Micro-fluidic brain organ chip as well as construction method and application thereof

Abstract

The invention relates to the technical field of micro-fluidic brain organ chips, and particularly discloses a micro-fluidic brain organ chip as well as 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 the following steps: packaging a chip, and carrying out amino silanization to realize bonding; s4, brain organ chip construction: (1) BBB reconstruction; and (2) inoculating brain microvascular endothelial cells and pericytes. According to the invention, the whole simulation of the brain structure, including blood-brain barrier, cerebrospinal fluid-brain barrier, brain tissue and a cerebral blood circulation anatomical physiological system which passes through the brain tissue after the blood-brain barrier and is collected to brain veins, can be realized. The method can be applied to research on the influence of absorbed substances in blood on the blood brain barrier; the method can be used for brain disease pathogenesis and peripheral system influence; and the kit can be used for replacing experimental animals in the drug screening process.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic brain organ chip, and in particular to a microfluidic brain organ chip, a construction method and applications thereof. Background Art

[0002] The molecular exchange between the brain and the periphery is limited by specific cellular and biochemical mechanisms that exist at several interfaces: the blood-brain barrier (BBB), the CSF-brain barrier, the blood-CSF barrier, and in addition, the metabolites in the brain tissue and its ventricular choroid plexus are ultimately collected in the veins of the brain. Cerebrospinal fluid is continuously produced and absorbed back into the veins, playing the role of lymph in the central nervous system, which constitutes the overall material exchange system between the brain and the periphery. The destruction of these barriers may lead to the occurrence of various diseases, such as stroke, multiple sclerosis or neuropsychiatric disorders. The destruction of these barriers may be caused by various factors, such as hypoxia, inflammation or stress factors such as intestinal flora imbalance, chronic stress, trauma, etc.

[0003] The BBB is a highly selective, semipermeable barrier that ensures the separation of circulating blood from the brain and central nervous system (CNS). This important physiological barrier enables brain cells to carry out their physiological functions by regulating the delicate brain microenvironment and maintaining brain homeostasis in 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 serves as an interface between the peripheral circulation and the CNS, with transporters and tight junctions (TJs) regulating 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, lining the blood vessels of the CNS vasculature. Pericytes, supporting glial cells (astrocytes and microglia), basement membranes, and extracellular matrix (ECM) also play crucial roles in maintaining the integrity of the BBB. BBB dysfunction is associated with increased BBB permeability and dysregulated influx and efflux, leading to the infiltration of toxins and immune cells into the CNS. This damage has been linked to various neurological diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis.

[0004] The CSF-brain barrier (CSFBB) consists of a single layer of ependymal cells that line the inner periphery of the ventricles and spinal canal. The ependymal layer is composed of multiciliated cuboidal to columnar epithelial cells that are held together near the apical surface by cell junction proteins to form the CSFBB. The primary function of the ependymal layer is to facilitate the overall flow of the CSF and the movement of debris through the highly coordinated beating of apical cilia. It also serves as a protective barrier that shields the juxtaposed basal subependymal gray matter and periventricular white matter tissue from CSF-containing toxins. The ependymal-CSFBB interface is considered only a partial barrier because it is selectively permeable 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 the 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 are differentiated 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, the use of organoids for disease modeling is limited by the limited diffusion of nutrients and oxygen and the difficulty in delivering them to the internal regions of the organoids, which has affected the shift to more controlled microenvironments in in vitro models.

[0006] A variety of model organisms, including Caenorhabditis elegans, fruit flies, rodents, and non-human primates, have been widely used to study the central nervous system in the past few decades. Although the study of model organisms has deepened people's understanding of brain function and diseases and provided important insights for the diagnosis and treatment of human neurological diseases, animal models cannot truly recapitulate some key features of the human brain, including neurodevelopmental trajectories, brain structures, and certain human-specific neural circuits and cell types.

[0007] Microfluidic organ chips are biomimetic systems that culture cells, tissues or organs on microfluidic devices to simulate the physiology and main functions of human organs. Therefore, the emergence of brain organ chips can effectively simulate the functional units of the human brain. Due to the integration of advanced microfluidic technology, perfusable devices with multiple chambers can be designed and multiple types of cells can be co-cultured, while combining flow systems that simulate blood circulation. Therefore, compared with traditional 2D in vitro models, brain organ chips can better simulate the highly dynamic microenvironment of the brain. In addition, compared with animal models, brain organ chips can be more easily based on brain physiological anatomy, with a well-defined and highly controllable microenvironment, so that the role of specific factors can be isolated and determined.

