drug transport research. The formation of the endothelial barrier allows for more accurate simulation of drug delivery and metabolism, contributing to a deeper understanding of drug distribution, metabolism, and excretion pathways in the body.

[0060] 4. The skin tissue constructed using the vascularized tissue culture chip provided by this invention includes subcutaneous blood vessels, dermis and epidermis, while the liver tissue includes vascularized liver lobule structures with drug metabolism function, exhibiting highly biomimetic skin and liver physiology.

[0061] 5. The vascularized tissue culture chip provided by this invention is suitable for studying drug hepatotoxicity under systemic and transdermal drug delivery conditions, as well as adverse reactions of drugs to skin tissue after first-pass metabolism in the liver. Its highly biomimetic characteristics enable complex toxicological assessments, which is of great significance for accelerating new drug development, reducing reliance on animal experiments, and improving the accuracy of drug safety assessments. Furthermore, it provides a powerful research platform for multiple biomedical research fields such as personalized medicine, disease modeling, drug screening, toxicity testing, and disease model research. Attached Figure Description

[0062] Figure 1 This is an exploded view of the vascularized tissue culture chip of the present invention.

[0063] Figure 2 This is a schematic diagram of the structure of the vascularized tissue culture chip of the present invention.

[0064] Figure 3 This is a cross-sectional view of the vascularized tissue culture chip of the present invention.

[0065] Figure 4 This is a schematic diagram of the top side of the fifth sub-chip of the vascularized tissue culture chip of the present invention.

[0066] Figure 5 This is a schematic diagram of the bottom side of the fifth sub-chip of the vascularized tissue culture chip of the present invention.

[0067] Figure 6 This is a schematic diagram of the adjusting column of the present invention.

[0068] Figure 7 This is a schematic diagram of the disc spring of the present invention.

[0069] Figure 8 This is a diagram showing the cell distribution in liver tissue.

[0070] Figure 9 HE staining image of skin tissue.

[0071] Figure 10 This is a three-dimensional image of vascular tissue under a confocal microscope.

[0072] Figure 11 A three-dimensional image of skin tissue survival under a confocal microscope, showing whether or not medication was applied.

[0073] Figure 12 This is a graph showing the survival analysis of skin cells during drug testing.

[0074] Figure 13 This is a protein characterization diagram of skin tissue under drug testing.

[0075] Figure 14This is a diagram showing the distribution of cells in skin tissue during drug testing.

[0076] Figure 15 HE staining image of skin tissue under drug testing conditions.

[0077] Figure 16 This is a diagram illustrating the skin barrier properties under drug testing.

[0078] Figure 17 This is a diagram showing the survival status of liver tissue.

[0079] Figure 18 This is a graph showing liver cell survival analysis under drug testing.

[0080] Figure 19 This is a graph showing the analysis of drug-metabolizing enzymes in liver tissue during drug testing.

[0081] Figure 20 This is a graph showing liver tissue function assessment under drug testing. DETAILED DESCRIPTION

[0082] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.

[0083] Example 1 like Figures 1-7 As shown, a vascularized tissue culture chip includes a first sub-chip 1 and a fifth sub-chip 5 stacked on top of each other. The first sub-chip 1 is provided with an inlet 101 and an outlet 103, and two culture chambers 102 are provided between the inlet 101 and the outlet 103. The fifth sub-chip 5 includes a connecting channel 501, through which the inlet 101 and the outlet 103 are connected. The lower end of each culture chamber 102 is connected to the connecting channel 501. A porous membrane 3 is also provided between the first sub-chip 1 and the fifth sub-chip 5, and the porous membrane 3 covers the lower end of each culture chamber 102.

[0084] The inlet 101, connecting channel 501, and outlet 103 form a U-shaped channel. The culture chamber 102 is located on the line connecting the inlet 101 and the outlet 103 and is located inside the U-shaped structure. Skin tissue is cultured in the first culture chamber 102, and liver tissue is cultured in the second culture chamber 102. The connecting channel 501 simulates blood vessels. The culture chamber 102 gradually approaches the connecting channel 501 in the depth direction. That is to say, the distance between the cells at different heights in the culture chamber 102 and the connecting channel 501 is different, which more realistically simulates the positional relationship between blood vessels and cells.

[0085] The porous membrane 3 has a multi-microporous structure that can block cell permeation. Under the action of transport pressure, nutrients permeate from the bottom end to the top end of the culture chamber 102 through the connecting channel 501, while cell metabolic waste can permeate from the bottom end of 102 to the bottom end into the channel 501, simulating the material exchange between blood and tissue.

