CN117683701A - Neurovascular unit model with blood brain barrier function, construction method and application thereof - Google Patents
Neurovascular unit model with blood brain barrier function, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a neurovascular unit model with a blood brain barrier function, and a construction method and application thereof. The neurovascular unit model comprises a first channel, a second channel, a brain region microenvironment layer, a first blood brain barrier layer and a second blood brain barrier layer; the first channel and the second channel are positioned at two sides of the brain region microenvironment layer, and the first blood brain barrier layer is positioned between the first channel and the brain region microenvironment layer; the second blood brain barrier layer is located between the second channel and the brain region microenvironment layer. The invention uses cell printing technology and organ chip technology to make neurovascular unit model with parallel perfusion channel and functional blood brain barrier. The blood brain barrier of the model can detect transmembrane impedance, permeability, barrier function gene expression and the like. The parallel multichannel design of the model simulates peripheral blood perfusion and intra-brain blood perfusion, and can be used for screening chemotherapeutics crossing blood brain barriers.
Description
Technical Field
The invention relates to the technical field of neurovascular unit cell culture, in particular to a neurovascular unit model with a blood brain barrier function, and a construction method and application thereof.
Background
The research of the neurovascular unit in-vitro model has important significance in the fields of neuropathology, tumor biology, drug development and the like. The in vitro model may help researchers to learn more about the biological properties of neurovascular units, drug sensitivity, and the effectiveness of therapeutic strategies. The following are some current trends and status in the in vitro model studies of neurovascular unit cells, particularly central nervous tumors:
three-dimensional cell culture model: traditional two-dimensional cell culture models are difficult to accurately simulate the complex microenvironment of tumors. Thus, researchers are increasingly inclined to use three-dimensional cell culture models such as tumor spheres (tumor spheres) or tumor organ-like cultures (tumor organs). These models are more able to mimic the growth and interaction of tumor cells in vivo.
Gene editing technology application: CRISPR-Cas9 isogenic editing techniques enable researchers to construct and edit tumor-associated genetic variations in vitro models to better understand the impact of these variations on tumor growth and therapeutic sensitivity.
Immune system simulation: in recent years, researchers have begun to mimic the effects of the immune system in vitro models to better study tumor immune escape mechanisms and develop immunotherapeutic strategies.
Drug screening and personalized medicine: glioma in vitro models are used for drug screening to find new therapeutic drugs or to verify the efficacy of existing drugs. In addition, some studies have attempted to combine in vitro models with patient personalized genetic information to achieve personalized medicine and therapy.
Transcriptomics and proteomics studies: using high throughput transcriptomics and proteomics techniques, researchers can explore in depth the gene expression and protein composition of tumor cells in vitro models, revealing more information about tumor biology.
The common three-dimensional neurovascular unit tumor cell culture model comprises the following components:
tumor spheres (Tumor spheres): tumor spheroids are a three-dimensional, spherical structure formed by the self-organization of tumor cells. This model more mimics the dense growth of tumor cells in vivo and the interactions between cells. Tumor spheroids can be self-assembled into spheres by suspending tumor cells in a culture medium. The tumor sphere can be used for researching tumor growth, invasion, drug resistance and the like.
Tumor organoid culture (Tumor organs): tumor organoid culture is a more advanced three-dimensional model that mimics the structure and function of the entire tissue. By culturing tumor tissue sections or individual cells under specific culture conditions, they can be self-assembled into organ-like structures with multicellular types. Tumor organ-like culture can more accurately mimic the diversity and complexity of tumor tissue.
Bioprinting technology (Bioprinting): the bioprinting technology is a method of printing cells, biological materials and growth factors in a three-dimensional structure. The method can construct more accurate cell arrangement and structure, and is helpful for studying cell-cell interaction, drug reaction and the like.
Matrix embedding model: in this model, tumor cells are embedded in a matrix-like scaffold, mimicking cell growth in a complex extracellular matrix. The model can better simulate the microenvironment of tumor cells.
Microfluidic chip (Microfluidic Chips): the microfluidic chip may create a tiny cell culture environment that mimics the microenvironment conditions in vivo. The model can be used for researching migration, invasion, angiogenesis and the like of tumor cells.
