A human blood-cerebrospinal fluid barrier model and its preparation method and application

Abstract

The present invention discloses a human blood-cerebrospinal fluid barrier model and its preparation method and application, the method comprising: preparing a microfluidic chip by a reverse molding method using a biocompatible material, wherein the microfluidic chip contains an upper channel and a lower channel arranged adjacently; arranging a porous film between the upper channel and the lower channel of the microfluidic chip, and fixing the microfluidic chip with a clamp to obtain a culture device; placing the culture device in a matrix material so that the matrix material coats the periphery of the upper channel and the lower channel; injecting a human choroid plexus epithelial cell suspension into the upper channel, injecting a human brain microvascular endothelial cell suspension into the lower channel, and after the cells are attached, applying fluid stimulation to the upper channel and the lower channel and culturing, and obtaining a human blood-cerebrospinal fluid barrier model. The model of the present invention has a high degree of bionics and is easy to detect, and is suitable for optical imaging instruments such as confocal microscopes.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering and organ chip, and in particular to a human blood-cerebrospinal fluid barrier model and a preparation method and application thereof. Background Art

[0002] BCSFB is located in the lateral ventricle, the third ventricle, and the fourth ventricle. It is composed of tightly connected, polarized choroid plexus epithelial cells (CPECs) and brain microvascular endothelial cells (BMECs) with fenestration structures. In addition to the physical barrier function, choroid plexus epithelial cells are the main site of cerebrospinal fluid secretion and reabsorption, and can secrete a variety of cytokines to maintain the stability of the nervous system. Studies have found that the use of human cerebrospinal fluid is closely related to neural development processes such as neurogenesis, gliogenesis, synapse formation, and neurite outgrowth, indicating that choroid plexus epithelial cells are essential for maintaining the normal function of the nervous system. In addition, choroid plexus epithelial cells express chemokines such as intercellular adhesion molecule-1 (ICAM-1) and CCL20, allowing lymphocytes to conduct continuous immune surveillance of cerebrospinal fluid and the brain through this pathway. The above research results all suggest that BCSFB is an important interface of the neuro-immune-endocrine axis in the central nervous system, which has significant clinical significance and research value. In addition, more and more evidence shows that changes in the morphology or molecular profile of choroid plexus epithelial cells are associated with a variety of neurological diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. In-depth research on BCSFB requires reliable model support, but existing models have many limitations.

[0003] At present, BCSFB models mainly include animal models and in vitro models. Due to the large differences between animal models and real human physiological conditions, and the fact that BCSFB is located deep in the brain and difficult to observe, its preclinical experimental research is limited; in vitro models are mainly constructed using two-dimensional cells, and the model structure is too simple to reproduce the physiological functions of BCSFB.

[0004] Therefore, in order to overcome the problems existing in the above-mentioned traditional models, it is urgently necessary to develop a human blood-cerebrospinal fluid barrier model with a high degree of bionics and easy to detect, as well as its construction method and application, which is suitable for optical imaging instruments such as confocal microscopes, so as to provide new technical support for in-depth research on the regulatory mechanism of BCSFB and the clinical treatment of related diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a human blood-cerebrospinal fluid barrier model and its preparation method and application. The barrier model obtained by the present invention is a three-layer structure, which is closer to the actual physiological situation compared with the structure of previous blood-cerebrospinal fluid barrier models; due to stable unidirectional fluid stimulation, the epithelial part of the barrier model can be seen to form a directional cell arrangement, and the vascular endothelial part forms a tight connection, which has a high degree of bionics and is easy to detect, and is suitable for optical imaging instruments such as confocal microscopes.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a method for preparing a human blood-cerebrospinal fluid barrier model is provided, the method comprising:

[0008] The biocompatible material is prepared by a reverse molding method to obtain a microfluidic chip, wherein the microfluidic chip contains an upper channel and a lower channel that are adjacently arranged;

[0009] A porous film is arranged between the upper channel and the lower channel of the microfluidic chip, and the microfluidic chip is fixed with a clamp to obtain a culture device;

[0010] The culture device is placed in a matrix material so that the matrix material coats the periphery of the upper channel and the lower channel; a human choroid plexus epithelial cell suspension is injected into the upper channel, and a human brain microvascular endothelial cell suspension is injected into the lower channel. After the cells are attached, fluid stimulation is applied to the upper channel and the lower channel and culture is performed to obtain a human blood-cerebrospinal fluid barrier model.