[0086] In a preferred embodiment, a first sealing gasket 2 and a second sealing gasket 4 are respectively provided on the upper and lower sides of the porous membrane 3. The first layer sub-chip 1 and the first sealing gasket 2 are closed and clamped together to clamp the first sealing gasket 2 and the second sealing gasket 4 to clamp the porous membrane 3. The first sealing gasket 2 is provided with a plurality of second through holes 202, each second through hole 202 being aligned with each culture chamber 102. The second sealing gasket 4 is provided with a through groove 401. The first sealing gasket 2 and the second sealing gasket 4 are also provided with a first through hole 201 and a third through hole 203, the first through hole 201 and the third through hole 203 being aligned with the liquid inlet 101 and the liquid outlet 103, respectively.

[0087] The first sub-chip 1, the first sealing gasket 2, and the second sealing gasket 4 are provided with a plurality of aligned bolt holes 104, while the bolt holes 104 on the fifth sub-chip 5 are threaded holes. The bolt is inserted from the first sub-chip 1, passes through the first sealing gasket 2 and the second sealing gasket 4, and the end is threadedly connected to the fifth sub-chip 5, locking the first sub-chip 1 to the fifth sub-chip 5 and improving the sealing effect.

[0088] In the preferred embodiment, a sink 402 is provided at the through waist groove 401, and the porous membrane 3 is placed in the sink 402.

[0089] The area of ​​the through groove 401 is slightly smaller than that of the porous membrane 3. The sink groove 402 serves to position the porous membrane 3, preventing misalignment during installation. It also presses down on the outer edge of the porous membrane 3 to prevent horizontal movement.

[0090] In the preferred embodiment, the connecting channel 501 is provided with multiple top pillars 502, the top pillars 502 are provided with flow channels 503 at their upper ends, and the upper ends of the top pillars 502 abut against the lower end of the porous membrane 3.

[0091] Since the porous membrane 3 is generally soft, the top posts 502 at both ends can press against the lower end of the second sealing gasket 4 near the outer edge of the porous membrane 3 to compress it. The top posts 502 at the inner end directly press against the lower end of the porous membrane 3 to prevent the porous membrane 3 from sagging and deforming after being filled with cells, gels and other nutrients.

[0092] The nutrient solution can also flow through the contact point between the top of the top column 502 and the porous membrane 3 in the flow channel 503.

[0093] In the preferred embodiment, the top column 502 is provided with multiple rows, and the connecting channel 501 has multiple rotatable adjusting columns 6 arranged in a straight line in the center. The adjusting column 6 is provided with a strip baffle 601. One end of the adjusting column 6 is provided with a rotating shaft 602. The rotating shaft 602 is rotatably connected to the fifth layer sub-chip 5 and passes through the fifth layer sub-chip 5. The end of the rotating shaft 602 is provided with an annular groove 603, and multiple disc springs 7 are also provided. The disc springs 7 are provided with U-shaped slots 702. The bottom end of the U-shaped slots 702 is provided with a snap-fit ​​part 701. The snap-fit ​​part 701 snaps into the annular groove 603, and the outer edge of the disc springs 7 abuts against the bottom end of the fifth layer sub-chip 5.

[0094] The outer edge of the disc spring 7 has multiple grooves 703 axially provided, which can improve the deformation capacity of the disc spring 7.

[0095] The bottom of the connecting channel 501 has multiple threaded holes and smooth holes. The threaded holes are used to install the top posts 502, and the smooth holes are used to install the adjusting posts 6. The third sealing gasket 504 is coated with adhesive and covers all the holes. After the adhesive hardens, small holes are punched at each hole, and the top posts 502 and adjusting posts 6 are installed to achieve a sealing effect. The disc spring 7 is elastic. After locking the adjusting post 6, it causes the adjusting post 6 to rotate while pressing the third sealing gasket 504, thus also ensuring a seal.

[0096] Another annular groove can be machined on the rotating shaft 602 and fitted with a sealing ring.

[0097] The end of the rotating shaft 602 can be machined with concave and convex stripes to increase friction and facilitate finger rotation.

[0098] Adjusting the regulating column 6 below the culture chamber 102 changes the flow area within the connecting channel 501, thereby adjusting the liquid level of the culture medium in the corresponding culture chamber 102. For example, reducing the flow area, according to Bernoulli's principle, increases the flow velocity at the lower end of a culture chamber 102 in the connecting channel 501, resulting in decreased pressure. This pressure is insufficient to support the original liquid level, causing the liquid level in the upper culture chamber 102 to drop. Since the first sub-chip 1 and the fifth sub-chip 5 are made of transparent material, the liquid level in the culture chamber 102 can be directly observed, and the end of the bottom regulating column 6 can be rotated until the desired height is reached. Alternatively, an arrow can be engraved on the lower end of the regulating column 6, and graduations can be engraved on the bottom end of the fifth sub-chip 5 to quantitatively adjust the rotation angle of the regulating column 6.

