Microfluidic brain organ chip, construction method and application thereof

Abstract

The present application relates to the technical field of microfluidic brain organ chip, and specifically discloses a microfluidic brain organ chip, a construction method and application thereof; the chip body comprises an upper layer chip, a porous film and a lower layer chip; the upper layer chip is provided with a brain tissue culture unit and an artery unit; the lower layer chip is provided with a vein unit; the construction method comprises: chip packaging, amino silanization is performed to realize bonding; S4 brain organ chip construction, (1) BBB reconstruction; (2) brain microvessel endothelial cell and pericyte inoculation are performed. The present application can realize overall simulation of the cerebral structure, including the blood-brain barrier, the cerebrospinal fluid-brain barrier, the brain tissue and the simulation of the cerebral blood circulation anatomical physiological system after the blood-brain barrier through the brain tissue and collected to the cerebral vein. It can be applied to the research on the influence of blood-absorbed substances on the blood-brain barrier; can be used for the pathogenesis of brain diseases and the influence on the peripheral system; and can replace the use of experimental animals in the drug screening process.

Description

Technical Field

[0001] This invention relates to the field of microfluidic brain organ-on-a-chip technology, specifically to a microfluidic brain organ-on-a-chip, its construction method, and its applications. Background Technology

[0002] Molecular exchange between the brain and its periphery is restricted by specific cellular and biochemical mechanisms at several interfaces: the blood-brain barrier (BBB), the CSF-brain barrier, and the blood-CSF barrier. Furthermore, metabolic products from brain tissue and the choroid plexus of the ventricles eventually drain into the cerebral veins. Cerebrospinal fluid is constantly produced and reabsorbed back into the veins, functioning as lymph in the central nervous system. All of this constitutes the overall system of material exchange between the brain and its periphery. Disruption of these barriers can lead to various diseases, such as stroke, multiple sclerosis, or neuropsychiatric disorders. This disruption can be triggered by a variety of factors, including hypoxia, inflammation or gut microbiota dysbiosis, chronic stress, and stress factors such as trauma.

[0003] The brain's peripheral blood vessel (BBB) ​​is a highly selective semi-permeable barrier that ensures the separation of circulating blood from the brain and central nervous system (CNS). This crucial physiological barrier enables brain cells to perform their physiological functions by modulating the delicate brain microenvironment and maintaining brain homeostasis within the CNS. The BBB is a dynamic and complex structure capable of rapid modulation, responsible for controlling the transport of molecules and ions. The BBB acts as an interface between the peripheral blood circulation and the CNS; transport proteins and tight junctions (TJs) regulate the passage of nutrients while protecting the brain from toxins and pathogens. Therefore, the expression and function of tight junction proteins are often used as indicators of BBB integrity. Brain microvascular endothelial cells are the core anatomical component of the BBB, arranged within the vessels of the CNS vascular system. Pericytes, supporting glial cells (astrocytes and microglia), the basement membrane, and the extracellular matrix (ECM) also play vital roles in maintaining BBB integrity. BBB dysfunction is associated with increased BBB permeability and inflow / outflow dysregulation, leading to the infiltration of toxins and immune cells into the CNS. This damage is associated with a variety of neurological disorders, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis.

[0004] The CSF-brain barrier (CSFBB) is composed of a single layer of ependymal cells arranged around the periphery of the ventricles and spinal canal. The ependymal layer consists of multiciliated cubic to columnar epithelial cells anchored together near their apical surfaces by cell junction proteins, forming the CSFBB. The primary function of the ependymal layer is to facilitate the overall flow of CSF and the movement of debris through highly coordinated tibial pulsation. It also acts as a protective barrier, protecting the adjacent subependymal gray matter and periventricular white matter from CSF-containing toxins. The ependymal CSFBB interface is considered only a partial barrier because it exhibits selective permeability to water, CSF proteins, and exogenous tracers through its gap junctions.

[0005] Traditional Transwell-based in vitro cell models are readily available but cannot replicate the physiological complexity of brain-brain block (BBB) ​​on a three-dimensional (3D) scale. Human brain tissue can be modeled in vitro using organoids, in which human induced pluripotent stem cells (iPSCs) or embryonic cells differentiate into neural cell types that mimic brain physiology in a 3D structure. Brain organoids have become a common tool for studying brain development and disease. However, when using organoids for disease modeling, the diffusion of nutrients and oxygen is limited, and delivery to the internal regions of the organoid is difficult, limiting the potential for in vitro models to be adapted to more controlled microenvironments.

[0006] Over the past few decades, various model organisms, including *Caenorhabditis elegans*, fruit flies, rodents, and non-human primates, have been widely used to study the central nervous system. While research on model organisms has deepened our understanding of brain function and diseases and provided important insights for the diagnosis and treatment of human neurological disorders, animal models cannot accurately represent some key features of the human brain, including neural developmental trajectories, brain structure, and certain human-specific neural circuits and cell types.

[0007] Microfluidic organ-on-a-chip (OIA) is a biomimetic system that culturees cells, tissues, or organs on a microfluidic device to mimic the physiology and major functions of human organs. Therefore, the advent of brain OIA can effectively simulate the functional units of the human brain. Due to the integration of advanced microfluidic technology, multi-chambered perfusion devices can be designed to co-culture multiple cell types, while incorporating flow systems that simulate blood circulation. Therefore, compared to traditional 2D in vitro models, brain OIA can better simulate the highly dynamic microenvironment of the brain. Furthermore, compared to animal models, brain OIA can more easily be based on brain physiology and anatomy, possessing a well-defined and highly controllable microenvironment, thus allowing for the isolation and identification of the effects of specific factors.