The three-dimensional cell culture models have important values in the aspects of researching tumor biology, drug screening, personalized medicine and the like. However, each model has its advantages and limitations, such as the lack of a functional blood brain barrier for some models, and researchers need to select appropriate models based on the study problem, in combination with other technical means, to better mimic the complexity of the tumor.
CN114480122a discloses a microfluidic chip for establishing a co-culture model of a blood brain barrier and glioma of a neurovascular unit. The chip consists of a vascular channel, a brain parenchymal channel, a three-dimensional tumor channel and a medium channel. The model integrates the three-dimensional culture microenvironment of the blood brain barrier and the neurovascular glioma, but has the following disadvantages:
(1) The model channels are generally rectangular interfaces, and this geometry results in non-uniform flow and shear stress distribution, which in turn results in differences in morphology and behavior of endothelial cells along the channels;
(2) The barrier structure of the model is a hydrogel interface at the junction of a vascular channel and a brain parenchymal channel, and the batch quality control of the interface is poor;
(3) The barrier structure of this model makes it difficult to measure barrier tightness, such as trans-membrane resistance TEER;
(4) The first culture chamber of the model is the co-culture (two-dimensional environment) of two cells, the second culture chamber is the fibrinogen and thrombin mixed solution (three-dimensional environment) of astrocytes, the third culture chamber is the matrigel mixed solution (three-dimensional environment) of neurovascular unit glioma cells, and the contact between the cells of the second culture chamber and the third culture chamber is limited (the glioma cells and the brain parenchymal cells in the brain and the blood vessels are in intricate contact).
(5) The barrier of the model is only one. The brain in the human body has a barrier to enter the brain and a barrier to leave the brain, and the bionic effect is poor.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a neurovascular unit model with a blood brain barrier function, and a construction method and application thereof. The method solves the problems that the bionic effect of a blood brain barrier model in the prior art is poor, and the nerve vascular unit cell complexity environment cannot be simulated better.
In a first aspect of the present invention, there is provided a neurovascular unit model having a blood brain barrier function, the neurovascular unit model comprising a first channel, a second channel, a brain region microenvironment layer, a first blood brain barrier layer, and a second blood brain barrier layer;
the first channel and the second channel are respectively positioned at two sides of the brain region microenvironment layer, and the first blood brain barrier layer is positioned between the first channel and the brain region microenvironment layer; the second blood brain barrier layer is located between the second channel and the brain region microenvironment layer.
In one embodiment of the present invention, the first blood brain barrier layer comprises a support membrane, wherein one side of the support membrane is planted with vascular endothelial cells, and the opposite side is planted with perivascular cells and astrocyte derivatives;
preferably, the support film is a porous structure, more preferably a PET porous film;
preferably, the preparation method of the brain microvascular pericyte and astrocyte derivatives comprises the following steps: preparing thermosensitive hydrogel containing brain microvascular pericytes and astrocytes, and hydrolyzing to form brain microvascular pericytes and astrocytes derivatives.
In one embodiment of the invention, the thickness of the temperature sensitive hydrogel containing brain microvascular pericytes and astrocytes is 0.2mm but not limited to 0.2mm.
In a specific embodiment of the present invention, the brain region microenvironment layer is printed from a cellular ink material, and the cellular ink material includes a cellular material and a hydrogel material;
the hydrogel material comprises one or more of methacryloylated gel, fibrinogen, gelatin, laminin, glutamine transaminase and thrombin;
the cell material comprises cerebrovascular endothelial cells, brain microvascular peripheral cells, brain astrocytes, brain microglial cells, brain oligodendrocyte cells and neural stem cells.
In one embodiment of the invention, the second blood brain barrier layer has the same structure as the first blood brain barrier layer.
In a specific embodiment of the present invention, the first channel is provided with a first channel port, the second channel is provided with a second channel port, and the first channel and the second channel are both used for peripheral blood perfusion; the brain region microenvironment layer is provided with a third channel, and the third channel is used for blood perfusion in the brain.