[0011] Furthermore, the biocompatible material includes polyethylene terephthalate (PDMS) prepolymer; or at least one of type I collagen, matrigel, GelMA, sodium alginate, fibrinogen, HAMA, HA-CA, silk fibroin, chitosan and gelatin.

[0012] Furthermore, the method of preparing the microfluidic chip by using the biocompatible material through the inverted molding method comprises:

[0013] Obtaining a male mold having an upper channel and a lower channel disposed adjacently;

[0014] Pouring a biocompatible material onto the male mold, vacuum drying and curing, and then peeling off to obtain a female membrane;

[0015] The negative film is cut into a shape and size suitable for subsequent assembly to obtain the microfluidic chip containing the upper channel and the lower channel.

[0016] Furthermore, the length, width and height of the upper channel and the lower channel are 9000-11000 μm, 800-1200 μm and 20-200 μm respectively.

[0017] Furthermore, the material of the porous film is polyethylene terephthalate, and the porosity of the porous film is set to 1×10 5 ~1×10 7 Pieces / cm 2 .

[0018] Furthermore, the matrix material includes one of Matrigel, laminin and fibronectin.

[0019] Furthermore, the cell density of the human choroid plexus epithelial cell suspension and the human brain microvascular endothelial cell suspension are both (5-10)×10 6 cells / mL.

[0020] Furthermore, in the fluid stimulation, the flow rate of the fluid is 10 to 200 μL / h.

[0021] In the second aspect of the present invention, a human blood-cerebrospinal fluid barrier model prepared by the method is provided.

[0022] In the third aspect of the present invention, the application of the human blood-cerebrospinal fluid barrier model in immunology, pharmacodynamics, and physiological and pathological analysis is provided.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] The present invention provides a human blood-cerebrospinal fluid barrier model and its preparation method and application. The reverse engineering construction strategy is used to simplify the human blood-cerebrospinal fluid barrier into a co-culture system of choroid plexus epithelium and endothelium with a pore-window structure. Biocompatible materials are used in the middle to provide support to form a sandwich structure, thereby bionic construction of the human blood-cerebrospinal fluid barrier interface. Organ chip technology is used to construct epithelial and endothelial dual channels that are consistent with the actual physiological conditions of the human body, and a porous film is used to separate the upper and lower channels. Microfluidic technology is used to control the internal flow field of the model, and by simulating the actual physiological fluid characteristics, cell self-organization is promoted to form an in vitro human blood-cerebrospinal fluid barrier model; the model has the following advantages:

[0025] (1) This model has stable fluid stimulation, can provide stable support for endothelial cells and choroid plexus epithelial cells, can form a complete human blood-cerebrospinal fluid barrier, and form a barrier function similar to the physiological level in vitro. It has significant advantages over traditional animal experiments and two-dimensional cell culture models;

[0026] (2) The microfluidic chip of the present invention has a mature mass-production standardized production process and is easy to carry out subsequent commercial production. The chip structure and size of the present invention can be designed and customized according to specific needs, and can be combined with the orifice plate system. By changing the fluid perfusion method in the channel, it can be easily transformed into a high-throughput system;

[0027] (3) The human blood-cerebrospinal fluid barrier model of the present invention can be directly taken out of the chip and subsequently stained and imaged without destroying its physiological structure, and has good compatibility with existing biological analysis and imaging instruments (such as high content systems) used in the field of drug development;

[0028] (4) The technical methods involved are simple to operate and highly reproducible, and can provide useful references for the construction of physiological and pathological models of tissue engineering. The present invention shows great development potential and application prospects in the construction of heterogeneous tissue / organ models, and is expected to be used in in vitro drug screening and disease formation mechanism research in the future, bringing hope to human research and treatment of nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A flow chart and a physical diagram of the construction of a human blood-cerebrospinal fluid barrier model provided in an embodiment of the present invention;

[0031] Figure 2 The cultivation and characterization of the human blood-cerebrospinal fluid barrier model provided in the embodiment of the present invention; wherein Figure 2 A is the barrier model cultivation flow chart; Figure 2 B is the fluid distribution inside the chip; Figure 2 C is the bright field image of the barrier model; Figure 2 D is the immunofluorescence identification result of cell populations above and below the barrier;

