An in-vitro blood-brain barrier model with tight junction structure and application thereof

Abstract

The application provides an in-vitro blood-brain barrier model with a tight junction structure and an application thereof. The in-vitro blood-brain barrier model comprises a tubular well plate support, a mesh substrate support for sealing the lower end of the well plate support, a cell membrane sheet loaded on the substrate support and having a tight junction structure, and a hydrogel structure for packaging the cell membrane sheet, the substrate support and the well plate support into an entirety. The blood-brain barrier model of the application is close to the blood-brain barrier under physiological conditions in terms of tissue structure, transmembrane resistance value, permeability, toxic drug reaction and the like, and provides an important research basis for drug development and disease diagnosis and treatment of nervous system diseases, and improves the accuracy of blood-brain barrier drug research and development. Moreover, the blood-brain barrier model has the characteristics of simple operation and easy popularization under the premise of ensuring the effectiveness and accuracy of the blood-brain barrier model, and has great commercial value.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and biomanufacturing technology under biomedical engineering, and specifically relates to an in vitro blood-brain barrier model with a tight junction structure and its application. Background Technology

[0002] The blood-brain barrier (BBB) ​​is a barrier that selectively prevents certain substances from entering the brain from the bloodstream through the blood vessels. Essentially, the BBB is a complex cellular or tissue structure existing between peripheral blood and brain tissue. It controls the passage and exchange of substances between the blood and cerebrospinal fluid, regulating and maintaining the homeostasis of the brain's internal environment. The cells constituting the BBB are primarily endothelial cells. These endothelial cells form tight junction structures under the action of various tight junction proteins, and interact with glial cells and pericytes to form this unique barrier system.

[0003] The blood-brain barrier (BBB) ​​has low permeability, making it a natural barrier to drug delivery into the brain and hindering effective drug transport. Therefore, research and development of drug delivery to the brain must consider the ability to effectively penetrate the BBB. Furthermore, abnormal development and functional loss of the BBB can disrupt the homeostasis of the brain microenvironment, leading to neurological dysfunction and triggering numerous neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease. Therefore, the development and in-depth study of in vitro models of the BBB will provide an important theoretical foundation for drug development and disease diagnosis and treatment of neurological diseases.

[0004] Existing blood-brain barrier models mainly include: in vivo animal models, in vitro models based on microfluidic chip technology, and Transwell chamber-based models. In vivo animal models primarily use the blood-brain barrier of animals such as mice, rats, and rabbits for research. However, due to species differences between small animals and humans, the accuracy of these models is difficult to guarantee. In vitro microfluidic chip-based models can better reproduce the microenvironment of the human blood-brain barrier. However, the high operational difficulty and technical barriers of microfluidic chip-based models hinder their widespread application. In vitro Transwell chamber-based models are easy to operate, and using human cells can minimize species differences. However, conventional Transwell chamber models struggle to effectively promote the formation of tight junctions between cells, making it difficult to guarantee the effectiveness of the blood-brain barrier.

[0005] Therefore, it is necessary to provide an effective blood-brain barrier model that is easy to operate, has low technical barriers, and can promote the formation of tight connections between cells. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an in vitro blood-brain barrier model with a tightly connected structure. This blood-brain barrier model can effectively simulate the function of the blood-brain barrier in vivo and has the characteristics of simple operation and low technical threshold, thus possessing great commercial value.

[0007] The present invention also provides the application of the above-mentioned in vitro blood-brain barrier model with a tight connection structure in the development of blood-brain barrier-related drugs.

[0008] An in vitro blood-brain barrier model with a tight junction structure includes:

[0009] Tubular orifice plate support;

[0010] A mesh substrate support for sealing the bottom of a perforated plate support;

[0011] A cell membrane sheet loaded on the substrate scaffold and having a tightly connected structure;

[0012] And the cell membrane sheet, substrate scaffold, and well plate scaffold are encapsulated into a single hydrogel structure.

[0013] After the in vitro blood-brain barrier model with a tightly connected structure is constructed, the lower end (cell membrane sheet) needs to be placed in cell culture medium to maintain cell viability. The cross-section of the well plate scaffold can be circular, square, rectangular, triangular, or irregular.

[0014] Cell membrane sheets, composed of one or more blood-brain barrier-associated cells, can be used to mimic the tight junction structure between blood-brain barrier cells.