In a second aspect of the present invention, there is provided a method for preparing a neurovascular unit model having a blood brain barrier function, comprising the steps of:
s1, constructing a first blood brain barrier layer and a second blood brain barrier layer: planting brain microvascular endothelial cells on one side of the support membrane, and planting brain microvascular pericytes and astrocyte derivatives on the opposite side;
s2, constructing a brain region microenvironment layer: the cell ink material is used as a raw material and is prepared by cell printing and pin inserting technology;
s3 assembly of blood brain barrier structure: the first blood brain barrier layer and the second blood brain barrier layer are respectively arranged at two sides of the brain region microenvironment layer;
s4, perfusion culture is carried out, and nutrition supply of a neurovascular unit microenvironment is simulated;
preferably, further comprising S31 constructing and assembling the first channel and the second channel between steps S3 and S4;
more optionally, the first channel and the second channel are formed by 3D printing on silica gel;
more preferably, the first channel is mounted on a side of the first blood brain barrier layer remote from the brain region microenvironment layer and the second channel is mounted on a side of the second blood brain barrier layer remote from the brain region microenvironment layer.
In a specific embodiment of the present invention, in step S1, the planting method includes sedimentation and cell printing.
In the present invention, the planting method includes, but is not limited to, cell sedimentation based on the action of gravity, and other treatment methods based on a combination of one or more of the cell assembly techniques of inkjet cell printing, which can achieve the same effect, are also included. Preferably, a cell sedimentation technique based on gravity is used.
In step S2, the cell printing and pin method preparation includes: printing a layer of cell ink material, inserting a steel needle, continuing printing the cell ink material until the cell ink material is full of a cavity formed by the whole outer frame, and performing ultraviolet irradiation; adding thrombin, culturing to crosslink and solidify the cell ink material; taking out the steel needle to form a third channel;
preferably the printing temperature is 21-25 ℃; preferably, the number of third channels is at least 2.
In step S3, the method of assembling includes: sequentially installing a first channel, a first packaging layer, a first blood brain barrier layer, a brain region microenvironment layer, a second blood brain barrier layer, a second packaging layer, a second channel and a third packaging layer on the surface of the substrate;
preferably the substrate is a glass sheet; the first packaging layer and the second packaging layer are stainless steel sheets; the third packaging layer is a PMMA plate;
in the step S4, the flow rate of perfusion culture is 240 mu L/min, and the flow rate is 1mm/S;
preferably, the perfusion culture is fluid driven by a peristaltic pump.
In a third aspect, the invention provides an application of the neurovascular unit model with the blood-brain barrier function in screening brain tumor drugs and detecting transmembrane impedance, permeability and barrier function gene expression.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses cell printing technology and organ chip technology to make neurovascular unit model with parallel perfusion channel and functional blood brain barrier. The blood brain barrier of the model can detect transmembrane impedance, permeability, barrier function gene expression and the like. The parallel multichannel design of the model simulates peripheral blood perfusion and intra-brain blood perfusion, and can be used for screening chemotherapeutics crossing blood brain barriers. The invention provides a new method for constructing the personalized neurovascular unit model.
Drawings
FIG. 1 is a schematic diagram of a neurovascular unit model with blood brain barrier function according to the present invention.
FIG. 2 is a schematic diagram of a neurovascular unit model construction process with blood brain barrier function according to the present invention.
FIG. 3 is a schematic diagram of the construction of an endothelial barrier (blood brain barrier) according to the present invention.
FIG. 4 is a schematic representation of cell planting according to the present invention.
Fig. 5 is a schematic diagram of a printing lower layer channel B according to the present invention.
Fig. 6 is a schematic view of the present invention encapsulating the underlying barrier and channel B.
Fig. 7 is a schematic diagram of the present invention for mounting an outline frame of a "brain region microenvironment structure".
Fig. 8 is a schematic diagram of a printed perfusable "brain region microenvironment architecture" according to the invention.
Fig. 9 is a schematic view of the invention for scraping excess material.
Fig. 10 is a schematic view of the structure of the upper barrier of the package of the present invention.
Fig. 11 is a schematic diagram of a printed upper layer channel a according to the present invention.
Fig. 12 is a schematic diagram of the packaging upper layer channel a and the mounting perfusion interface of the present invention.
FIG. 13 is a schematic illustration of perfusion culture of the present invention.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Technical terms:
the temperature sensitive hydrogel is a block copolymer of poly (N-isopropyl acrylamide) and polyethylene glycol, which is liquid at low temperature and coagulates at high temperature, and the change of the state is reversible with the temperature.
The "brain region microenvironment layer" has the same meaning as the "neurovascular unit brain region microenvironment" and "brain region microenvironment structure".