[0008] However, existing brain organ-on-a-chip models only simulate a single barrier, such as the blood-brain barrier structure (a single blood vessel layer-brain tissue structure). Moreover, many existing single-barrier simulations are developed based on the needs of a certain disease research and are not complete compared with the actual human brain anatomical barriers. Furthermore, due to the lack of a complete cerebral blood circulation system, especially the lack of interaction between multiple brain barrier systems, they are even less able to fully reproduce the complex physiological functions of the human brain. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a microfluidic brain organ-on-a-chip, its construction method, and its application, enabling a complete simulation of the blood-brain barrier, the cerebrospinal fluid-brain barrier, and the cerebral blood circulation system that flows through the brain and into the veins after passing through the blood-brain barrier.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A microfluidic brain organ-on-a-chip includes an upper chip, a porous membrane, and a lower chip. The upper chip has a venous channel inlet port, a venous channel outlet port, a brain tissue culture unit, an arterial unit, a ventricle unit, a brain tissue culture unit inlet, and a brain tissue culture unit outlet. The lower chip has a venous channel inlet shallow groove, a venous channel outlet shallow groove, a venous unit, a venous inlet connecting channel, and a venous outlet connecting channel. The venous unit is located below the brain tissue culture unit, and the porous membrane is located between the venous unit and the brain tissue culture unit. The position of the venous channel inlet port corresponds vertically to the position of the venous channel inlet shallow groove. The position of the venous channel outlet port corresponds vertically to the position of the venous channel outlet shallow groove. The venous channel inlet shallow groove is connected to the venous unit through the venous inlet connecting channel, and the venous channel outlet shallow groove is connected to the venous unit through the venous outlet connecting channel. The arterial unit is located above the brain tissue culture unit, and the ventricle unit is located to the right of the brain tissue culture unit.

[0012] Optionally, the porous film is made of PC or PET material.

[0013] Optionally, the material used for the chip body may be PDMS, PMMA, PC, or COC.

[0014] Optionally, the lower layer of the porous membrane is seeded with brain microvascular endothelial cells and pericytes.

[0015] Optionally, the brain tissue culture unit is filled with a porous microfluidic medium, in which neurons, astrocytes, and microglia are embedded.

[0016] Optionally, the porous microfluidic medium is made of basement membrane extract hydrogel, fibrinogen, gelatin, laminin, or collagen; the arterial unit is inoculated with brain microvascular endothelial cells and pericytes.

[0017] Optionally, the fluid between the ventricle unit and the brain tissue culture unit is configured for bidirectional interaction, and the fluid does not overflow into its artery, i.e., the fluid in the artery is configured for unidirectional flow.

[0018] A microfluidic brain organ-on-a-chip fabrication method, wherein a microfluidic brain organ-on-a-chip is fabricated as described above, and the brain organ-on-a-chip is made using PDMS or thermoplastic plastic;

[0019] The PDMS chip manufacturing process includes:

[0020] S1 Pre-processing

[0021] Mix the PDMS prepolymer and curing agent at a mass ratio of 10:1 and stir until fully mixed; remove air bubbles from the mixed PDMS prepolymer using a vacuum method; pour the defoamed PDMS prepolymer onto a mold, ensuring that there are no air bubbles present; then transfer it to an oven for heating and curing.

[0022] S2 drilling

[0023] Drill holes at the corresponding positions and vertical angles using a punch of the appropriate size on the upper-layer chip of the PDMS.

[0024] S3 chip package

[0025] Since PDMS and PET film cannot be directly bonded by oxygen plasma treatment, bonding is achieved by aminosilanizing the PET film.

[0026] The aminosilanization process is as follows:

[0027] (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water, preheat it to 80°C, clean the PDMS chip with isopropanol and dry it with nitrogen or compressed air, and clean both sides of the PDMS chip to remove any obvious debris.

[0028] (2) Treat the PET film with oxygen plasma for 30 seconds and then transfer it to a preheated 5% APTES solution for 30 minutes;

[0029] (3) Wash the PET film with deionized water and dry it at room temperature;

[0030] (4) Simultaneously treat the aminosilanized PET film and the PDMS chip with oxygen plasma for 30 seconds and then bond them together;

[0031] (5) Place the assembled PDMS chip into an oven and heat it at 60°C for 24 hours;

[0032] S4 brain organ-on-a-chip construction

[0033] The assembled PDMS chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use.

[0034] (1) To construct neural tissue on a brain organochip, neural progenitor cells, astrocytes, and microglia were embedded in BME hydrogel and then injected into the brain tissue culture unit of the chip. Neural progenitor cells were suspended in neural expansion medium supplemented with 2 mM glutamine and 0.02 μg / ml fibroblast growth factor. To obtain resting astrocytes and microglia, they were cultured for 1 day in serum-free astrocyte culture medium and astrocyte ACM conditioned medium, respectively, before injection. Neural progenitor cells, astrocytes, and microglia were mixed in a ratio of 8:4:1 (n / n / n), and then a cell mixture was prepared according to a BME hydrogel prepolymer to cell volume ratio of 4:1 (v / v). The cell mixture prepared according to this ratio contained neural progenitor cells, astrocytes, and microglia. The proportion of progenitor cells developing into mature neurons exceeded 80%, ultimately achieving a cell ratio of neurons, astrocytes, and microglia within the range of 5-6:4-5:1 (n:n:n). Before gel injection, a pin device consistent with the structure of the ventricle unit was inserted into the ventricle unit, and then the gel-cell mixture was injected into the brain tissue culture unit of the chip placed on ice. After injection, the chip was transferred to a 37°C cell culture incubator and cultured for 30 minutes to gel. After gelation, a serum-free mixed culture medium prepared with NEM:AM:ACM at a volume ratio of 8:4:1 (v / v / v) was injected into the venous unit, arterial unit, and ventricle unit, and the culture medium was changed daily. From day 3, neural differentiation medium was replaced with NEM. In the later stages, the neural differentiation medium was changed every other day until the BBB was reconstituted.