[0015] Preferably, the perforated plate support is a cylindrical tube structure. To facilitate processing and installation and improve the sealing effect of the blood-brain barrier model, the substrate support is a circular sheet structure that matches the cylindrical tube structure of the perforated plate support.

[0016] Preferably, the upper end of the well plate support is provided with a positioning part for positioning the well plate support. The function of the positioning part is to position the blood-brain barrier model so that the lower end of the well plate support with cell membrane sheets can be suspended in the cell culture medium. In this way, the cell membrane sheets can divide the cell culture medium into two parts, the inner and outer parts of the blood-brain barrier model, to simulate the inner and outer parts of the blood-brain barrier, thereby realizing the reproduction of the physiological function of the blood-brain barrier in vitro. Among them, the inner part (cell culture medium) of the blood-brain barrier model is the upper chamber, and the outer part (cell culture medium) is the lower chamber.

[0017] As a further preferred embodiment, the positioning part is a positioning rod perpendicular to the central axis of the well plate holder and fixedly connected to the upper end of the well plate holder. When the well plate holder is placed in a container containing cell culture medium, the outer end of the positioning rod (the end not connected to the well plate holder) is positioned at the top of the container. The size of the well plate holder is set according to the size of the container.

[0018] As a further preferred embodiment, the positioning rods are provided in three parts, which are evenly distributed around the upper circumference of the perforated plate support.

[0019] To improve the stability of the positioning part structure, as a further preferred embodiment, the positioning part further includes a positioning ring sleeved on the outside of the perforated plate support. The positioning rod is connected to the upper end of the perforated plate support and the positioning ring respectively, and the radius of the positioning ring is smaller than the sum of the radius of the perforated plate support and the length of the positioning rod.

[0020] Preferably, the lower end of the perforated plate support is provided with a support portion for mounting the substrate support. The support portion is an annular mounting platform disposed along the inner wall of the lower end of the perforated plate support, and the inner diameter of the mounting platform is larger than the outer diameter of the substrate support.

[0021] Preferably, the substrate scaffold is a polycaprolactone (PCL) scaffold printed using near-field direct writing high-precision 3D printing, wherein the distance between two adjacent filaments in the same direction is 0.1–10 mm. More preferably, it is 0.8–1.2 mm.

[0022] Preferably, the hydrogel structural material is a substance that changes from a liquid state to a gel state after being stimulated by external conditions (such as temperature, light, etc.).

[0023] As a further preferred embodiment, the hydrogel structural material is one or more selected from gelatin, gelatin derivatives, hyaluronic acid, hyaluronic acid derivatives, alginate compounds, Pluronic F-127, fibrinogen, collagen, silk fibroin, chitosan, agarose, polyethylene glycol, and polyethylene oxide. Even more preferably, methacrylic anhydride-modified gelatin (GelMA) is preferred.

[0024] Preferably, the method for culturing the cell membrane sheet with a tightly connected structure includes:

[0025] Cells resuspended in culture medium are seeded onto bottom support liquid in a culture container, and after culturing on the surface of bottom support liquid, cell membrane sheets with tight junction structures are formed.

[0026] The density of the bottom supporting liquid is greater than that of the culture medium, and it is not miscible with the culture medium.

[0027] In the above-mentioned cell membrane culture method, a bottom-supporting liquid is used as the growth support for cells. When cultured in a liquid matrix, the extracellular matrix secreted by the cells cannot adhere to the liquid matrix, but can only adhere to other adjacent cells, thereby causing the cells to secrete more extracellular matrix and form a tightly connected cell membrane with high cell density.

[0028] As a further preferred embodiment, the bottom support liquid includes, but is not limited to, one or more of the following: fluorinated oil, fluoroalkane compounds, siloxane compounds (such as silicone oil, uncured polydimethylsiloxane), and ester compounds (such as dimethyl carbonate, dimethyl sulfate).

[0029] The main reason for choosing hydrophobic liquids, such as fluorinated oils, as the bottom support liquid is that the hydrophobic effect of the fluorinated oils in the liquid substrate provides radial-inward surface tension to the cells from all directions. The shape of the cells is controlled by the Yang-Laplace equation; that is, under the constraint of the conservation of the volume of each cell, the potential energy is minimized, so that the cells become three-dimensional spheres. The three-dimensional spherical cells spontaneously self-assemble to form a hexagonal tightly connected structure.