[0099] In a preferred embodiment, computer-aided design software is used to draw the microchannel structure and microstructure of the first sub-chip 1 and the fifth sub-chip 5, and microfabrication technology is used to process the substrate surfaces of the first sub-chip 1 and the fifth sub-chip 5.

[0100] The first sub-chip 1, the first sealing gasket 2, the porous membrane (3), the second sealing gasket 4 and the fifth sub-chip 5 are aligned and sealed in sequence to form a vascularized tissue culture chip.

[0101] In a preferred embodiment, the processing methods for the first sub-chip 1 and the fifth sub-chip 5 include 3D printing, laser etching, CNC milling, or hot pressing.

[0102] In the preferred embodiment, the pore size of the porous membrane is 0.4~1 μm.

[0103] Example 2 In vitro tissue construction was performed using the vascularized tissue culture chip described in Example 1.

[0104] The method for constructing the vascularized tissue culture chip includes the following steps: S1. After cleaning the first sub-chip 1, first sealing gasket 2, second sealing gasket 4 and fifth sub-chip 5 of the vascularized tissue culture chip with clean water, soak them in 75% alcohol for 30 minutes. Take out the sub-chips and place them in a clean bench to air dry naturally. Place the first sub-chip 1, first sealing gasket 2, second sealing gasket 4 and fifth sub-chip 5 face up in a biosafety cabinet and irradiate with ultraviolet light for 30 minutes. Turn them over and continue to irradiate with ultraviolet light for 30 minutes to complete the sterilization. The required screws, nuts and washers are placed in an autoclave and sterilized at 120°C for 30 minutes to complete the sterilization process. In the clean bench, the experimenter wears sterile gloves and performs aseptic operations to assemble the sub-chips and accessories of each layer. After sterilization by irradiating with ultraviolet light for 30 minutes, a vascularized tissue culture chip is obtained. S2. Preparation of vascular tissue cell suspension: Umbilical vein endothelial cells were suspended in a 2.5 mg / mL bovine fibrin solution. Thrombin (final concentration 3 U / mL) was added dropwise under ice bath conditions, followed by rapid mixing to obtain 30 μL of a 5 × 10⁻⁶ oz solution. 6 A suspension of vascular tissue cells per mL; S3. Vascular tissue construction: Use a pipette to add the vascular tissue cell suspension obtained in step S2 into the first and second culture chambers of the vascularized tissue culture chip in step S1, and place it in an incubator at 37 ℃ and 5% CO2 for 30 min to allow the vascular tissue cell suspension to solidify. S4. Skin tissue cell suspension: Keratinocytes were suspended in keratinocyte culture medium to prepare a 100 μL solution with a concentration of 2 × 10⁻⁶ cells / mL. 6A keratinocyte suspension of 1 × 10⁶ cells / mL was prepared by suspending fibroblasts in a solution of 2.5 mg / mL type I rat tail collagen and 2.5 mg / mL bovine fibrin in a 1:1 volume ratio. The pH was adjusted to approximately 7.4 under ice bath conditions. Thrombin (final concentration 3 U / mL) was added dropwise and the mixture was rapidly stirred. This yielded a 100 μL solution with a concentration of 1 × 10⁶ cells / mL. 6 Fibroblast suspension of cells / mL; S5. Skin tissue construction: The fibroblast suspension prepared in step S4 is injected into the first culture chamber of the vascular tissue culture chip in step S3, and placed in an incubator at 37°C and 5% CO2 for 30 min to allow the fibroblast suspension to solidify. The dermal tissue of the skin is directly above the vascular tissue. Then, the keratinocyte suspension prepared in step S4 is seeded on the surface of the solidified fibroblast suspension and allowed to stand for 1 h to allow the cells to adhere. S6. Culture medium I (using high-glucose DMEM culture medium as the base medium, with the addition of 0.1% antibiotics, 30-50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum) is injected into the U-shaped channel of the vascularized tissue culture chip in step S5 through the inlet. When the chip is placed horizontally at rest, the liquid level of culture medium I is kept no higher than the horizontal line of the skin tissue surface. Culture medium II (using high-glucose DMEM culture medium as the base medium, with the addition of 0.1% antibiotics and 10% fetal bovine serum) is added from the top of the first culture chamber to the second culture chamber, ensuring that the liquid level in the second culture chamber is no higher than the horizontal line of the skin tissue surface. Dynamic culture is performed for 2 days using a rocker arm drive. During the dynamic culture process, the rocker arm amplitude is 15°, the shaking frequency is 15-50 rpm, and the motor power is 25 W. When the rocker arm is running, the culture medium circulates left and right in the U-shaped channel, simulating the blood circulation in the body. S7. Induction of epidermal differentiation: After 2 days of dynamic culture, the cell confluence reached 70%. The culture medium II in the first culture chamber was removed, and culture medium III (culture medium III was based on high glucose DMEM culture medium, with the addition of 0.1% antibiotics, 1.8 mM calcium chloride, 20~50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum) was injected into the U-shaped channel of the vascularized tissue culture chip through the inlet to replace culture medium I. When placed horizontally at rest, the liquid level of culture medium III was kept no higher than the horizontal line of the upper surface of the skin tissue to construct a skin tissue with a gas-liquid interface culture microenvironment. Dynamic culture was continued using a rocker arm. After 14 days of dynamic culture, vascularized skin tissue with a stratum corneum was formed. On day 10 of dynamic culture for inducing epidermal differentiation, liver tissue cell suspension was prepared for liver tissue construction. S8. Liver tissue cell suspension: Hepatocytes, sinusoidal endothelial cells, hepatic stellate cells, and hepatic macrophages were mixed and suspended in a type I rat tail collagen solution with a concentration of 2.5 mg / mL. Under ice bath conditions, the pH was rapidly adjusted to 7-7.4 to obtain 50 μL of hepatocyte suspension with a concentration of 4 × 10⁻⁶ cells / mL. 6 The number of cells / mL was 0.7 × 10⁻⁶ / mL, and the density of hepatic sinusoidal endothelial cells was 0.7 × 10⁻⁶ / mL. 6 The concentration of hepatic stellate cells was 0.2 × 10⁶ cells / mL. 6 The concentration of hepatic macrophages was 0.1 × 10⁶ cells / mL. 6 Liver tissue cell suspension with cells per mL; S9. Liver Tissue Construction: On the 10th day of dynamic culture for inducing epidermal differentiation, the liver tissue cell suspension obtained in step S8 was added to the second culture chamber of the vascularized tissue culture chip in step S7. The liver tissue cell suspension was placed directly above the vascularized tissue and placed in an incubator at 37°C and 5% CO2 for 30 min to allow the liver tissue cell suspension to solidify. Culture medium IV (culture medium IV is based on DMEM culture medium, with the addition of 0.1% antibiotic, 1 nM insulin, 2 mM glutamine, 50 μM hydrocortisone succinate and 10% fetal bovine serum) was added to the second culture chamber. When placed horizontally, the liquid level in the second culture chamber should not be higher than the horizontal line of the upper surface of the skin tissue. Dynamic culture was continued using a rocker arm. After 4 days of dynamic culture, functionally mature vascularized liver tissue could be formed.