[0035] (2) After co-culturing brain tissue nerve cells for 4 days, brain microvascular endothelial cells and pericytes were inoculated:

[0036] First, a porous membrane containing a mixture of 400 μg / mL collagen IV and 100 μg / mL fibronectin was prepared with DPBS and coated, and incubated at 37°C for at least 4 hours.

[0037] The coating solution was then removed, and the membrane was rinsed with DPBS. Brain microvascular endothelial cells and pericytes were suspended in endothelial cell culture medium and pericyte culture medium, respectively. Brain microvascular endothelial cells and pericytes were mixed at a ratio of 9:1 (n / n), and the cell suspension was injected into the venous unit of the chip. The chip was gently inverted to allow the brain microvascular endothelial cells and pericytes to attach to the bottom of the porous membrane. After 3 hours of culture, unattached cells were removed. Brain microvascular endothelial cells and pericytes were seeded into the arterial unit in the same manner. The ECM and PM mixed culture medium in the blood-side channels (venous unit) and the top of the BME gel was changed every other day. The ventricular unit was injected with a mixed NDM, AM, and ACM culture medium. The chip was cultured for another 3 days to form a BBB.

[0038] The culture medium level in the ventricle unit is lower than that in the arterial unit, but not lower than that at the inlet and outlet of the venous channel; that is, the culture medium level in the arterial unit > the culture medium level in the ventricle unit > the culture medium level at the inlet and outlet of the venous channel, so as to generate a pressure difference to drive the flow of culture medium, so that the culture medium in the arterial unit flows through the porous microfluidic medium to the ventricle unit and the venous unit; the culture medium in the ventricle unit flows to the porous microfluidic medium and eventually flows back to the vein, without overflowing to the top of the porous microfluidic medium.

[0039] Optionally, in the S1 preprocessing step,

[0040] The PDMS prepolymer and curing agent are mixed using a toothed roller device, and the mixed PDMS prepolymer is vacuumed and the air bubbles are eliminated using a double negative pressure defoaming device.

[0041] The toothed rolling roller device includes a left support and a right support, which are connected by a crossbar. Three or more rolling rollers are arranged between the left support and the right support, and the outer wall of each rolling roller has tooth marks. The three rolling rollers are arranged in a triangle to form a group, wherein the first rolling roller and the second rolling roller are arranged at the lower part at intervals along the same horizontal line, and a third rolling roller is arranged between the first rolling roller and the second rolling roller. The third rolling roller is arranged above the first rolling roller and the second rolling roller, and the tooth marks of the third rolling roller mesh with the tooth marks of the first rolling roller and the second rolling roller, respectively.

[0042] The dual negative pressure defoaming device includes a base, a separation plate, and a top cover; the separation plate is detachably disposed between the base and the top cover; the bottom surface of the separation plate is provided with a plurality of defoaming holes; the defoaming holes are conical in shape, with the diameter of the upper opening being larger than the diameter of the lower opening; a first suction pipe and a second suction pipe are respectively provided on one side of the base and the separation plate, and a first valve and a second valve are respectively provided on the first suction pipe and the second suction pipe.

[0043] Applications of microfluidic brain organ-on-a-chip, as described above, include: studies involving the effects of substances absorbed into the bloodstream (food, toxins, drugs) on the blood-brain barrier and the brain, as well as studies on the brain's response to these substances.

[0044] Or it can be used to study the effects of intrathecal (subarachnoid space, cerebrospinal fluid) administration on the cerebrospinal fluid-brain barrier and the brain;

[0045] Or research on the effects of changes in the internal and external environment of the body on the brain;

[0046] It may be used to study the pathogenesis of various brain diseases and the brain's effects on the periphery;

[0047] And alternatives to the use of laboratory animals in the drug screening process.

[0048] The beneficial effects of this invention compared to the prior art are as follows:

[0049] 1. The present invention sets up a brain tissue culture unit, a ventricle unit and corresponding arterial and venous structures respectively. By embedding nerve cells in a porous microfluidic medium and filling the brain tissue culture unit, the physiological structure and function of brain tissue are more closely simulated.

[0050] 2. Due to the porous structure of the porous microfluidic medium, liquids can pass through freely. By seeding brain microvascular endothelial cells and pericytes on both sides of the porous microfluidic medium, and defining the upper layer of the medium as an arterial structure and the lower layer as a venous structure, the liquid in the upper layer permeates through the brain microvascular endothelial cells and pericytes into the porous microfluidic medium, ultimately flowing into the lower structure. This structure allows for the simulation of the blood-brain barrier and the construction of a cerebral blood circulation system that flows through the brain and into the veins. It also facilitates standardized and simplified chip fabrication, ensuring controllable fabrication quality and significantly improving the chip's yield. Furthermore, this structure facilitates smooth control of subsequent liquid flow, maximizing the chip's performance.

[0051] 3. The ventricle unit is placed on the side of the brain tissue culture unit, and the two units are connected by a connecting channel to achieve mutual exchange between the porous microfluidic medium and the liquid inside the ventricle unit. At the same time, by controlling the height of the liquid in the overall chip, the liquid in the ventricle unit is prevented from overflowing to the top of the porous microfluidic medium and can flow into the venous structure, thus realizing the construction of an exchange system between peripheral substances and the brain.