[0030] As a further preferred option, the bottom support liquid is one or a mixture of 3M Novec HFE series fluorinated oil (such as HFE7500), 3M Fluorinert FC series fluorinated oil, TECCEM Fluoronox series fluorinated oil, silicone oil, uncured polydimethylsiloxane, dimethyl carbonate, and dimethyl sulfate.

[0031] As a further preferred embodiment, the amount of bottom support liquid added to the culture container is greater than 0.08 mL / cm². 2 As a further preferred embodiment, the amount of bottom support liquid added is 0.3–0.7 mL / cm³. 2 .

[0032] As a further preferred option, the cells are thawed cryopreserved cells or passaged and digested cells.

[0033] As a further preferred embodiment, the cells are cells related to the blood-brain barrier model, including but not limited to one or more of endothelial cells, glial cells, and pericytes. Endothelial cells are even more preferred.

[0034] As a further preferred option, the cell seeding concentration during cell culture is 2 × 10⁻⁶. 4 ~2×10 8 pcs / cm 2 A further preferred value is 1×10. 6 pcs / cm 2 ~2×10 6 pcs / cm 2 .

[0035] As a further preferred option, the cell culture temperature is 35–39°C, and the culture time is 1–28 days. Even more preferred is that the culture temperature is 37°C, the culture time is 1–14 days, and the cultured cells form membranous cell sheets with tight junctions.

[0036] As a further preferred option, the cells were cultured in a 5% carbon dioxide incubator after seeding.

[0037] As a further preferred embodiment, the culture medium is changed every 10-15 hours during cell culture, with the volume of the replaced cell culture medium being 70-90% of the original volume. Even more preferably, the culture medium is changed every 12 hours during culture.

[0038] As a further preferred option, the new culture medium should be preheated before being replaced.

[0039] As a further preferred option, the bottom support liquid is added to the culture container after sterilization.

[0040] As a further preferred option, the bottom support liquid is sterilized using one or more of the following sterilization methods: chemical sterilization, radiation sterilization, dry heat sterilization, moist heat sterilization, and filtration sterilization. Even more preferred is ultraviolet light sterilization.

[0041] The culture container can be a culture plate, a culture dish, or any container suitable for general cell culture. Culture containers can also be made of custom materials, shapes, and structures. Preferably, the culture container is a commercially available multi-well culture plate.

[0042] As a further preferred method, the substrate scaffold is first embedded in the bottom supporting liquid, followed by cell seeding. After cell culture is complete and cell membranes form, the substrate scaffold is lifted upwards and removed. During this process, the cell membranes adhere to the substrate scaffold, resulting in a substrate scaffold loaded with cell membranes. Since the formed cell membranes are relatively fragile, it is difficult to remove them intact without external force. This technical solution uses a mesh-like substrate scaffold as the supporting structure for the cell membranes. With the support of the substrate scaffold, the cell membranes are removed intact, and the substrate scaffold and cell membranes are applied as a whole in a blood-brain barrier model.

[0043] To facilitate the removal of the substrate support, as a further preferred option, a collection support can be added as an auxiliary tool for picking up and placing the substrate support. The collection support includes an annular structure at the bottom and a lifting rod connected to the annular structure. The inner side of the annular structure is provided with an annular boss for installing the substrate support.

[0044] The lifting rod is parallel to the central axis of the ring structure, with its lower end connected to the ring structure and its upper end equipped with a bent handle. When the collection rack is placed in the culture container, the handle can be hung on top of the culture container. The size of the collection rack is set according to the size of the culture container.

[0045] When using it, first install the substrate support on the collection support and then put it into the bottom support liquid. After the cell membrane sheet is formed, remove the collection support directly, and then remove the substrate support and cell membrane sheet from the collection support.

[0046] As a further preferred embodiment, the collection bracket is a 3D-printed polylactic acid (PLA) bracket.

[0047] Preferably, the substrate support and the collection support are added to the culture container after sterilization. More preferably, sterilization is performed using one or more of the following methods: chemical sterilization, radiation sterilization, dry heat sterilization, moist heat sterilization, and filtration sterilization. Even more preferably, ultraviolet light sterilization is used.

[0048] Preferably, after removing the substrate scaffold carrying cell membranes from the collection scaffold, it is installed on the well plate scaffold. The substrate scaffold carrying cell membranes is then suspended in a container containing cell culture medium through the positioning part of the well plate scaffold to obtain the in vitro blood-brain barrier model.