[0032] Figure 3 The present invention provides a pathological evaluation and characterization result of a human blood-cerebrospinal fluid barrier model; wherein, Figure 3 A is the flow rate of the two channels, Figure 3 B is the shear force condition of two channels;

[0033] Figure 4 The immunofluorescence results in Experiment 2; Figure 4 A is the expression of ZO-1 after chip construction. Figure 4B shows the changes of ZO-1 on the chip after treatment with the inflammatory factor TNF-α. Figure 4 C is the change in the chip's permeability to standard IgG before and after treatment with inflammatory factors; Figure 4 D is the change in the chip's permeability to the standard dextran before and after treatment with inflammatory factors. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0035] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0036] In addition, in the description of the present application, "multiple" and "several" mean two or more, unless otherwise clearly and specifically defined.

[0037] The overall idea of ​​the technical solution of this application is as follows:

[0038] According to a typical embodiment of the present invention, a method for preparing a human blood-cerebrospinal fluid barrier model is provided, the method comprising:

[0039] Step S1, preparing a microfluidic chip by a reverse molding method using a biocompatible material, wherein the microfluidic chip contains an upper channel and a lower channel that are adjacently arranged;

[0040] The biocompatible material includes polyethylene terephthalate (PDMS) prepolymer; or at least one of type I collagen, matrigel, GelMA, sodium alginate, fibrinogen, HAMA, HA-CA, silk fibroin, chitosan and gelatin.

[0041] The step S1 specifically includes:

[0042] Step S101, obtaining a male mold having an upper channel and a lower channel arranged adjacent to each other;

[0043] The length, width and height of the upper channel and the lower channel are 9000-11000 μm, 800-1200 μm and 20-200 μm respectively.

[0044] If the channel height is less than 20 μm, the internal hydraulic impedance is too large, the loading process is difficult, and the barrier structure is easily destroyed during the loading process; if the height is greater than 200 μm, there are disadvantages such as excessive loss of loaded cells;

[0045] Step S102, pouring the biocompatible material onto the positive mold, vacuum drying and curing, and then peeling off to obtain a negative membrane;

[0046] As a specific implementation method, a vacuum dryer is used to ensure that there is no air between the PDMS and the mold, and the PDMS is placed in an oven at 80° C. for curing for 1 hour; the cured PDMS is peeled off from the positive mold to obtain a negative film.

[0047] Step S103, cutting the negative film into a shape and size suitable for subsequent assembly to obtain the microfluidic chip containing the upper channel and the lower channel.

[0048] As a specific implementation, if it is combined with a well plate system, the negative membrane is cut into a size suitable for the well plate system.

[0049] Step S2, disposing a porous film between the upper channel and the lower channel of the microfluidic chip, and fixing the microfluidic chip with a clamp to obtain a culture device;

[0050] In the step S2,

[0051] The material of the porous film is polyethylene terephthalate (PET), and the porosity of the porous film is set to 1×10 5 ~1×10 7 Pieces / cm 2 .

[0052] If the porosity of the porous film is less than 1×10 5 Pieces / cm 2 , the barrier function is limited by the porous film, which is inconsistent with the physiological condition; if the porosity of the porous film is greater than 1×10 7 Pieces / cm 2 , there are disadvantages such as difficulty in cell attachment; the porosity of the porous film of the present invention refers to the ratio of the volume of pores to the apparent volume of the film;

[0053] The culture device can be assembled as a whole, and can be physically assembled using a clamp or the like, or chemically assembled using a plasm or the like; it can be reversibly assembled or irreversibly assembled.

[0054] Step S3, placing the culture device in a matrix material so that the matrix material coats the periphery of the upper channel and the lower channel; injecting a human choroid plexus epithelial cell suspension into the upper channel, and injecting a human brain microvascular endothelial cell suspension into the lower channel, and after the cells are attached, applying fluid stimulation to the upper channel and the lower channel and culturing them to obtain a human blood-cerebrospinal fluid barrier model.

[0055] The matrix gel coating can be performed using a variety of gels for single or mixed coating, and the upper and lower channels can be coated simultaneously or separately. Only one channel can be modified, or the two channels can be modified differently to adapt to different research situations.