[0052] 4. This invention enables the overall simulation of brain structure, including the blood-brain barrier, the cerebrospinal fluid-brain barrier, and the cerebral blood circulation system that flows from the blood-brain barrier into the veins. The simulation of the blood-brain barrier includes the physical and metabolic barriers that regulate molecular exchange between blood and the brain. The simulation of the cerebrospinal fluid-brain barrier includes the selective exchange of substances between cerebrospinal fluid and extracellular fluid in the brain. Ultimately, through the simulation of the cerebral blood circulation system, it achieves the organic connection between blood, the blood-brain barrier, brain tissue, and cerebrospinal fluid in cerebral circulation. It also has the feasibility of expanding the simulation of the structural and functional characteristics of the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-blood barrier. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the exploded structure of the brain organ-chip of the present invention;

[0054] Figure 2 This is a cross-sectional view of the brain organ-chip of the present invention;

[0055] Figure 3 This is a schematic diagram of the structure of the first embodiment of the toothed rolling roller device of the present invention;

[0056] Figure 4 yes Figure 3 A diagram illustrating the usage status of the document;

[0057] Figure 5 This is a schematic diagram of the structure of a second embodiment of the toothed rolling roller device of the present invention;

[0058] Figure 6 This is a cross-sectional view of the rolling roller of the present invention;

[0059] Figure 7 This is a schematic diagram of the usage state of the dual negative pressure defoaming device of the present invention;

[0060] Figure 8 This is a cross-sectional view of the dual negative pressure defoaming device of the present invention;

[0061] Figure 9 yes Figure 8 Enlarged diagram of area A in the middle. Detailed Implementation

[0062] The above description is a detailed explanation of this patent in conjunction with specific embodiments, and it should not be construed that the specific embodiments of this patent are limited to the above description. For those skilled in the art, any substitutions or modifications made to the described embodiments without departing from the concept of this patent should be considered within the scope of protection of this patent. In this specification, specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

[0063] A microfluidic brain organ-on-a-chip, such as Figures 1-2As shown, the chip body is divided into an upper chip 1, a porous film 2, and a lower chip 3. The upper chip includes a vein channel inlet via 11, a vein channel outlet via 12, a brain tissue culture unit 13, an arterial unit 14, a ventricle unit 15, a brain tissue culture unit inlet 16, and a brain tissue culture unit outlet 17. The lower chip includes a vein channel inlet shallow groove 31, a vein channel outlet shallow groove 32, a vein unit 33, a vein inlet connection channel 34, and a vein outlet connection channel 35. The vein unit 33 is located below the brain tissue culture unit 13, and the arterial unit 14 is located above the brain tissue culture unit 13. A porous membrane 2 is disposed between the brain tissue culture unit 13 and the venous channel inlet 11; the position of the venous channel inlet shallow groove 31 corresponds vertically to the position of the venous channel outlet shallow groove 32; the venous venous unit 15 is disposed to the right of the brain tissue culture unit 13; the venous channel inlet shallow groove 31 is connected to the venous unit 33 through the venous inlet connecting channel 34, and the venous channel outlet shallow groove 32 is connected to the venous unit 33 through the venous outlet connecting channel 35. The porous membrane 2 is made of PC or PET material. In this embodiment, the height of the upper chip is set to 4-6 mm, the diameter of the venous channel inlet aperture 11 and the venous channel outlet aperture 12 is set to 3-4 mm, the height of the brain tissue culture unit 13 is set to 0.2-0.5 mm, and the length and width are set to 4 mm and 5 mm respectively, the diameter of the arterial unit 14 is set to 3 mm, the width and length of the ventricular unit 15 are rounded rectangles of 3 mm and 6 mm respectively, the diameter of the brain tissue culture unit inlet 16 and the brain tissue culture unit outlet 17 is set to 1-2 mm; the diameter of the venous channel inlet aperture 11 and the venous channel outlet aperture 12 is set to 3-4 mm; the feature depth in the lower chip is set to 0.4-0.6 mm; the diameter of the venous channel inlet shallow groove 31 and the venous channel outlet shallow groove 32 is set to 3-4 mm; the length and width of the venous unit 33 are set to 4 mm and 5 mm respectively; the width of the venous inlet connecting channel 34 and the venous outlet connecting channel 35 is set to 1-2 mm; and the pore size of the porous membrane 2 is 3-8 μm.

[0064] Considering the overall experimental design and subsequent application requirements, the chip can be made of PDMS material, which has good air permeability, high transparency and is easy to process. Alternatively, depending on the experimental conditions, materials with good biocompatibility and thermoplastic mechanical processing, such as PMMA, PC, and COC, can be selected.

[0065] During the process, brain microvascular endothelial cells are seeded in the lower layer of the porous membrane 2. The brain tissue culture unit is filled with a porous microfluidic medium containing neurons, astrocytes, and microglia, representing the brain parenchyma and capillaries distributed within it. The arterial unit is seeded with brain microvascular endothelial cells to serve as the arterial structure. For the porous microfluidic medium, basement membrane extract (BME) hydrogel, fibrinogen, gelatin, laminin, collagen, etc., can be selected. In practical applications, BME hydrogel is preferred as the porous microfluidic medium. The upper structure on the right is the ventricular structure. The fluid in the ventricular structure interacts bidirectionally with the fluid in the brain tissue culture unit, but the fluid does not overflow into the artery; that is, the fluid within the artery is designed for unidirectional flow.