[0049] Taking cell culture in a 12-well plate as an example, the construction process of the above-mentioned in vitro blood-brain barrier model with tight junction structure is as follows:

[0050] (1) Preparation of cell membrane sheets with tight junction structures

[0051] Prepare a clean, sterilized 12-well culture plate, and place a clean, sterilized, custom-sized collection rack and substrate rack (the substrate rack is installed on the collection rack) into it in sequence.

[0052] The outer diameter of the custom-sized collection holder is slightly smaller than the inner diameter of the 12-well culture plate; its height is slightly greater than the depth of the 12-well culture plate.

[0053] Bottom support liquid (the liquid level should at least cover the substrate support) and cell culture medium containing cells are added sequentially to a 12-well culture plate for incubation. After incubation, a cell membrane with a tight junction structure forms on the surface of the bottom support liquid.

[0054] The bottom supporting liquid has a density greater than that of the culture medium and is not miscible with the culture medium.

[0055] (2) Establishing an in vitro blood-brain barrier model

[0056] Prepare another clean and sterilized 6-well culture plate. Slowly remove the collection scaffold from the 12-well culture plate, during which the cell membrane sheets adhere to the substrate scaffold. Separate the substrate scaffold containing the cell membrane sheets from the collection scaffold and place it into a clean and sterilized custom-sized well plate scaffold.

[0057] The well plate support needs to be suspended on the well plate, and the distance between the bottom of the well plate support (i.e., the cell membrane sheet) and the well plate is 1 to 10 mm.

[0058] The substrate scaffold carrying cell membrane sheets was encapsulated with hydrogel and the well plate scaffold. The encapsulated well plate scaffold was then placed in a 6-well culture plate containing cell culture medium for culture to maintain cell viability.

[0059] Preferably, the collection bracket is a 3D-printed polylactic acid (PLA) bracket with an outer diameter of 21.2 mm and a height of 19 mm.

[0060] Preferably, the substrate support is a circular mesh structure with a diameter of 15-21 mm and a wire spacing of 0.1-10 mm.

[0061] Preferably, the substrate is a polycaprolactone (PCL) scaffold printed by near-field direct writing high-precision 3D printing, with a diameter of 19 mm and a filament pitch of 1 mm.

[0062] Preferably, the perforated plate support is a 3D-printed polylactic acid (PLA) support with an outer diameter of 35mm, a height of 17mm, and a distance of 5mm between the bottom of the perforated plate support and the perforated plate.

[0063] Application of an in vitro blood-brain barrier model with a tightly connected structure as described in any of the above claims in the development of blood-brain barrier-related drugs.

[0064] Preferably, the bottom (cell membrane) of the blood-brain barrier model is suspended in a cell culture medium, and a blood-brain barrier-related drug is added to the cell culture medium (upper chamber) inside the blood-brain barrier model. The concentration of the drug in the cell culture medium (lower chamber) outside the blood-brain barrier model is detected to determine the penetration effect of the drug in the blood-brain barrier model.

[0065] The present invention discloses a method for establishing an in vitro blood-brain barrier model with a tightly connected structure. The method involves preparing blood-brain barrier-related endothelial cells into cell membrane sheets with a tightly connected structure, and encapsulating the cell membrane sheets with a custom-sized well plate scaffold and a substrate scaffold using hydrogel to form a whole. The well plate scaffold is divided into upper and lower parts to simulate the inner and outer parts of the blood-brain barrier. This method can reproduce the physiological function of the blood-brain barrier in vitro and observe the permeability of drugs in the blood-brain barrier.

[0066] The present invention discloses a method for establishing an in vitro blood-brain barrier model with a tightly connected structure, which constructs a platform that simulates the in vivo blood-brain barrier. This platform can be applied to life science and clinical medical research, providing an important theoretical basis for drug development and disease diagnosis and treatment of nervous system diseases.

[0067] The present invention provides a method for establishing an in vitro blood-brain barrier model with a tightly connected structure, comprising the following steps: (1) culturing cell sheets using a cell culture method that produces adherent cells with a tightly connected structure; (2) printing a cell sheet substrate scaffold; (3) printing a well plate scaffold of a customized size; and (4) encapsulating the cell sheet, substrate scaffold, and well plate scaffold with hydrogel.