[0105] Biomimetic testing was performed on the vascularized skin tissue and vascularized liver tissue described in Example 2.

[0106] 1. Analysis of the constructed liver tissue interface Before constructing liver tissue, hepatic parenchymal cells, sinusoidal endothelial cells, and hepatic stellate cells were pre-stained with fluorescent dyes (DiI, DiO, etc.) or transfected with fluorescent proteins (GFP, mCherry, etc.). After 4 days of dynamic culture, functionally mature vascularized liver tissue was formed. Different cell types were distinguished using red, green, and blue fluorescence, and cell distribution and the formation of tissue interfaces (liver plates, hepatic sinusoids, etc.) were observed under a confocal microscope.

[0107] The results are as follows Figure 8 As shown, hepatocytes formed tissue-like cell clusters, sinusoidal endothelial cells formed a vascularized network, and LX2 cells were sparsely distributed in the cell culture area.

[0108] 2. Analysis of the constructed skin tissue interface Skin tissue was removed from the vascularized tissue culture chip, sectioned, and stained with H&E. Figure 9 As shown, the interface between the dermis and epidermis is highly biomimetic.

[0109] 3. Vascularization assessment The distribution, density, orientation, length, and branching of microvascular networks in vascularized skin and liver tissues were quantitatively analyzed under confocal microscopy to assess the degree of biomimicry in vascularization.

[0110] Figure 10 This is a three-dimensional diagram of the vascular structure, clearly showing the constructed three-dimensional vascularized tissue. The blood vessels form a network and have a luminal structure.

[0111] The above tests show that the vascularized tissue culture chip and the method for constructing skin and liver tissues in vitro using the method provided by this invention can successfully culture vascularized skin tissue and vascularized liver tissue.

[0112] Example 3 The vascularized tissue culture chip constructed in Example 2 was used to test the adverse skin reactions and hepatotoxicity of the drug.

[0113] Diclofenac sodium is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic, anti-inflammatory, and antipyretic effects. However, diclofenac sodium also has some side effects. This embodiment utilizes the completed vascularized tissue culture chip provided in Example 2 to study the adverse skin reactions and hepatotoxicity of diclofenac sodium under injection administration.

[0114] Simulated injection administration: In the use of the vascularized tissue culture chip constructed in Example 2, diclofenac sodium was added through inlet 101 to simulate systemic administration, and after dynamic culture for a specified time, the skin tissue and liver tissue were tested.