[0008] However, existing brain organ chip models only simulate a single barrier, such as the blood-brain barrier structure (a single blood vessel layer-brain tissue structure), and many existing single barrier simulations are only developed based on the research needs of a certain disease. Compared with the actual human brain anatomy, they are not complete. Moreover, due to the lack of a complete cerebral blood circulation system, especially the lack of mutual integration between multiple barrier systems in the brain, they are not enough to fully reproduce the complex physiological functions of the human brain. Summary of the invention

[0009] In response to the above technical problems, the present invention provides a microfluidic brain organ chip, a construction method and its application, so as to realize a complete simulation of the blood-brain barrier, the cerebrospinal fluid-brain barrier and the cerebral blood circulation system after the blood-brain barrier that passes through the brain and collects into the veins.

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

[0011] A microfluidic brain organ chip, the chip body comprises an upper chip, a porous film and a lower chip; the upper chip is provided with a venous channel inlet through hole, a venous channel outlet through hole, 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 is provided with 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 arranged below the brain tissue culture unit, and the porous film is arranged between the venous unit and the brain tissue culture unit; the position of the venous channel inlet through hole corresponds to the position of the venous channel inlet shallow groove in the vertical direction; the position of the venous channel outlet through hole corresponds to the position of the venous channel outlet shallow groove in the vertical direction; 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 arranged above the brain tissue culture unit; the ventricle unit is arranged on the right side of the brain tissue culture unit.

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

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

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

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

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

[0017] Optionally, the liquid between the ventricular unit and the brain tissue culture unit is set to bidirectionally interact, and the liquid does not overflow into the artery thereof, that is, the liquid in the artery is set to flow unidirectionally.

[0018] A method for constructing a microfluidic brain organ chip, constructing the microfluidic brain organ chip as described above, wherein the brain organ chip is made of PDMS or thermoplastic plastic;

[0019] The PDMS chip production process includes:

[0020] S1 Pre-processing

[0021] The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1 and stirred until fully mixed; the mixed PDMS prepolymer is vacuumed to eliminate bubbles; the debubbled PDMS prepolymer is poured onto the mold to ensure that there are no bubbles in the PDMS prepolymer; then it is transferred to an oven and heated to cure;

[0022] S2 Punch Hole

[0023] Use a puncher of corresponding size to punch holes at a vertical angle at the corresponding position on the PDMS upper chip;

[0024] S3 chip package

[0025] Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, bonding was achieved by aminosilanization of the PET film;

[0026] The aminosilanization process is:

[0027] (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water and preheat it to 80°C. Clean the PDMS chip with isopropanol and blow dry it with nitrogen or compressed air. 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 transfer it to a preheated 5% APTES solution for 30 minutes;

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

[0030] (4) The aminosilanized PET film and the PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and bonded;

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

[0032] S4 Brain Organ Chip Construction

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

[0034] (1) In order to construct neural tissue on a brain organ chip, 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 a neural expansion medium supplemented with 2 mM glutamine and 0.02 μg / ml fibroblast growth factor, and in order to obtain resting astrocytes and microglia, they were cultured in serum-free astrocyte culture medium and astrocyte ACM conditioned medium for 1 day respectively before injection; neural progenitor cells, astrocytes and microglia were mixed in a quantitative ratio of 8:4:1 (n / n / n), and then a cell mixture was prepared in a ratio of BME hydrogel prepolymer to cell volume of 4:1 (v / v), in which the 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 in a ratio of 4:1 (v / v) between the BME hydrogel prepolymer and the cell volume, and in the cell mixture prepared according to this ratio, the neural progenitor cells, astrocytes and microglia were mixed in a ratio of 8:4:1 (n / n / n), and the neural progenitor cells, astrocytes and microglia were mixed in a ratio of 8:4:1 (n / n / n), and then in a ratio of 4:1 (v / v) between the BME hydrogel prepolymer and the cell volume, and in the cell mixture prepared according to this ratio, the neural progenitor cells, astrocytes and microglia were mixed in a ratio of 8:4:1 (n / n / n), and the neural progenitor cells, astrocytes and microglia were mixed in a ratio of 8:4:1 (n / n / n), and in the cell mixture prepared according to the ratio, the neural progenitor cells, astrocytes and microglia were mixed in a ratio of 8:4:1 (n / n / n), and in the cell mixture prepared according to the ratio, the The proportion of mature neurons developed from progenitor cells exceeded 80%, and the cell ratio of neurons, astrocytes and microglia was finally achieved in the range of 5-6:4-5:1 (n:n:n); before injecting the gel, a plug 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 and cultured for 30 minutes to gel; after gelation, a serum-free mixed culture medium prepared with NEM:AM:ACM in a volume ratio of 8:4:1 (v / v / v) was injected into the venous unit, arterial unit and ventricular unit, and then the culture medium was replaced every day; from the third day, NEM was replaced with neural differentiation medium; the neural differentiation medium was replaced every other day until the BBB was reconstructed;