[0068] The cell sheet, composed of one or more blood-brain barrier-related cells, can be used to simulate the tight junctions between blood-brain barrier cells. The cell sheet substrate scaffold is used to retrieve the cell sheet and provide mechanical support. The well plate scaffold can be suspended in well plates of different sizes, dividing the well plate into upper and lower chambers. This invention integrates the construction, characterization, barrier function assessment, and drug evaluation of an in vitro blood-brain barrier model, and can be used for in vitro simulation of the blood-brain barrier and for testing blood-brain barrier-related drugs. Compared with existing blood-brain barrier models, this invention resolves the contradiction between accuracy and simplicity in constructing in vitro blood-brain barrier models, more closely approximating the real in vivo environment, improving experimental efficiency, and showing promising application prospects in the research and drug development of blood-brain barrier-related diseases.

[0069] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0070] (1) The blood-brain barrier model of the present invention eliminates species differences compared with the in vivo blood-brain barrier animal model. The experimental data obtained through this platform (blood-brain barrier model) are more consistent with the real situation of the human body, thus improving the accuracy of blood-brain barrier drug development.

[0071] (2) Compared with in vitro blood-brain barrier models based on microfluidic chips, the blood-brain barrier model of the present invention reduces the difficulty of operation and the technical threshold. While ensuring the effectiveness and accuracy of the blood-brain barrier model, it has the characteristics of simple operation and easy promotion, and has great commercial value.

[0072] (3) Compared with the blood-brain barrier model of ordinary Transwell chamber in vitro, the blood-brain barrier model of the present invention promotes the formation of tight junctions between cells and has a tissue structure similar to the real situation of human tissue, thus forming an effective blood-brain barrier model.

[0073] (4) This invention is the first to construct a blood-brain barrier in vitro that is close to physiological conditions in terms of tissue structure, transmembrane resistance, permeability, and drug-toxicity response, providing an important research platform for drug development and diagnosis and treatment of nervous system diseases. Attached Figure Description

[0074] Figure 1This is a schematic diagram illustrating the process of preparing cell membrane sheets with tightly connected structures and establishing an in vitro blood-brain barrier model according to an embodiment of the present invention; wherein, 1 is a sterile 12-well culture plate, 2 is a collection scaffold, 3 is a substrate scaffold, 4 is a bottom support liquid, 5 is cells, 6 is cell culture medium, 7 is a sterile 6-well culture plate, 8 is a well plate scaffold, and 9 is a hydrogel.

[0075] Figure 2 This is a schematic diagram of the structure of the in vitro blood-brain barrier model according to an embodiment of the present invention;

[0076] Figure 3 This is a schematic diagram of the structure of the collection bracket and the perforated plate bracket in an embodiment of the present invention; wherein, Figure 3 In the image, 'a' represents the oblique view, top view, and side view of the collection bracket. Figure 3 In the diagram, b represents the oblique view, top view, and side view of the orifice plate support; where 10 is a ring structure, 11 is a lifting rod, 12 is a handle, and 13 is an annular boss; 80 is a circular tube structure, 81 is a positioning rod, 82 is a positioning ring, and 83 is an annular mounting platform.

[0077] Figure 4 The images shown are physical photos of the substrate support in the embodiments of the present invention, as well as magnified micrographs of the edges and center portions.

[0078] Figure 5 This is a physical diagram of the process in an embodiment of the present invention; wherein, Figure 5 Image a shows a physical diagram of a cell membrane sheet with a tight junction structure. Figure 5 Image b is a top view of a cell membrane sheet with a tightly connected structure. Figure 5 Image c shows the actual cell membrane sheet with a tight junction structure collected by a collection scaffold after preparation. Figure 5 In the middle d, there is a physical image of the cell membrane sheet on the substrate scaffold after the preparation of the cell membrane sheet with a tight connection structure (substrate scaffold carrying the cell membrane sheet); Figure 5 The image in the middle (e) is a physical diagram of an in vitro blood-brain barrier model.

[0079] Figure 6 These are micrographs and electron microscope images of cell membrane sheets with tightly connected structures in embodiments of the present invention; wherein, Figure 6 Image a is a 4x micrograph of a cell membrane sheet with a tight junction structure on a substrate scaffold. Figure 6 Image b is a 10x magnification micrograph of a cell membrane sheet with a tight junction structure on a substrate scaffold. Figure 6 Image c is a 300x electron microscope image of a cell membrane sheet with a tight junction structure on a substrate scaffold. Figure 6 In the middle d, there is a 4000x electron microscope image of a cell membrane sheet with a tight junction structure on a substrate scaffold.