[0066] This microfluidic brain organochip can achieve a comprehensive simulation of brain structure, including the blood-brain barrier, the cerebrospinal fluid-brain barrier, and the cerebral blood circulation system that flows from the blood-brain barrier into the veins. The simulation of the blood-brain barrier includes the physical and metabolic barriers that regulate molecular exchange between blood and the brain. The simulation of the cerebrospinal fluid-brain barrier includes the selective exchange of substances between cerebrospinal fluid and extracellular fluid in the brain. Ultimately, through the simulation of the cerebral blood circulation system, it achieves the organic connection between blood, the blood-brain barrier, brain tissue, and cerebrospinal fluid in cerebral circulation. It also has the feasibility of expanding the simulation of the structural and functional characteristics of the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-blood barrier.

[0067] Microfluidic brain organ-on-a-chip construction method

[0068] The chip design is created using SolidWorks software. Chips can be fabricated using PDMS and thermoplastics. For thermoplastic chips, chip modules can be obtained through injection molding or CNC machining, and then assembled using thermoforming bonding. For PDMS chips, molds can be obtained by CNC machining of PMMA, or through 3D printing. The PDMS chip fabrication process is as follows:

[0069] The PDMS chip manufacturing process includes:

[0070] S1 Pre-processing

[0071] The PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1. The mixture is stirred using an existing stirring device (such as a stirring rod or fork) or the toothed roller device of this invention until fine bubbles are generated to ensure thorough mixing (the PDMS prepolymer and curing agent must be thoroughly mixed, otherwise chip quality will be affected). Because stirring the PDMS prepolymer and curing agent naturally generates bubbles, using existing stirring devices such as stirring rods not only introduces excessive air into the liquid but also results in low mixing efficiency (PDMS prepolymer is a viscous liquid). To reduce the amount of air introduced during stirring, improve the efficiency of subsequent vacuum degassing operations, and enhance the mixing efficiency, the toothed roller device of this invention is preferred in this embodiment to achieve the above-mentioned technical effects. The first embodiment of the toothed roller device 4 is as follows... Figures 3-4 As shown, the device includes a left support and a right support 41, connected by a crossbar 49. Three or more rolling rollers are arranged between the left and right supports 41, and the outer walls of the rolling rollers have toothed grooves. The three rolling rollers are arranged in a triangular formation, with the first rolling roller 42 and the second rolling roller 48 spaced apart at the bottom along the same horizontal line. A third rolling roller 43 is positioned between the first and second rolling rollers, above them, and its toothed grooves engage with those of the first rolling roller 42 and the second rolling roller 48. This arrangement serves to: by pushing the first and second rolling rollers 42 and 48 to roll, the third rolling roller 43 is driven, and through the engagement and rotation of the toothed grooves of the three rolling rollers, the PDMS prepolymer and curing agent are agitated and mixed. Figure 4 As shown, in use, three rolling rollers are submerged below the liquid surface to achieve mixing, effectively preventing excessive air from entering the liquid. Agitation is achieved through the toothed grooves of the three rotating rollers, significantly improving the mixing efficiency of the two components. For ease of operation, in this embodiment, as... Figure 3 As shown, it also includes a left connecting rod and a right connecting rod 45. The left connecting rod and the right connecting rod 45 are rotatably connected to the left bracket and the right bracket 41 respectively via a rotating shaft 44. The upper parts of the left connecting rod and the right connecting rod 45 are connected by a connecting rod. An operating rod 46 is connected to the middle of the connecting rod. A handle 47 is provided on the top of the operating rod 46 to facilitate the above-mentioned pushing operation.

[0072] like Figure 5As shown, in a second embodiment of the toothed roller device 4, the height of the three rollers can be adjusted according to the depth of the liquid to ensure that the height of the three rollers is adjusted with the depth of the target liquid, ensuring that all three rollers are submerged in the liquid. The difference between this embodiment and the first embodiment lies only in that: a first adjusting elongated hole 413, a second adjusting elongated hole 416, and a third adjusting elongated hole 411 are respectively provided on the left support and the right support 41; the first adjusting elongated hole 413 and the second adjusting elongated hole 416 are arranged horizontally, while the third adjusting elongated hole 411 is arranged vertically; as shown... Figure 6 As shown, taking the first adjusting elongated hole 413 as an example, a T-shaped bushing 415 is movably disposed within the first adjusting elongated hole 413, and a thread is provided on the outside of the T-shaped bushing 415. An upper nut 414 is provided on the outside to lock and fix the T-shaped bushing 415. The rolling shaft of the first rolling roller 42 can be rotatably inserted into the T-shaped bushing 415, that is, the position of the first rolling roller 42 is adjusted by adjusting the positions of the T-shaped bushings 415 on both sides. In some embodiments, the first adjusting elongated hole 41... 3. The second and third adjustment elongated holes 416 and 411 are also provided with scales 412 to improve the accuracy of adjustment. In use, if the liquid level is shallow, the gap between the first rolling roller 42 and / or the second rolling roller 48 is widened and the position of the third rolling roller 43 is lowered, so that the third rolling roller 43 engages with the first rolling roller 42 and the second rolling roller 48 respectively, thereby reducing the height of the three rolling rollers and ensuring that the three rolling rollers can always be submerged below the liquid level for stirring.