Example 1 construction of neurovascular unit model with blood brain Barrier function
The construction method is shown in fig. 2, the structure of the neurovascular unit model is shown in fig. 1, and the specific steps are as follows:
1. the blood brain barrier (including the first blood brain barrier layer and the second blood brain barrier layer) was constructed, and the construction flow thereof is shown in fig. 3.
1) Cell planting
Human brain microvascular endothelial cells hCMEC/D3 (EC) were planted on the A-side of the PET porous membrane by sedimentation method, cell density was 1×10 5 cells/cm 2 . The schematic diagram is shown in fig. 4.
2) Placing the mixture into an incubator for culturing for 6 hours;
3) Turning over and waiting for cell printing;
4) Cell printing
Printing cells on the B surface of the PET porous membrane, wherein the cells are human brain microvascular cells (PC) +human Astrocytes (AC); cell density pc=4×10 5 cells/cm 3 ，AC＝4×10 5 cells/cm 3 The method comprises the steps of carrying out a first treatment on the surface of the The planting method is that printing cells, uniformly mixing and printing a layer; the ink material is 7.5% Gelatin; the printing temperature is 21 ℃ of the spray head and 18 ℃ of the bottom plate; the needle is 25G;
5) Placing in a 37 ℃ incubator for 2 hours, changing gel into sol at the moment, settling cells, and aggregating near the PET porous membrane;
6) Adding culture medium, and continuing culturing.
2. Printing lower layer channel B (first channel)
The liquid channel B was printed on a 160 μm glass sheet as shown in fig. 5. The glass is used for facilitating photographing by a confocal microscope. The material used was SE1700 (a silica gel), a: b=10: 0.6; the printing temperature is normal temperature; the needle is 21G, and the needle is conical;
3. the lower barrier (blood brain barrier a or first blood brain barrier layer) is encapsulated and the flow is shown in fig. 6.
1) Mounting the structure printed in the step 2 in a stainless steel plate under aseptic conditions;
2) Mounting a stainless steel sheet A (a first packaging layer) containing a preset hole;
3) Cutting off the first blood brain barrier layer constructed in the step 1 by using a scalpel and scissors subjected to aseptic treatment, and mounting the first blood brain barrier layer on a neurovascular unit microenvironment device (a preset hole of a stainless steel sheet A);
4. an outline of the "brain region microenvironment structure" (brain region microenvironment layer) is installed as shown in fig. 7.
1) Inserting a stainless steel needle (23G) into the mounting hole of the stainless steel plate;
2) The outer frame stainless steel plate of the brain region micro-environment structure is arranged on the structure assembled in the step 3;
5. printing a perfusable "brain region microenvironment structure" (brain region microenvironment layer).
As shown in fig. 8, the specific steps are as follows:
1) Preparation of a photocurable hydrogel material GelMA+, preferably with a material composition of 5% GelMA (methacryloylated gel) +2.5mg/mL Fibrinogen (Fibrinogen) +5% Gelatin (Gelatin) +20. Mu.g/mL Laminin) +3U/mL transglutaminase (glutamine transaminase);
2) GelMA+ ink containing cells (preferably, the cellular components are cerebrovascular endothelial cells, brain microvascular pericytes, brain astrocytes, brain microglial cells, brain oligodendrocytes, neural stem cells) was loaded into a 3mL screw-port syringe (BD 302113), and then a autoclaved 25G stainless steel needle was mounted;
3) Mounting a 3mL screw port syringe onto the cell print head;
4) Printing a layer of cell ink material at the temperature of 25 ℃ of a spray head;
5) Pushing the stainless steel needle (23G) of step 4 in, and axially centering (inserting a 250 μm diameter silver needle into the inner tube of the stainless steel needle);
6) Printing a cell ink material at the temperature of 21 ℃ of a spray head to fill the area where the stainless steel needle is positioned; printing cell ink material at the temperature of 25 ℃ of the spray head to fill the whole 'neurovascular unit micro-environment structure' cavity; uv crosslinking (wavelength=405 nm), 50mW irradiation for 30s; spraying a culture medium containing 100U/mL thrombin on the ink material, and incubating for 30min in a 37 ℃ incubator to crosslink and solidify the material;
7) The stainless steel needle is gently pulled out (incompletely pulled out), the distance between the needles in the axial direction is 5mm, and a channel C/D (third channel) is formed
6. And (5) scraping off redundant materials. Excess material (a portion above the plane of the stainless steel outer frame) was scraped off using a sterile-treated scalpel, as shown in fig. 9.