[0080] Figure 7 Here is an electron micrograph of the hydrogel in an embodiment of the present invention; wherein, Figure 7 In the image, a is a 100x electron microscope image of the hydrogel surface; Figure 7 Image b is a 300x electron microscope image of the hydrogel surface; Figure 7 In the middle, c is a 50x electron microscope image of the interior of the hydrogel; Figure 7 In the image, d is a 200x electron microscope image of the interior of the hydrogel;

[0081] Figure 8 These are test results verifying the sealing performance of the perforated plate stent (blood-brain barrier model) in this embodiment of the invention.

[0082] Figure 9 Test results of transmembrane resistance of the blood-brain barrier under different blood-brain barrier construction conditions;

[0083] Figure 10 Test results of fluorescent substances of different molecular weights crossing the blood-brain barrier under different blood-brain barrier construction conditions;

[0084] Figure 11 These are the results of blood-brain barrier permeability tests before and after treatment with a blood-brain barrier toxic drug (methamphetamine) in this embodiment of the invention.

[0085] Figure 12 These are the test results of blood-brain barrier-related drugs (dopamine, L-dopamine) crossing the blood-brain barrier in embodiments of the present invention; wherein, Figure 12 In figure 'a', it is a schematic diagram of the passage of drugs related to the blood-brain barrier across the blood-brain barrier. Figure 12 In Figure b, the results of the detection of blood-brain barrier-related drugs crossing the blood-brain barrier are shown. Detailed Implementation

[0086] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and experimental example are commercially available. The specific embodiments described herein are only for explaining this invention and are not intended to limit this invention.

[0087] like Figure 1 As shown, a method for establishing an in vitro blood-brain barrier model with a tightly connected structure includes the following steps:

[0088] a. Prepare a clean and sterilized 12-well culture plate, and place a clean and sterilized custom-sized collection rack and substrate rack (the substrate rack is installed on the collection rack) into it in sequence.

[0089] b. Add bottom support liquid (the liquid level should cover the substrate support) and cell culture medium containing endothelial cells to the 12-well culture plate in sequence for incubation.

[0090] c. After culturing for 24 hours, slowly remove the collection scaffold from the 12-well culture plate. During the removal process, the cell membranes adhere to the substrate scaffold. Separate the substrate scaffold containing the cell membranes from the collection scaffold.

[0091] d. Prepare another clean and sterilized 6-well culture plate. Place the substrate scaffold carrying the cell membrane sheets into the clean and sterilized custom-sized well plate scaffold. Encapsulate the substrate scaffold carrying the cell membrane sheets and the well plate scaffold with hydrogel.

[0092] e. The encapsulated plate support is placed into a 6-well culture plate containing cell culture medium for culture, wherein the cell membrane is suspended in the cell culture medium, that is, both the upper and lower sides of the cell membrane are immersed in the cell culture medium.

[0093] f. The constructed in vitro blood-brain barrier model with a tight junction structure allows for the addition of blood-brain barrier-related drugs from the upper chamber (the cell culture medium on the upper side of the cell membrane, i.e., the cell culture medium inside the blood-brain barrier model) and the detection of the drug's penetration effect on the blood-brain barrier from the lower chamber (the cell culture medium inside a 6-well culture plate).

[0094] Product (blood-brain barrier model) appearance and structure:

[0095] The above process can construct an in vitro blood-brain barrier model with a tightly connected structure, whose structure is mainly composed of... Figure 2 As shown, a plate support is suspended on a 6-well culture plate, with a 5mm gap between the bottom of the support and the 6-well culture plate. The bottom of the plate support carries the substrate support and the cell membrane sheet formed by endothelial cells, all three being encapsulated with hydrogel.

[0096] Collection support structure such as Figure 3 As shown in Figure a, the collection support includes an annular structure 10 at the bottom and a lifting rod 11 connected to the annular structure 10. The inner side of the annular structure 10 is provided with an annular boss 13 for mounting the substrate support. The lifting rod 11 is parallel to the central axis of the annular structure 10, with its lower end connected to the annular structure 10 and its upper end provided with a bent handle 12. When the collection support is placed in the culture container, the handle can be hung on the top of the culture container.