[0073] The mixed PDMS prepolymer can be placed in a vacuum desiccator for bubble removal, but using this traditional method, bubble removal generally requires waiting for more than 1 hour (bubbles must be completely removed, otherwise defects such as bubble holes will appear after curing, leading to chip defects). In this embodiment, the dual negative pressure debubbling device of the present invention is preferred, which can significantly improve the bubble removal speed and effect, and greatly improve work efficiency. Figures 7-9As shown, the dual negative pressure defoaming device 6 includes a base 63, a separation disc 62, and a top cover 61. The separation disc 62 is detachably disposed between the base 63 and the top cover 61, and its contact surfaces can be provided with silicone rings to improve sealing. The bottom surface 621 of the separation disc 62 is provided with a plurality of extrusion holes 622. The extrusion holes 622 have a conical structure, and the diameter d1 of the upper opening is larger than the diameter d2 of the lower opening. The purpose of this design is to enable the mixed PDMS prepolymer to flow downwards and defoam through the extrusion holes. The structure of 622 pushes out small air bubbles uniformly mixed in the liquid upwards, significantly improving the efficiency of eliminating small air bubbles in the liquid; a first suction pipe 67 and a second suction pipe 66 are respectively provided on one side of the chassis 63 and the separation plate 62, and a first valve 65 and a second valve 64 are respectively provided on the first suction pipe 67 and the second suction pipe 66; a first pressure gauge 611, an exhaust valve (not shown), a safety valve (not shown), etc. are provided on the upper cover 61; a second pressure gauge 651 is provided on the first suction pipe 67. In use, the mixed liquid to be degassed is poured onto the bottom surface 621. Then, the air pump 7 is used to evacuate air from above the separation plate 62 and the base plate 63. Gravity can be used to force the mixed liquid to flow downwards from the degassing hole 622. Alternatively, the following control method can be selected: the pressure on the upper and lower sides of the liquid is checked by the first pressure gauge 611 and the second pressure gauge 651. Then, the pressure difference between the upper and lower sides is controlled by the first valve 65 and the second valve 64, that is, the air pressure on the lower side of the liquid is controlled to be lower than the air pressure on the upper side, thereby increasing the downward flow speed of the liquid. The gas in the liquid is squeezed upwards and then pumped to the upper surface of the liquid. The downward flowing liquid is also subject to the vacuum of the base plate 63 to continue to degas the bubbles. Through the above principle, the bubble degassing speed and degassing effect in this step can be significantly improved, greatly improving the quality and efficiency of chip fabrication, and with good repeatability.

[0074] Pour the defoamed PDMS prepolymer onto the mold and perform vacuum defoaming again until no bubbles are present in the PDMS prepolymer. Transfer it to an 80℃ oven and heat to cure for 1 hour. Finally, carefully peel the cured PDMS off the mold and cut it into PDMS blocks of appropriate size for subsequent use.

[0075] S2 drilling

[0076] Use a punch of the appropriate size to drill holes at the corresponding positions and at a vertical angle on the upper-layer chip of the PDMS.

[0077] S3 chip package

[0078] Since PDMS and PET films cannot be directly bonded via oxygen plasma treatment, the PET film needs to undergo aminosilanization to achieve bonding. In the aminosilanization process, a 5% 3-aminopropyltriethoxysilane (APTES) solution is first prepared with water and preheated to 80°C using a hot plate. The PDMS chip is then cleaned with isopropanol and dried with nitrogen or compressed air. Both sides of the PDMS chip are cleaned with Scotch tape to remove any visible debris. The PET film is then treated with oxygen plasma for 30 seconds (80W) and transferred to the preheated 5% APTES solution for 30 minutes. Next, the PET film is rinsed with deionized water and dried at room temperature. The aminosilanized PET film and the PDMS chip are simultaneously treated with oxygen plasma for 30 seconds (80W) and then bonded. Finally, the assembled PDMS chip is placed in an oven and heated at 60°C for 24 hours.

[0079] Brain organ-on-a-chip construction

[0080] The assembled brain organ-on-a-chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use.

[0081] To construct neural tissue on a microfluidic chip, neural progenitor cells, astrocytes, and microglia were embedded in BME hydrogel and then injected into the chip's brain tissue culture unit. Neural progenitor cells were suspended in neural expansion medium (NEM) supplemented with 2 mM glutamine and 0.02 μg / ml fibroblast growth factor. To obtain resting astrocytes and microglia, they were cultured for 1 day, respectively, in serum-free astrocyte medium and astrocyte conditioned medium (ACM), before injection. The density of each cell suspension was approximately 8 × 10⁻⁶. 6Cells / mL. Neural progenitor cells, astrocytes, and microglia were mixed in a number ratio of 8:4:1 (n / n / n). Then, a cell mixture was prepared according to a BME hydrogel prepolymer to cell volume ratio of 4:1 (v / v). In this cell mixture, the proportion of neural progenitor cells developing into mature neurons exceeded 80%, ultimately achieving a neuronal, astrocyte, and microglia ratio within the range of 5-6:4-5:1 (n:n:n), similar to the ratio in the human brain. Before injecting the gel, a pin device consistent with the structure of the ventricle unit was inserted into the ventricle unit to prevent gel from flowing into the ventricle unit during loading. The gel-cell mixture was then injected into the brain tissue culture unit of the chip placed on ice. After injection, the chip was transferred to a 37°C cell culture incubator and cultured for 30 minutes to gel. After gelation, a serum-free mixed culture medium prepared with NEM, AM, and ACM at a volume ratio of 8:4:1 (v / v / v) was injected into the intravenous, arterial, and ventricular units, and the medium was changed daily. From day 3 onwards, NEM was replaced with neural differentiation medium (NDM). This medium was then changed every other day until BBB reconstitution.

[0082] After co-culturing brain tissue neural cells for 4 days, brain microvascular endothelial cells and pericytes were seeded. First, a porous membrane was coated with a mixture containing 400 μg / mL collagen IV and 100 μg / mL fibronectin prepared with DPBS and incubated at 37°C for at least 4 hours. The coating solution was then removed, and the membrane was rinsed with DPBS. Brain microvascular endothelial cells and pericytes were then suspended in endothelial cell culture medium (ECM) and pericyte culture medium (PM), respectively, at a cell density of approximately 1 × 10⁻⁶ cells / mL. 6 Cells / mL. Brain microvascular endothelial cells and pericytes were mixed at a ratio of 9:1 (n / n), and the cell suspension was injected into the venous unit of the chip. The chip was gently inverted to allow the brain microvascular endothelial cells and pericytes to attach to the bottom of the porous membrane. After 3 hours of culture, unattached cells were removed with fresh mixed medium (ECM:PM, 9:1 (v / v)). Brain microvascular endothelial cells and pericytes were seeded into the arterial unit in the same manner. The ECM and PM mixed medium in the blood-side channels (venous unit and BME gel top) was replaced every other day. The ventricular unit was injected with a mixed medium of NDM, AM, and ACM. The chip was cultured for another 3 days to form a BBB.