7. The upper barrier structure (blood brain barrier B or second blood brain barrier layer) is encapsulated. As shown in fig. 10. The method comprises the following specific steps:
1) Cutting the barrier structure (second blood brain barrier layer) constructed in step 1 with a sterile scalpel, scissors,
2) Is arranged on a neurovascular unit microenvironment device (brain region microenvironment layer);
3) Mounting a stainless steel sheet B (second packaging layer) with a preset hole;
8. the upper layer channel a (second channel) is printed. The channel a was printed on PMMA (third encapsulation layer) as shown in fig. 11.
The material used is SE1700 silica gel, A: b=10: 0.6; the printing temperature is normal temperature; the needle was 21G, a conical needle was used.
9. And (5) packaging the upper layer channel A (the second channel) and installing a perfusion interface. As shown in fig. 12, the specific steps are as follows:
1) 1.6mm (1/16 inch) medical grade PP (polypropylene) luer female connector, PMMA plate is sterilized by immersing in 70% alcohol, and sterilized by ultraviolet irradiation;
2) In a sterile environment, using glue to bond the luer female connector to the PMMA plate, and then performing ultraviolet and ozone sterilization treatment;
3) Fixing a PMMA plate (third encapsulation layer) onto the device using M2 screws;
4) The cone end of the luer female connector is connected with a sterile silica gel hose with the diameter of 0.5 mm or 0.8 mm;
5) The luer male connector is connected with a stainless steel needle head, and the pagoda end is connected with a sterile silica gel hose with the diameter of 0.5 mm or 0.8 mm;
10. and (5) perfusion culture. As shown in fig. 13, the specific steps are as follows:
1) A 100mL glass bottle (Shu cattle, high boron silicon material) is selected as a liquid storage cavity for perfusion culture;
2) A sterile silicone hose of 0.5 x 0.8mm (inner diameter x wall thickness) connects the reservoir chamber and the neurovascular unit microenvironment device;
3) The flow rate of about 240 mu L/min and the flow rate of about 1mm/s in the A/B channel of the neurovascular unit microenvironment can be realized by using a BT100-2J driver and a DG10 pump head with the rotating speed of 16 rpm.
4) The perfusion culture was continued for 10 days, and half (20 mL) of the medium was renewed on day 5.
Example 2 application of neurovascular unit model with blood brain Barrier function
The chemotherapeutic drugs (5-Fluorouracil, vorinostat, paclitaxel and cisplatin) with the same concentration are perfused in the A/B channel, and the drug concentration is extracted from the culture medium of the C/D channel, so that the in-vivo drug diffusion simulation capability and the therapeutic capability of the chemotherapeutic drugs crossing the blood brain barrier can be obtained; for example, this model was used to simulate blood brain barrier permeability experiments with post-intravenous pentafluoroethane (50 μg/mL, peak of blood drug concentration following general clinical injection) in vivo, and drug concentrations in brain (C/D) channels were found to reach steady values after 8 hours of perfusion. The model was used to simulate blood brain barrier permeability experiments of vorinostat (peak of blood concentration after intravenous administration of 0.24 μg/mL, general clinical injection) in vivo, and it was found that the drug concentration of brain region (C/D) channels reached a stable value after 16 hours of perfusion.
Similarly, the medium of the same concentration of the chemotherapeutic drug and the isotope-labeled glucose is perfused in the A/B channel, and after 12 hours and 24 hours of perfusion, cells are extracted for researching the cell center metabolic flow, and then the mechanism of the chemotherapeutic drug interfering the cell metabolic flow is revealed. Or the antibacterial drugs with the same concentration are irrigated in the A/B channel, and the culture medium is extracted from the culture medium of the C/D channel, so that the in-vivo antibacterial drug transmembrane therapeutic capability and permeability can be obtained.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. A neurovascular unit model with blood brain barrier function, characterized by: the neurovascular unit model comprises a first channel, a second channel, a brain region microenvironment layer, a first blood brain barrier layer and a second blood brain barrier layer;
the first channel and the second channel are respectively positioned at two sides of the brain region microenvironment layer, and the first blood brain barrier layer is positioned between the first channel and the brain region microenvironment layer; the second blood brain barrier layer is located between the second channel and the brain region microenvironment layer.