[0097] Orifice plate support structure as follows Figure 3As shown in Figure b, the well plate support is a circular tube structure 80. A positioning ring 82 is fitted onto the upper end of the circular tube structure 80, along with three positioning rods 81 that fix the upper end of the circular tube structure 80 to the positioning ring 82. The positioning rods 81 are perpendicular to the central axis of the circular tube structure 80. The three positioning rods 81 are evenly distributed circumferentially along the circular tube structure 80, and the radius of the positioning ring 81 is less than the sum of the radius of the circular tube structure 80 and the length of the positioning rods 81. An annular mounting platform 83 for mounting the substrate support is provided on the inner wall of the lower end of the circular tube structure 80. The substrate support carrying cell membrane sheets, mounted on the annular mounting platform 83, is suspended in the cell culture medium by a positioning section composed of the positioning ring 82 and the three positioning rods 81.

[0098] The structure and micrograph of the substrate support are shown below. Figure 4 As shown, the substrate support is a circular mesh structure.

[0099] Figure 5 Figures a and b show a front view and a top view of a physical sample of a cell membrane sheet with a tightly connected structure. Figure 5 Image c shows a photograph of a cell membrane sheet with a tight junction structure after it has been collected by a collection scaffold. Figure 5 Image d shows a physical image of a cell membrane sheet with a tightly connected structure on a substrate scaffold after fabrication. Figure 5 As can be seen from d, the membrane-like cell sheets form a tight connection at a macroscopic size and are completely attached to the substrate scaffold. Figure 5 The image in the middle (e) is a physical diagram of an in vitro blood-brain barrier model.

[0100] Product characteristics:

[0101] The tight junction structure of the endothelial cell membrane in this embodiment was verified using microscopic and electron microscopic images. Figure 6 It can be seen that, at the microscopic level, the membrane-like cell sheets form tight connections and are completely attached to the substrate scaffold.

[0102] Electron microscopy images confirmed that the hydrogel used for encapsulation in this embodiment does not generate a barrier function; that is, the barrier function is generated by the constructed cell membrane sheet. Figure 7 It can be seen that, at the microscopic level, the hydrogel used for encapsulation has a loose and porous hollow structure, which allows the material to pass through.

[0103] The edge sealing performance of the hydrogel used for encapsulation in this embodiment was verified by fluorescence permeation experiment.

[0104] The specific experimental procedure was as follows: Hydrogel was used to encapsulate the well plate scaffold and the substrate scaffold carrying cell membrane sheets, respectively, with and without obstructions. Then, fluorescent dye was added to the upper chamber. After a period of time, the fluorescence intensity in the lower chamber was measured to reflect the degree of fluorescence leakage. The detection results are as follows: Figure 8 As shown.

[0105] Depend on Figure 8 The results show that after 12 hours, the edges encapsulated by the hydrogel did not show significant penetration, demonstrating good sealing performance.

[0106] Product functionality testing:

[0107] The electrodes of the Millicell-ERS volt-ohmmeter transmembrane resistance meter were immersed in the liquid (cell culture medium) in the upper and lower chambers, respectively. The transmembrane resistance of the blood-brain barrier model constructed in this example was measured to determine the differences in transmembrane resistance between the model and the hydrogel control group, the hydrogel + monolayer cell control group, and the in vivo blood-brain barrier under the same conditions. The results are as follows: Figure 9 As shown. Figure 9 The results showed that the transmembrane resistance of the blood-brain barrier model constructed in this embodiment (shown as hydrogel + cell membrane sheet in the figure) was as high as approximately 1900 Ω / cm. 2 The data were similar to those of the human blood-brain barrier, but showed significant differences compared to the control group.

[0108] The permeability (barrier function) of the blood-brain barrier model constructed in this example was determined by permeation testing with substances of different molecular weights, compared with the hydrogel control group and the hydrogel + monolayer cell control group under the same conditions. The results are as follows: Figure 10 As shown. Figure 10 The results showed that the permeability of the blood-brain barrier model constructed in this embodiment (shown as hydrogel + cell membrane sheet in the figure) was significantly different from that of the control group, indicating that it had good barrier function.