[0083] Regarding the amount of culture medium added, the level of the culture medium in the ventricle unit should be lower than that in the arterial unit, but not lower than that at the inlet and outlet of the venous channel. That is, the arterial unit culture medium level > the ventricle unit culture medium level > the venous channel inlet and outlet culture medium level, to create a pressure difference that drives the flow of the culture medium, allowing it to flow from the arterial unit through the porous microfluidic medium to the ventricle and venous units. The ventricle unit culture medium flows into the porous microfluidic medium and ultimately returns to the vein, without overflowing onto the top of the porous microfluidic medium.

[0084] Applications of microfluidic brain organ-on-a-chip

[0085] This proposed brain organ-on-a-chip has a wide range of applications. Because the chip simulates the arterial-brain-venous cerebral blood flow system and integrates the blood-brain barrier and cerebrospinal fluid-brain barrier, it reconstructs the physiological structure of the brain relatively completely in vitro, demonstrating potential feasibility for expansion into complex brain tissues, the blood-cerebrospinal fluid barrier, and the cerebrospinal fluid-venous barrier. This chip can be applied to research on brain physiological functions, including studies on the effects of substances absorbed into the bloodstream (food, toxins, drugs) on the blood-brain barrier and the brain, and studies on the brain's responses to these substances; or to studies on the effects of intrathecal (subarachnoid, cerebrospinal fluid) drug administration on the cerebrospinal fluid-brain barrier and the brain; or to studies on the effects of changes in the internal and external environment on the brain, such as the influence of the cerebral circulatory system on the brain, making it possible to study the effects of internal environmental factors on the brain, such as the effects of hormones (glucocorticoids produced under chronic stress) or other bioactive substances (lipopolysaccharides) on the function of the brain structural barrier; or it can be used to study the pathogenesis of various brain diseases and the brain's peripheral effects; and to replace the use of experimental animals in drug screening processes.

Claims

1. A microfluidic brain organ-on-a-chip, characterized in that: The chip body includes an upper chip, a porous film, and a lower chip. The upper chip has a venous channel inlet port, a venous channel outlet port, a brain tissue culture unit, an arterial unit, a ventricle unit, a brain tissue culture unit inlet, and a brain tissue culture unit outlet. The lower chip has a venous channel inlet shallow groove, a venous channel outlet shallow groove, a venous unit, a venous inlet connecting channel, and a venous outlet connecting channel. The venous unit is located below the brain tissue culture unit, and the porous film is located between the venous unit and the brain tissue culture unit. The position of the venous channel inlet port corresponds vertically to the position of the venous channel inlet shallow groove. The position of the venous channel outlet port corresponds vertically to the position of the venous channel outlet shallow groove. The venous channel inlet shallow groove is connected to the venous unit through the venous inlet connecting channel, and the venous channel outlet shallow groove is connected to the venous unit through the venous outlet connecting channel. The arterial unit is located above the brain tissue culture unit, and the ventricle unit is located to the right of the brain tissue culture unit. The brain tissue culture unit is filled with a porous microfluidic medium, in which neurons, astrocytes, and microglia are embedded. The porous microfluidic medium is made of basement membrane extract hydrogel, fibrinogen, gelatin, laminin, or collagen. The fluid between the ventricle unit and the brain tissue culture unit is configured for bidirectional interaction, and the fluid does not overflow into the artery; that is, the fluid in the artery is configured for unidirectional flow.

2. The microfluidic brain organ-on-a-chip according to claim 1, characterized in that: The porous film is made of PC or PET material.

3. The microfluidic brain organ-on-a-chip according to claim 1, characterized in that: The chip body is made of PDMS, PMMA, PC, or COC.

4. A microfluidic brain organ-on-a-chip according to claim 1, characterized in that: The lower layer of the porous membrane is seeded with brain microvascular endothelial cells and pericytes.

5. A microfluidic brain organ-on-a-chip according to claim 1, characterized in that: The arterial unit was inoculated with brain microvascular endothelial cells and pericytes.