[0056] The upper channel is coated with Matrigel 80 μg / mL, and the lower channel is coated with Laminin (laminin) 50 μg / mL and Fibronectin (fibronectin) 80 mg / mL. The matrix material includes one of Matrigel, laminin and fibronectin.

[0057] The cell density of the human choroid plexus epithelial cell suspension and the human brain microvascular endothelial cell suspension is (5-10)×10 6 If the cell seeding density is low, it is difficult for the cells to completely adhere to the film interface to form a barrier. If the cell seeding density is high, cells will accumulate during the barrier formation process, affecting the observation imaging and barrier function.

[0058] In the fluid stimulation, the flow rate of the fluid is 10 to 200 μL / h.

[0059] If the continuous fluid stimulation flow rate is less than 10 μL / h, there will be insufficient stimulation and it will be difficult to promote the formation of the barrier structure; if the continuous fluid stimulation flow rate is greater than 200 μL / h, there will be excessive shear force and the barrier structure will be destroyed.

[0060] According to another typical embodiment of the present invention, a human blood-cerebrospinal fluid barrier model prepared by the method is provided.

[0061] The barrier model obtained by the present invention is a three-layer structure, which is closer to the actual physiological situation compared with the structure of the previous blood-cerebrospinal fluid barrier model; due to the stable unidirectional fluid stimulation, the epithelial part of the barrier model can be seen to form a directional cell arrangement, and the vascular endothelial part forms a tight connection.

[0062] In addition to the biological part, the human blood-cerebrospinal fluid barrier model can also be engineered as a whole with the microfluidic channel to realize the physiological function of the barrier structure. The upper and lower channel parts of its three-layer structure can be used for fluid perfusion and collection, and the effect of the fluid inside the entire model can be analyzed.

[0063] According to another typical embodiment of the present invention, there is provided the application of the human blood-cerebrospinal fluid barrier model in immunology, pharmacodynamics, and physiological and pathological analysis.

[0064] The human blood-cerebrospinal fluid barrier (BCSFB) model and its construction method and application. Through the reverse engineering construction strategy, the human blood-cerebrospinal fluid barrier is simplified to a sandwich structure of choroid plexus epithelial cells and endothelial cells with a perforated structure. Organ chip technology is used to construct epithelial and endothelial dual channels that are consistent with the actual human physiological conditions, and a porous film is used to separate the upper and lower channels. The model can form a complete human blood-cerebrospinal fluid barrier, which has a barrier function similar to human physiology. The technical method involved in the present invention is simple to operate and highly repeatable. It can be used for neurodrug development and preclinical neurotoxicity assessment, and has broad commercial application prospects.

[0065] A human blood-cerebrospinal fluid barrier model of the present application and its preparation method and application will be described in detail below with reference to the accompanying drawings.

[0066] Example 1: A human blood-cerebrospinal fluid barrier model and its preparation method

[0067] (1) The microfluidic chip was fabricated using the standard soft lithography method. First, a silicon master mold with a microfluidic structure was fabricated by soft lithography. Second, the upper and lower layers of the chip were fabricated using PDMS by the method of inverting the mold. Subsequently, the chip was thoroughly dusted with 3M tape, and a piece of porous polyethylene terephthalate (PET) film (film thickness 7 μm, pore diameter 0.4 μm, pore density 4×10 6 / cm 2 ), align the upper and lower channels, and fix the assembly with a fixture made of polymethyl methacrylate (PMMA). Finally, the assembled chip is fully disinfected for later use;

[0068] (2) The upper channel of the chip was coated with Matrigel 80 μg / mL, and the lower channel was coated with Laminin 50 μg / mL and Fibronectin 80 mg / mL, and placed in an incubator overnight. The channel was rinsed with PBS and culture medium before inoculating cells. 5 to 10 × 10 6 The BMECs suspension was gently injected into the lower channel with a 10 μL pipette tip at a density of 10 cells / mL, and the chip was quickly flipped over to place it in an inverted position in the incubator, so that the BMECs adhered to the top of the upper and lower channels of the PET membrane. After 1 hour, the chip was restored to the upright position, so that the unattached BMECs adhered to the other three sides of the channel to form a tubular structure. After 3 hours, 5 to 10 × 106 The CPECs suspension was gently injected into the upper channel with a 10 μL pipette tip and placed in an incubator overnight. The next day, the upper and lower channels were perfused and cultured.