[0115] The skin and liver tissues in the vascularized tissue culture chip constructed in Example 2 were set as blank control groups.

[0116] Example 4 The vascularized tissue culture chip constructed in Example 2 was used to test the adverse skin reactions and hepatotoxicity of the drug.

[0117] Diclofenac sodium is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic, anti-inflammatory, and antipyretic effects. However, diclofenac sodium also has some side effects. This embodiment utilizes the vascularized tissue culture chip provided in Example 2 to study the adverse skin reactions and hepatotoxicity of diclofenac sodium under oral administration.

[0118] Simulated oral administration: Orally administered drugs enter the digestive system and are metabolized in the liver tissue via the portal vein. Simulating oral administration in a constructed vascularized tissue culture chip involves adding diclofenac sodium above the second culture chamber of the cultured liver tissue. After being metabolized by the liver, it enters the U-shaped circulation and finally acts on the skin tissue. After dynamic culture for a specified time, the skin and liver tissues are analyzed. Example 5 The transdermal drug delivery test using the vascularized tissue culture chip constructed in Example 2 can be used to test adverse skin reactions and hepatotoxicity caused by drugs and cosmetics. It can also test the hepatotoxicity caused by large-scale, long-term drug use and predict the dose of topical drugs that may cause hepatotoxicity.

[0119] Drugs such as diclofenac sodium are currently formulated into topical preparations for relieving local pain, such as arthritis. The vascularized tissue culture chip provided by this invention can evaluate skin damage and hepatotoxicity caused by high-dose transdermal administration.

[0120] In the use of the vascularized tissue culture chip constructed in Example 2, 1% diclofenac sodium was added to the first culture chamber from above the culture chamber of the skin tissue culture. After the drug was absorbed through the skin, it entered the U-shaped channel and acted on the liver tissue. After dynamic culture for a certain period of time, the skin tissue and liver tissue were tested.

[0121] The skin and liver tissues in the vascularized tissue culture chip constructed in Example 2 were set as blank control groups.

[0122] In this embodiment of the disclosure, the drug testing of the vascularized skin-liver tissue co-culture models constructed in Examples 3-5 is evaluated using the following method: 1. Skin tissue examination 1.1 Cell viability analysis Skin tissue was extracted and placed in a 24-well plate, and cells were washed with PBS. Then, a PBS solution containing 2 μM Calcein-AM, 10 μg / mL Hoechst 33342, and 10 μg / mL PI was added (Calcein-AM / PI / Hoechst 33342 staining was used; apoptotic cells were stained with PI, viable cells with Calcein-AM, and cell nuclei with Hoechst 33342). The plate was incubated at 37°C for 30 minutes, followed by three washes with PBS. Finally, cell viability of the three-dimensional skin tissue on the vascularized tissue culture chip was analyzed using confocal microscopy on days 7, 14, 21, and 28 (daily monitoring is also possible upon request). The cytoplasm of viable cells showed green fluorescence, the nuclei of dead cells showed red fluorescence, and the nuclei of all cells showed blue fluorescence. Since the sample being tested is a three-dimensional skin tissue constructed in vitro, confocal scanning is required in the z-direction with a z-interval of 20 micrometers and a total height of h. The survival rate at z=1 / 4h, z=1 / 2h, and z=3 / 4h is calculated. The number of dead cells (red) and all cells (blue) is counted using ImageJ software, and the cell survival rate is calculated.

[0123] The results are as follows Figure 11 As shown, in skin tissue, the cell survival rate in the blank control group was 87%, while the cell survival rate in the experimental group that received simulated injection (10 μL of diclofenac) was 55%. Cell survival could also be directly observed using a transparent vascularized tissue culture chip. It was observed that the majority of cells in the control group were surviving, while the number of apoptotic cells in the experimental group after drug administration was significantly increased.

[0124] 1.2 Cell proliferation analysis Skin tissue was removed and placed in a 24-well plate. CCK8 test solution was added. After 2 hours, 100 μL of the test solution was transferred to a 96-well plate, and the absorbance was measured using a microplate reader. Figure 12 Yes, transdermal administration of diclofenac sodium can reduce cell proliferation as the dosage increases.

[0125] 1.3 Protein Characterization Immunofluorescence staining was used to characterize keratin K5 and K14 in keratinocytes, and Fibronectin and F-actin in fibroblasts. Results are as follows: Figure 13 As shown in the image, the green fluorescence represents F-actin, and the cytoskeleton network is clearly visible with uniform cell distribution. In this test, with oral administration, diclofenac sodium showed no significant changes regardless of whether it was administered orally, and almost no adverse reactions were observed in skin tissue.