[0035] (2) After 4 days of co-culture of brain tissue neural cells, brain microvascular endothelial cells and pericytes were inoculated:

[0036] First, a mixture containing 400 μg / mL collagen IV and 100 μg / mL fibronectin was prepared with DPBS to coat the porous membrane and incubated at 37°C for at least 4 h;

[0037] Then remove the coating solution and rinse the film with DPBS; suspend brain microvascular endothelial cells and pericytes in endothelial cell culture medium and pericyte culture medium respectively; mix brain microvascular endothelial cells and pericytes in a ratio of 9:1 (n / n), and then inject the cell suspension into the venous unit of the chip; gently flip the chip to allow brain microvascular endothelial cells and pericytes to attach to the bottom of the porous film; after culturing for 3 hours, remove the unattached cells; and inoculate brain microvascular endothelial cells and pericytes in the arterial unit in the same way; replace the ECM and PM mixed culture medium in the blood side channel, i.e., the venous unit and the top of the BME gel, every other day; inject the ventricular unit with a mixed culture medium of NDM, AM and ACM; the chip is cultured for another 3 days to form a BBB;

[0038] The culture medium level of the ventricular unit is lower than that of the arterial unit, and is not lower than the culture medium level at the inlet and outlet of the venous channel; that is, the arterial unit level> the ventricular unit level> the venous channel inlet and outlet level, so as to generate a pressure difference to drive the flow of the culture medium, so that the culture medium in the arterial unit flows through the porous microfluidic medium to the ventricular unit and the venous unit; the ventricular unit culture medium flows to the porous microfluidic medium, and finally flows back to the vein, and does not overflow to the top of the porous microfluidic medium.

[0039] Optionally, in the S1 preliminary processing step,

[0040] The PDMS prepolymer and the curing agent are mixed by a toothed rolling roller device, and the mixed PDMS prepolymer is vacuumed to eliminate bubbles by a double negative pressure degassing device;

[0041] The toothed rolling roller device comprises a left bracket and a right bracket, and the left bracket and the right bracket are connected by a cross bar; more than three rolling rollers are arranged between the left bracket and the right bracket, and the outer walls of the rolling rollers are provided with 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 same horizontal line at the lower part, and the third rolling roller is arranged between the first rolling roller and the second rolling roller, and 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 are respectively meshed with the tooth marks of the first rolling roller and the tooth marks of the second rolling roller;

[0042] The double negative pressure defoaming device includes a base plate, a separation plate and an upper cover; the separation plate is detachably arranged between the base plate and the upper cover; a plurality of squeezing holes are arranged on the bottom surface of the separation plate; the squeezing holes are in a conical structure, and the aperture of the upper opening is larger than the aperture of the lower opening; a first exhaust pipe and a second exhaust pipe are respectively arranged on one side of the base plate and the separation plate, and a first valve and a second valve are respectively arranged on the first exhaust pipe and the second exhaust pipe.

[0043] Applications of microfluidic brain organ chips, as described above, are applied to: research involving the effects of substances (food, toxins, drugs) absorbed into the blood on the blood-brain barrier and the brain, and research on the brain's response to these substances;

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

[0045] Or the study of 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 effects of the brain on the periphery;

[0047] and to replace the use of experimental animals in the drug screening process.

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

[0049] 1. The present invention separately sets up a brain tissue culture unit, a ventricular unit, and corresponding arterial and venous structures, and more closely simulates the physiological structure and function of brain tissue by embedding nerve cells in a porous microfluidic medium and filling it in the brain tissue culture unit.