2. A neurovascular unit model with blood brain barrier function as in claim 1, wherein: the first blood brain barrier layer comprises a support membrane, wherein one side of the support membrane is planted with vascular endothelial cells, and the opposite side is planted with perivascular cells and astrocyte derivatives;
preferably, the support film is a porous structure, more preferably a PET porous film;
preferably, the preparation method of the brain microvascular pericyte and astrocyte derivatives comprises the following steps: preparing thermosensitive hydrogel containing brain microvascular pericytes and astrocytes, and hydrolyzing to form brain microvascular pericytes and astrocytes derivatives.
3. A neurovascular unit model with blood brain barrier function as in claim 1, wherein: the brain region microenvironment layer is formed by printing a cell ink material, wherein the cell ink material comprises a cell material and a hydrogel material;
the hydrogel material comprises one or more of methacryloylated gel, fibrinogen, gelatin, laminin, glutamine transaminase and thrombin;
the cell material comprises cerebrovascular endothelial cells, brain microvascular peripheral cells, brain astrocytes, brain microglial cells, brain oligodendrocyte cells and neural stem cells.
4. A neurovascular unit model with blood brain barrier function as in claim 1, wherein: the second blood brain barrier layer has the same structure as the first blood brain barrier layer.
5. A neurovascular unit model with blood brain barrier function as in claim 1, wherein: the first channel is provided with a first channel opening, the second channel is provided with a second channel opening, and the first channel and the second channel are both used for peripheral blood perfusion; the brain region microenvironment layer is provided with a third channel, and the third channel is used for blood perfusion in the brain.
6. A method for preparing a neurovascular unit model with blood brain barrier function, which is characterized in that: the method comprises the following steps:
s1, constructing a first blood brain barrier layer and a second blood brain barrier layer: planting brain microvascular endothelial cells on one side of the support membrane, and planting brain microvascular pericytes and astrocyte derivatives on the opposite side;
s2, constructing a brain region microenvironment layer: the cell ink material is used as a raw material and is prepared by cell printing and pin inserting technology;
s3 assembly of blood brain barrier structure: the first blood brain barrier layer and the second blood brain barrier layer are respectively arranged at two sides of the brain region microenvironment layer;
s4, perfusion culture is carried out, and nutrition supply of a neurovascular unit microenvironment is simulated;
preferably, further comprising S31 constructing and assembling the first channel and the second channel between steps S3 and S4;
more optionally, the first channel and the second channel are formed by 3D printing on silica gel;
more preferably, the first channel is mounted on a side of the first blood brain barrier layer remote from the brain region microenvironment layer and the second channel is mounted on a side of the second blood brain barrier layer remote from the brain region microenvironment layer.
7. The method for preparing a neurovascular unit model with blood brain barrier function according to claim 6, wherein: in step S1, the planting method includes sedimentation method and cell printing method.
8. The method for preparing a neurovascular unit model with blood brain barrier function according to claim 6, wherein: in step S2, the cell printing and pin method preparation includes: printing a layer of cell ink material, inserting a steel needle, continuing printing the cell ink material until the cell ink material is full of a cavity formed by the whole outer frame, and performing ultraviolet irradiation; adding thrombin, culturing to crosslink and solidify the cell ink material; taking out the steel needle to form a third channel;
preferably the printing temperature is 21-25 ℃; preferably, the number of third channels is at least 2.
9. The method for preparing a neurovascular unit model with blood brain barrier function according to claim 6, wherein: in step S3, the method of assembling includes: sequentially installing a first channel, a first packaging layer, a first blood brain barrier layer, a brain region microenvironment layer, a second blood brain barrier layer, a second packaging layer, a second channel and a third packaging layer on the surface of the substrate;
preferably the substrate is a glass sheet; the first packaging layer and the second packaging layer are stainless steel sheets; the third packaging layer is a PMMA plate;
in the step S4, the flow rate of perfusion culture is 240 mu L/min, and the flow rate is 1mm/S;
preferably, the perfusion culture is fluid driven by a peristaltic pump.
10. Use of a neurovascular unit model with blood brain barrier function according to any one of claims 1-5 for screening brain tumor drugs and detecting transmembrane impedance, permeability and barrier function gene expression.
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