[0126] 1.4 Cell Distribution Analysis First, the cells were digested and centrifuged at 1500 rpm to remove the supernatant. Then, the cells were incubated with 2.5 μM DiI (red) or 2.5 μM DiO (green) fluorescent dye at 37 °C for 30 minutes. Finally, the cells were centrifuged at 1500 rpm for 3 minutes, the dye solution was removed, and the cells were resuspended in PBS. This process was repeated 3 times to obtain fluorescently labeled cells. Different types of cells were distinguished by different fluorescent colors, and the cell distribution was observed under a confocal microscope.

[0127] The results are as follows Figure 14 As shown, fibroblasts (DII) and keratinocytes (DIO) were observed, and cell distribution was observed. In the blank control group, the tissue interface structure forming the epidermis and dermis could be seen. In the diclofenac sodium transdermal drug delivery experimental group, the epidermal structure was observed to be destroyed.

[0128] 1.5 Organizational Interface Analysis HE staining of tissue sections was used to analyze the formation of dermal and epidermal structures within the skin tissue, and the thickness of the epidermis and dermis was quantitatively analyzed.

[0129] The results are as follows Figure 15 As shown, the blank control group showed the presence of epidermis, dermis, and a tendency to differentiate into the stratum corneum; the diclofenac sodium transdermal drug delivery experimental group showed severe damage to the skin structure and the absence of tissue interfaces.

[0130] 1.6 Skin Barrier Characteristics Analysis Cell connectivity was assessed using immunofluorescence staining (e.g., ZO-1), and permeability was assessed using a FITC-dextran permeability assay.

[0131] 100 μL of FITC-glucan (MW 70000) was added above the skin tissue culture area, i.e., the first culture chamber. Samples were taken in the U-shaped channel at 0 min, 60 min, and 120 min, and the fluorescence intensity was measured using a multi-functional microplate reader or fluorescence microscope. Three groups of experiments were set up: (1) vascularized tissue culture chip without skin tissue; (2) the second group of experiments differed from the first group only in that the vascularized tissue culture chip contained skin tissue; (3) the third group of experiments differed from the second group only in that the skin tissue in the vascularized tissue culture chip was interfered with by diclofenac sodium during the culture process. The results are as follows. Figure 16 As shown, the experimental results indicate that, compared with the first group of experiments, the presence of skin tissue reduces the amount of FITC-glucan that permeates into the underlying blood channels, demonstrating a significant barrier effect that can be used for drug delivery studies; compared with the second group of experiments, the skin barrier properties of the diclofenac sodium transdermal drug delivery experimental group were disrupted.

[0132] 2. Examination of liver tissue 2.1 Cell survival and proliferation analysis Calcein-AM / PI / Hoechst staining was used to analyze cell viability of 3D liver tissue on the aforementioned vascularized tissue culture chip using confocal microscopy or inverted fluorescence microscopy, and cell proliferation was assessed using CCK8 assay. Results are as follows: Figure 17 and Figure 18 As shown, the cell proliferation capacity of the injection-administered experimental group decreased with increasing dosage.

[0133] 2.2 Detection of drug-metabolizing enzymes The activities of CYP450 enzymes (CYP1A2, CYP2E1, CYP3A4, etc.) in hepatocytes were detected using a kit. The results are as follows: Figure 19 As shown, in the blank control group, CYP1A2, CYP2E1, and CYP3A4 gradually increased with the increase of culture days, which is due to cell proliferation. In contrast, the CYP enzyme activity in the oral antacid experimental group either decreased or remained unchanged compared to the blank control group.

[0134] 2.3 Liver tissue function assessment Culture medium was collected from the vascularized tissue culture chip of the transdermal drug delivery experimental group every 24 hours, and albumin, urea, total bile acids, etc. were detected using a detection kit. Results are as follows: Figure 20 As shown, in the blank control group, albumin gradually increased with increasing culture days, which is due to cell proliferation. Compared with the blank control group, the transdermal drug delivery experimental group showed decreased albumin secretion after drug administration, and the greater the drug dosage, the greater the cell damage.

[0135] The above tests clearly show that, regardless of whether it is administered by injection, oral administration, or transdermal administration, the adverse reactions of diclofenac sodium to the skin and the hepatotoxicity of the liver can be directly reflected. Therefore, the vascularized tissue culture chip and its construction method provided by this invention can be used for drug safety assessment, providing a platform with highly reliable and reproducible experimental data for drug screening.

[0136] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A vascularized tissue culture chip, characterized in that: It comprises a first layer of sub-chips (1) and a fifth layer of sub-chips (5) stacked in sequence from top to bottom; The first layer of sub-chip (1) is provided with a liquid inlet (101) and a liquid outlet (103), and two culture chambers (102) are provided between the liquid inlet (101) and the liquid outlet (103); A porous membrane (3) is also provided between the first layer sub-chip (1) and the fifth layer sub-chip (5), and the porous membrane (3) covers the lower end of each culture chamber (102); The upper surface of the fifth-layer sub-chip (5) is provided with a connecting groove (501), and the liquid inlet (101) and the liquid outlet (103) are connected through the connecting groove (501) to form a U-shaped channel; an array of top columns (502) are provided in the connecting groove (501), and a flow groove (503) is provided at the upper end of the top column (502), and the upper end of the top column (502) abuts against the lower end of the porous membrane (3).