[0069] (3) The model was placed in an incubator and a microinjection pump was used to perform long-term stable fluid stimulation on the two channels of the chip at a flow rate of 100 μL / h.

[0070] Example 2

[0071] In the embodiment of the present invention, the chip channel length is increased by 20000 μm, and the other structures and steps are the same as those in Embodiment 1.

[0072] Example 3

[0073] In the embodiment of the present invention, the assembly method of the culture device is changed to chemical modification bonding, and the other structures and steps are the same as those in embodiment 1.

[0074] Comparative Example 1

[0075] In this comparative example, the continuous perfusion rate was changed to 8 μL / h, and the other structures and steps were the same as those in Example 1.

[0076] Comparative Example 2

[0077] In this comparative example, the continuous perfusion rate was changed to 210 μL / h, and the other structures and steps were the same as those in Example 1.

[0078] Comparative Example 3

[0079] In this comparative example, the cell seeding density was changed to 1×10 6 cells / mL, and other structures and steps were the same as those in Example 1.

[0080] Comparative Example 4

[0081] In this comparative example, the cell seeding density was changed to 100×10 6 cells / mL, and other structures and steps were the same as those in Example 1.

[0082] Comparative Example 5

[0083] In this comparative example, no matrix material is used to coat the chip, and other structures and steps are the same as those in Example 1.

[0084] Comparative Example 6

[0085] In this comparative example, the porous film is changed to have a porosity of 0, and the other structures and steps are the same as those in Example 1.

[0086] Comparative Example 7

[0087] In this comparative example, the porous film is changed to have a porosity of 100, and the other structures and steps are the same as those in Example 1.

[0088] Experimental Example 1

[0089] The model building results of the above-mentioned Examples 1-3 and Comparative Examples 1-7 were statistically analyzed, and the barrier structures formed were observed under bright field, as shown in Table 1;

[0090] Table 1

[0091] Group Seeding density Porous Film Properties Perfusion rate Channel coating Remark Example 1 <![CDATA[10×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Encapsulation Barrier structure Example 2 <![CDATA[20×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Encapsulation Barrier structure Example 3 <![CDATA[10×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Encapsulation Barrier structure Comparative Example 1 <![CDATA[10×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 10μL / h Encapsulation No barrier structure Comparative Example 2 <![CDATA[10×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 200μL / h Encapsulation No barrier structure Comparative Example 3 <![CDATA[1×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Encapsulation No barrier structure Comparative Example 4 <![CDATA[100×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Encapsulation Poor structural morphology Comparative Example 5 <![CDATA[10×10 6 cells / ml]]> <![CDATA[4×10 6 / cm 2 ]]> 100μL / h Not covered Poor structural morphology Comparative Example 6 <![CDATA[10×10 6 cells / ml]]> 0 100μL / h Encapsulation No barrier structure Comparative Example 7 <![CDATA[10×10 6 cells / ml]]> / 100μL / h Encapsulation No barrier structure

[0092] From the data in Table 1, we can see that:

[0093] In Comparative Example 1, the flow rate of the fluid was 8 μL / h, which was less than the range of 10 to 200 μL / h in the embodiment of the present invention, and a barrier structure could not be formed. Due to insufficient fluid stimulation, the cell state in the model was similar to that of the traditional two-dimensional culture.

[0094] In Comparative Example 2, the flow rate of the fluid is 220 μL / h, which is greater than the range of 10 to 200 μL / h in the embodiment of the present invention, and a barrier structure cannot be formed. The perfusion rate is high, and the cells in the channel are affected by the shear force, especially the cells near the channel wall are flushed out of the flow channel with the fluid;

[0095] In comparative example 3, the cell seeding density was changed to 1×10 6 cells / mL, less than (5-10)×10 6 cells / mL range, if the cell seeding density is low, it is difficult for the cells to completely adhere to the film interface to form a barrier;

[0096] In Comparative Example 4, the cell seeding density was changed to 1×10 6 cells / mL, less than (5-10)×10 6 cells / mL range. If the cell seeding density is high, cell accumulation will occur during the barrier formation process, affecting the observation imaging and barrier function.