6. A microfluidic brain organ-on-a-chip construction method, characterized in that: Construct a microfluidic brain organ chip as described in any one of claims 1 to 5, wherein the brain organ chip is fabricated using PDMS; The PDMS chip manufacturing process includes: S1 Pre-processing Mix the PDMS prepolymer and curing agent at a mass ratio of 10:1 and stir until fully mixed; remove air bubbles from the mixed PDMS prepolymer using a vacuum method; pour the defoamed PDMS prepolymer onto a mold, ensuring that there are no air bubbles present; then transfer it to an oven for heating and curing. S2 drilling Drill holes at the corresponding positions and vertical angles using a punch of the appropriate size on the upper-layer chip of the PDMS. S3 chip package Since PDMS and PET film cannot be directly bonded by oxygen plasma treatment, bonding is achieved by aminosilanizing the PET film. The aminosilanization process is as follows: First, prepare a 5% 3-aminopropyltriethoxysilane solution with water, preheat it to 80°C, clean the PDMS chip with isopropanol and dry it with nitrogen or compressed air, and clean both sides of the PDMS chip to remove any obvious debris. The PET film was treated with oxygen plasma for 30 seconds and then transferred to a preheated 5% APTES solution for 30 minutes. Wash the PET film with deionized water and dry it at room temperature; Aminosilanized PET film and PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and then bonded together. The assembled PDMS chip was placed in an oven and heated at 60°C for 24 hours. S4 brain organ-on-a-chip construction The assembled PDMS chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use. (1) To construct neural tissue on a brain organ-on-a-chip, neural progenitor cells, astrocytes, and microglia were embedded in BME hydrogel and then injected into the brain tissue culture unit of the chip. The neural progenitor cells were suspended in neural expansion medium supplemented with 2 mM glutamine and 0.02 μg / ml fibroblast growth factor. To obtain resting astrocytes and microglia, they were cultured for 1 day in serum-free astrocyte culture medium and astrocyte ACM conditioned medium, respectively, before injection. The neural progenitor cells, astrocytes, and microglia were mixed in a number ratio of 8:4:1 (n / n / n). Then, a cell mixture was prepared according to a BME hydrogel prepolymer to cell volume ratio of 4:1 (v / v). The proportion of neural progenitor cells developing into mature neurons exceeded 80%, ultimately achieving a cell ratio of neurons, astrocytes, and microglia within the range of 5-6:4-5:1 (n:n:n). Before gel injection, a pin device consistent with the structure of the ventricular unit was inserted into the ventricular unit, and then the gel-cell mixture was injected into the brain tissue culture unit of the chip placed on ice. After injection, the chip was transferred to a 37°C cell culture incubator for 30 minutes to gel. After gelation, a serum-free mixed culture medium prepared with a NEM:AM:ACM ratio of 8:4:1 (v / v / v) was injected into the venous unit, arterial unit, and ventricular unit, and the culture medium was changed daily. From day 3 onwards, neural differentiation medium was replaced with neural differentiation medium. In the later stages, the neural differentiation medium was changed every other day until the BBB was reconstituted. (2) After co-culturing brain tissue nerve cells for 4 days, brain microvascular endothelial cells and pericytes were inoculated: First, a porous membrane containing a mixture of 400 μg / mL collagen IV and 100 μg / mL fibronectin was prepared with DPBS and coated, and incubated at 37°C for at least 4 hours. The coating solution was then removed, and the membrane was rinsed with DPBS. Brain microvascular endothelial cells and pericytes were suspended in endothelial cell culture medium and pericyte culture medium, respectively. Brain microvascular endothelial cells and pericytes were mixed at a ratio of 9:1 (n / n), and the cell suspension was injected into the venous unit of the chip. The chip was gently inverted to allow the brain microvascular endothelial cells and pericytes to attach to the bottom of the porous membrane. After 3 hours of culture, unattached cells were removed. Brain microvascular endothelial cells and pericytes were seeded into the arterial unit in the same manner. The ECM and PM mixed culture medium in the blood-side channels (i.e., the venous unit) and the top of the BME gel was changed every other day. The ventricular unit was injected with a mixed culture medium of NDM, AM, and ACM. The chip was cultured for another 3 days to form a BBB. The culture medium level in the ventricle unit is lower than that in the arterial unit, but not lower than that at the inlet and outlet of the venous channel; that is, the culture medium level in the arterial unit > the culture medium level in the ventricle unit > the culture medium level at the inlet and outlet of the venous channel, so as to generate a pressure difference to drive the flow of culture medium, so that the culture medium in the arterial unit flows through the porous microfluidic medium to the ventricle unit and the venous unit; the culture medium in the ventricle unit flows to the porous microfluidic medium and eventually flows back to the vein, without overflowing to the top of the porous microfluidic medium.

7. The microfluidic brain organ-on-a-chip construction method according to claim 6, characterized in that: In the S1 preprocessing step The PDMS prepolymer and curing agent are mixed using a toothed roller device, and the mixed PDMS prepolymer is vacuumed and the air bubbles are eliminated using a double negative pressure defoaming device. The toothed rolling roller device includes a left support and a right support, which are connected by a crossbar. Three or more rolling rollers are arranged between the left support and the right support, and the outer wall of each rolling roller has tooth marks. The three rolling rollers are arranged in a triangle to form a group, wherein the first rolling roller and the second rolling roller are arranged at the lower part at intervals along the same horizontal line, and a third rolling roller is arranged between the first rolling roller and the second rolling roller. The third rolling roller is arranged above the first rolling roller and the second rolling roller, and the tooth marks of the third rolling roller mesh with the tooth marks of the first rolling roller and the second rolling roller, respectively. The dual negative pressure defoaming device includes a base, a separation plate, and a top cover; the separation plate is detachably disposed between the base and the top cover; the bottom surface of the separation plate is provided with a plurality of defoaming holes; the defoaming holes are conical in shape, with the diameter of the upper opening being larger than the diameter of the lower opening; a first suction pipe and a second suction pipe are respectively provided on one side of the base and the separation plate, and a first valve and a second valve are respectively provided on the first suction pipe and the second suction pipe.

8. The application of microfluidic brain organ-on-a-chip, characterized by: The microfluidic brain organ chip as described in any one of claims 1 to 5 is applied to: studies involving the effects of substances absorbed into the bloodstream on the blood-brain barrier and the brain, and studies on the brain's response to these substances; Or it could be used to study the effects of intrathecal administration on the cerebrospinal fluid-brain barrier and the brain; Or research on the effects of changes in the internal and external environment of the body on the brain; Or it can be used to study the pathogenesis of various brain diseases and the brain's effects on the periphery; And alternatives to the use of laboratory animals in the drug screening process.

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