2. The vascularized tissue culture chip according to claim 1, characterized in that: The porous membrane (3) is provided with a first sealing pad (2) and a second sealing pad (4) on the upper and lower sides respectively; the upper plate (1) and the first sealing pad (2) are closed and clamped; the first sealing pad (2) and the second sealing pad (4) are used to clamp the porous membrane (3); the first sealing pad (2) is provided with a second through hole (202) aligned with each culture chamber (102); the second sealing pad (4) is provided with a through waist groove (401); the first sealing pad (2) and the second sealing pad (4) are also provided with a first through hole (201) and a third through hole (203); the first through hole (201) and the third through hole (203) are aligned with the liquid inlet (101) and the liquid outlet (103) respectively.

3. The vascularized tissue culture chip according to claim 1, characterized in that: The porous membrane (3) has a pore size of 0.4-1 μm and a porosity of 5%-20%.

4. The vascularized tissue culture chip according to claim 1, characterized in that: The through waist groove (401) is provided with a sink groove (402), and the porous membrane (3) is placed in the sink groove (402).

5. The vascularized tissue culture chip according to claim 1, characterized in that: A plurality of rotatable adjusting columns (6) arranged in a straight line are provided in the center of the connecting groove (501), the adjusting columns (6) are provided with a strip-shaped baffle (601), a rotating shaft (602) is provided at one end of the adjusting column (6), the rotating shaft (602) is rotatably connected to the fifth-layer sub-chip (5) and passes through the fifth-layer sub-chip (5), an annular groove (603) is provided at the end of the rotating shaft (602), and a plurality of disc springs (7) are also provided, the disc springs (7) are provided with a U-shaped slot (702), a clamping portion (701) is provided at the bottom end of the U-shaped slot (702), the clamping portion (701) is clamped with the annular groove (603), and the outer edge of the disc spring (7) abuts against the bottom end of the fifth-layer sub-chip (5).

6. The method for constructing a vascularized tissue culture chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, assembling and sterilizing the sub-chips and accessories of each layer of the vascularized tissue culture chip to obtain a vascularized tissue culture chip; S2, vascular tissue construction: inoculating the umbilical vein endothelial cell suspension into the first culture chamber and the second culture chamber of the vascularized tissue culture chip in step S1, placing it in an incubator for a period of time to allow the umbilical vein endothelial cell suspension to solidify and form a preliminary vascular tissue structure; S3, skin tissue construction: inoculating the fibroblast suspension into the first culture chamber of the vascularized tissue culture chip in step S2, covering the formed vascular tissue, placing it in an incubator and letting it stand for a period of time to solidify the fibroblast suspension, the dermis tissue of the skin is directly located above the vascular tissue, and then inoculating the keratinocyte suspension on the upper surface of the solidified fibroblast suspension, and letting it stand for a period of time to allow the keratinocytes to adhere; S4, dynamic culture: injecting culture solution I into the vascularized tissue culture chip in step S3 through the liquid inlet, adding culture solution II into the first culture chamber from the top of the first culture chamber, and performing dynamic culture; S5. Inducing epidermal differentiation: After the skin tissue is initially formed, the culture solution II in the first culture chamber is sucked out, and the culture solution III is injected from the liquid inlet to replace the culture solution in the U-shaped channel with the culture solution III, so as to construct an air-liquid interface culture microenvironment of the skin tissue, and dynamically culture to form a vascularized skin tissue with a stratum corneum; S6. Liver tissue construction: Inoculate the liver tissue cell suspension into the second culture chamber of the vascularized tissue culture chip in step S5, cover the vascular tissue with the liver tissue cell suspension, place it in an incubator and let it stand for a period of time to solidify the liver tissue cell suspension, add culture medium IV to the second culture chamber, and dynamically culture to form functionally mature vascularized liver tissue.

7. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: In the method for constructing the vascularized tissue culture chip, sterile technology is used throughout the entire process.

8. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: In step S1, before assembly, except for the porous membrane, other sub-chips and sealing gaskets are cleaned with clean water and then soaked in 75% alcohol for a certain period of time, the sub-chips are taken out and placed in a clean bench to dry naturally, and then all the sub-chips, sealing gaskets and porous membranes are placed in a biological safety cabinet for ultraviolet irradiation sterilization; the accessories used are sterilized by high-pressure steam sterilization at 120°C; After assembly, the sterilization includes ultraviolet irradiation sterilization or ethylene oxide sterilization.

9. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: In step S2, the concentration of the umbilical vein endothelial cell suspension is 1×10 6 ~5×10 6 Pieces / mL; In step S3, the concentration of the keratinocyte suspension is 1×10 6 ~2×10 6 Pieces / mL; In step S3, the concentration of the fibroblast suspension is 0.5×10 6 ~3×10 6 Pieces / mL; In step S6, the liver cell concentration in the liver tissue cell suspension is 2×10 6 ~4×10 6 / mL, the density of hepatic sinusoidal endothelial cells was 0.35×10 6 ~0.7×10 6 / mL, the concentration of hepatic stellate cells was 0.1×10 6 ~0.2×10 6 / mL, the concentration of liver macrophages was 0.05×10 6 ~0.1×10 6 Pieces / mL; The suspension of the umbilical vein endothelial cell suspension, fibroblast suspension, and liver tissue cell suspension comprises at least one of type I rat tail collagen solution, fibrin gel solution, hyaluronic acid solution, laminin solution, or a mixed solution of type I rat tail collagen and bovine fibrin; When the suspension of the umbilical vein endothelial cell suspension, fibroblast suspension and liver tissue cell suspension is a mixed solution of type I rat tail collagen and bovine fibrin, the volume ratio of type I rat tail collagen to bovine fibrin is 0.5-2:

1.

10. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: The culture medium I is based on high-glucose DMEM culture medium, supplemented with 0.1% antibiotics, 30-50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum; When the keratinocytes are cell lines, the culture medium II uses high-glucose DMEM culture medium as the base liquid, and adds 0.1% antibiotics and 10% fetal bovine serum; when the keratinocytes are primary extracted cells, the culture medium II is a commercial keratinocyte culture medium; When the keratinocytes are cell lines, the culture solution III uses high-glucose DMEM culture solution as the base solution, and adds 0.1% antibiotics, 1.8 mM calcium chloride, 20-50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum; when the keratinocytes are primary extracted cells, the culture solution III uses DMEM culture solution as the base solution, and adds 1.8 mM calcium chloride, 20-50 ng / ml endothelial growth factor EGF, 0.1% antibiotics, 0.2% commercial keratinocyte growth additive EDGS and 10% fetal bovine serum; The culture medium IV uses DMEM culture medium as the base liquid, supplemented with 0.1% antibiotics, 1 nM insulin, 2 mM glutamine, 50 μM hydrocortisone succinate and 10% fetal bovine serum.

11. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: The dynamic culture is placed on a rocker arm or connected to a peristaltic pump system for dynamic culture; When placed on the rocker for dynamic culture, the seesaw amplitude is 15°, the rocking frequency is 15-50 rpm, and the motor power is 25W; When connected to a peristaltic pump system for dynamic culture, both ends of the peristaltic pump are connected to the inlet and outlet of the vascularized tissue culture chip to directly form a fluid circulation, or to form a circulation with the aid of a culture bottle, with a flow rate of 1-5 μL / min.

12. The method for constructing a vascularized tissue culture chip according to claim 6, characterized in that: In step S4 and step S5, when the vascularized tissue culture chip is placed horizontally, the liquid level of the culture fluid in the U-shaped channel is not higher than the horizontal line height of the upper surface of the skin tissue, and the liquid level in the second culture chamber is not higher than the horizontal line height of the upper surface of the skin tissue.

13. The method for constructing a vascularized tissue culture chip according to any one of claims 6 to 12, characterized in that: The invention comprises the application of the constructed vascularized tissue culture chip in testing the adverse skin reaction and / or liver toxicity of drugs or cosmetics.

14. The method for constructing a vascularized tissue culture chip according to claim 13, characterized in that: The administration method of the drug includes oral administration, injection administration or transdermal administration.

15. The method for constructing a vascularized tissue culture chip according to any one of claims 13 to 14, characterized in that: The application specifically includes the following steps: for the drug to be injected, the fluid inlet through-hole enters the U-shaped channel in the constructed vascularized tissue culture organ chip, and the skin tissue and liver tissue are tested after dynamic culture for a certain period of time; For drugs administered orally, the drugs were added from the culture chamber of the liver tissue culture, and the skin and liver tissues were tested after a certain period of dynamic culture; For transdermal drugs, the drugs are added from above the culture chamber of the skin tissue culture, and the skin tissue and liver tissue are tested after a certain period of dynamic culture.

16. The method for constructing a vascularized tissue culture chip according to claim 13, characterized in that: The application specifically includes the following steps: adding the cosmetics from the top of the culture chamber for skin tissue culture, then infiltrating into the blood circulation system to reach the liver, and testing the skin tissue and liver tissue after dynamic culture for a certain period of time.

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