[0109] The response of the blood-brain barrier model constructed in this embodiment to blood-brain barrier toxic drugs was determined by a blood-brain barrier toxicity test, and the results are as follows: Figure 11 As shown. Figure 11 The results showed that the blood-brain barrier model constructed in this embodiment (shown as blood-brain barrier in the figure) had a significantly reduced barrier function after treatment with a blood-brain barrier toxic drug (methamphetamine), but its barrier function was significantly improved after 24 hours of spontaneous recovery. This is consistent with the response of the human blood-brain barrier to a blood-brain barrier toxic drug (methamphetamine).

[0110] The permeability (selective permeation function) of the blood-brain barrier model constructed in this embodiment to blood-brain barrier-related drugs was determined by a blood-brain barrier-related drug permeation test. The results are as follows: Figure 12 As shown. Figure 12 The results showed that, after dopamine and L-dopamine were added to the upper chamber, the L-dopamine content detected in the lower chamber of the blood-brain barrier model constructed in this embodiment was significantly higher than that of dopamine. This indicates that the blood-brain barrier model constructed in this embodiment has selective permeability to blood-brain barrier-related drugs, which is consistent with the results of selective permeability of blood-brain barrier-related drugs by the human blood-brain barrier.

[0111] In summary, the in vitro blood-brain barrier model with a tightly connected structure constructed in this invention can effectively simulate the function of the human blood-brain barrier, improve the accuracy and effectiveness of blood-brain barrier drug development, and is simple to operate and easy to promote, providing an important research foundation for the development of drugs for nervous system diseases and the diagnosis and treatment of diseases.

Claims

1. An in vitro blood-brain barrier model having a tight junction structure, characterized by, include: Tubular orifice plate support; A mesh substrate support for sealing the bottom of a perforated plate support; A cell membrane sheet loaded on the substrate scaffold and having a tightly connected structure; And the cell membrane sheet, substrate scaffold, and well plate scaffold are encapsulated into a single hydrogel structure; The orifice plate support is suspended on the orifice plate, and the distance between the bottom of the orifice plate support and the orifice plate is 1~10 mm. The method for culturing the cell membrane sheet with a tight junction structure includes: Cells resuspended in culture medium are seeded onto bottom support liquid in a culture container, and after culturing on the surface of bottom support liquid, cell membrane sheets with tight junction structures are formed. The density of the bottom supporting liquid is greater than that of the culture medium, and it is not miscible with the culture medium; The bottom support fluid is 3M Novec HFE series fluorinated oil, 3M Fluorinert FC series fluorinated oil, or TECCEM Fluoronox series fluorinated oil. The cells in question are endothelial cells.

2. The in vitro blood-brain barrier model having a tight junction structure according to claim 1, wherein The upper end of the perforated plate support is provided with a positioning part for positioning the perforated plate support.

3. The in vitro blood-brain barrier model having a tight junction structure according to claim 1, wherein The lower end of the perforated plate holder is provided with a support for mounting the substrate holder.

4. The in vitro blood-brain barrier model with a tightly connected structure according to claim 1, characterized in that, The hydrogel structural material is one or more of gelatin, gelatin derivatives, hyaluronic acid, hyaluronic acid derivatives, alginate compounds, Pluronic F-127, fibrinogen, collagen, silk fibroin, chitosan, agarose, polyethylene glycol, and polyethylene oxide.

5. The in vitro blood-brain barrier model with a tightly connected structure according to claim 1, characterized in that, When the cell sheet culture is performed, the cell is seeded at a concentration of 2 x 10 4 ~2 x 10 8 / cm 2 .

6. The in vitro blood-brain barrier model with a tightly connected structure according to claim 1, characterized in that, First, the substrate scaffold is buried in the bottom support liquid, then cells are seeded. After the cell culture is completed and cell membranes are formed, the substrate scaffold is lifted upwards and removed. During this process, the cell membranes adhere to the substrate scaffold, resulting in a substrate scaffold loaded with cell membranes.

7. The application of an in vitro blood-brain barrier model with a tightly connected structure as described in any one of claims 1 to 6 in the development of drugs for nervous system diseases.

8. The application according to claim 7, characterized in that, The bottom of the blood-brain barrier model is suspended in a cell culture medium. A drug for neurological diseases is added to the cell culture medium inside the blood-brain barrier model. The concentration of the drug in the cell culture medium outside the blood-brain barrier model is detected to determine the penetration effect of the drug in the blood-brain barrier model.

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