[0097] In comparative example 5, the chip was not coated with a matrix material, and it can be seen that the barrier formation effect under bright field is quite different from the result after coating;

[0098] In Comparative Example 6, the porous film was changed to a porosity of 0, and the barrier function was limited by the porous film, which was inconsistent with the physiological condition;

[0099] In Comparative Example 7, the porous film was changed to a porosity of 100%, which resulted in disadvantages such as difficulty in cell attachment;

[0100] In Examples 1 to 3 of the present invention, the formation of barrier structures can be seen in bright field observation. Compared with traditional animal models and two-dimensional cell culture models, the present invention provides a human blood-cerebrospinal fluid barrier model with high biomimetic properties, high uniformity, and ease of imaging, providing an innovative research tool for related research.

[0101] Experimental Example 2: Characterization of the Construction of Human Blood-Cerebral Spinal Fluid Model

[0102] 1. The process of constructing an engineered blood-cerebrospinal fluid barrier model is as follows Figure 1 As shown, choroid plexus epithelial cells and brain microvascular endothelial cells are seeded in the upper and lower layers of the chip channels, respectively, to form a physiologically similar structure.

[0103] 2. Example 1 Characterization of blood-cerebrospinal fluid barrier model construction: The barrier formation after fluid stimulation was compared with that after static culture, and the relevant markers ZO-1 and Cld-5 were detected. The results are as follows Figure 2 , the finite element simulation was used to determine the flow field changes inside the chip. The results are as follows Figure 3 . It is shown that the embodiment of the present invention successfully constructed a human blood-cerebrospinal fluid barrier model.

[0104] 3. The human blood-cerebrospinal fluid barrier model constructed in Example 1 was treated with TNF-α, and the barrier function was detected. The immunofluorescence results showed that the barrier structure was destroyed and the barrier function was significantly lost compared with the control group. The results are as follows: Figure 4 As shown, it is shown that the present invention can be used for clinical and pharmacological research.

[0105] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0106] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0107] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a human blood-cerebrospinal fluid barrier model, characterized in that: The method comprises: The biocompatible material is prepared by a reverse molding method to obtain a microfluidic chip, wherein the microfluidic chip contains an upper channel and a lower channel which are adjacent to each other; the length, width and height of the upper channel and the lower channel are 9000-11000 μm, 800-1200 μm and 20-200 μm respectively; A porous film is arranged between the upper channel and the lower channel of the microfluidic chip, and the microfluidic chip is fixed by a clamp to obtain a culture device; the material of the porous film is polyethylene terephthalate, and the porosity of the porous film is set to 1×10 5 ~1×10 7 Pieces / cm 2 ; The culture device is placed in a matrix material so that the matrix material coats the periphery of the upper channel and the lower channel; a human choroid plexus epithelial cell suspension is injected into the upper channel, and a human brain microvascular endothelial cell suspension is injected into the lower channel. After the cells are attached, fluid stimulation is applied to the upper channel and the lower channel and culture is performed to obtain a human blood-cerebrospinal fluid barrier model; the matrix material is one of matrix glue Matrigel, laminin and fibronectin; the cell density of the human choroid plexus epithelial cell suspension and the human brain microvascular endothelial cell suspension is (5-10)×10 6 cells / mL; in the fluid stimulation, the flow rate of the fluid is 10-200 μL / h, and the biocompatible material is at least one of polyethylene terephthalate PDMS prepolymer, type I collagen, matrigel, GelMA, sodium alginate, fibrinogen, HAMA, HA-CA, silk fibroin, chitosan and gelatin.

2. The method for preparing the human blood-cerebrospinal fluid barrier model according to claim 1, characterized in that: The method of preparing the microfluidic chip by using the biocompatible material through the inverted molding method comprises: Obtaining a male mold having an upper channel and a lower channel disposed adjacently; Pouring a biocompatible material onto the male mold, vacuum drying and curing, and then peeling off to obtain a female membrane; The negative film is cut into a shape and size suitable for subsequent assembly to obtain the microfluidic chip containing the upper channel and the lower channel.

3. A human blood-cerebrospinal fluid barrier model prepared by the method according to any one of claims 1-2.

4. Use of the human blood-cerebrospinal fluid barrier model according to claim 3 in drug screening.

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