[0050] 2. Since the porous microfluidic medium itself has a porous structure, it allows liquid to pass freely. By inoculating brain microvascular endothelial cells and pericytes on the upper and lower sides of the porous microfluidic medium, the upper layer of the porous microfluidic medium is defined as an arterial structure, and the lower layer is defined as a venous structure. Therefore, the upper layer of liquid enters the porous microfluidic medium through the brain microvascular endothelial cells and pericytes, and finally flows into the lower structure. Such a structure can simulate the blood-brain barrier and construct a cerebral blood circulation system that passes through the brain and converges to the veins. At the same time, it is conducive to the standard preparation of the chip in the later stage and the preparation is simple, which makes the preparation quality controllable and significantly improves the qualified rate of chip preparation. At the same time, the above structure is conducive to the smoothness of the related liquid flow control in the later stage, and maximizes the relevant performance of the chip.

[0051] 3. The ventricular unit is set on the side of the brain tissue culture unit, and the two units are connected through the connecting channel to achieve the exchange of liquid between the porous microfluidic medium and the ventricular unit. At the same time, by controlling the height of the liquid in the entire chip, the liquid in the ventricular unit does not overflow to the top of the porous microfluidic medium, and can flow into the venous structure, so as to realize the construction of an exchange system between peripheral substances and the brain.

[0052] 4. The present invention can achieve overall simulation of brain structure, including blood-brain barrier, cerebrospinal fluid-brain barrier, and cerebral blood circulation system after the blood-brain barrier that passes through the brain and collects into the veins. The simulation of the blood-brain barrier includes the physical and metabolic barriers that regulate the molecular exchange between the blood and the brain. The simulation of the cerebrospinal fluid-brain barrier includes the selective exchange of substances between the cerebrospinal fluid and the extracellular fluid of brain cells. Ultimately, through the simulation of the cerebral blood circulation system, the organic connection between the blood, blood-brain barrier, brain tissue and cerebrospinal fluid in the simulated cerebral circulation is achieved. At the same time, it has the feasibility of expanding the structural and functional characteristics of the simulated blood-cerebrospinal fluid and cerebrospinal fluid-blood barrier. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0056] Figure 4 yes Figure 3 Schematic diagram of the usage status;

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

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

[0059] Figure 7 It is a schematic diagram of the use state of the double negative pressure defoaming device of the present invention;

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

[0061] Fig. 9 yes Figure 8 A magnified schematic diagram of area A in the middle. DETAILED DESCRIPTION

[0062] The above content is a detailed description of this patent in combination with specific implementation methods, and it cannot be determined that the specific implementation methods of this patent are limited to the above description. For ordinary technicians in the technical field to which this patent belongs, without departing from the concept of this patent, several substitutions or modifications made to the above-described implementation methods should be deemed to belong to the protection scope of this patent. In the description of this specification, in this specification, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In the absence of contradictions, those skilled in the art can combine and combine 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 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 is provided with a venous channel inlet through hole 11, a venous channel outlet through hole 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 is provided with a venous channel inlet shallow groove 31, a venous channel outlet shallow groove 32, a venous unit 33, a venous inlet connecting channel 34, and a venous outlet connecting channel 35; the venous unit 33 is arranged below the brain tissue culture unit 13, and the arterial unit 14 is arranged above the brain tissue culture unit 13; the venous unit A porous film 2 is arranged between the brain tissue culture unit 13 and the brain tissue culture unit 13; the position of the venous channel entrance through hole 11 corresponds to the position of the venous channel entrance shallow groove 31 in the vertical direction, and the position of the venous channel outlet through hole 12 corresponds to the position of the venous channel outlet shallow groove 32 in the vertical direction; the ventricular unit 15 is arranged on the right side of the brain tissue culture unit 13; the venous channel entrance shallow groove 31 is connected to the venous unit 33 through the venous entrance 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 film 2 is made of PC or PET. In this embodiment, the height of the upper chip is set to 4-6mm, the diameters of the venous channel inlet through hole 11 and the venous channel outlet through hole 12 are set to 3-4mm, the height of the brain tissue culture unit 13 is set to 0.2-0.5mm, the length and width are set to 4mm and 5mm respectively, the diameter of the arterial unit 14 is set to 3mm, the width and length of the ventricular unit 15 are rounded rectangles of 3mm and 6mm respectively, the diameters of the brain tissue culture unit inlet 16 and the brain tissue culture unit outlet 17 are set to 1-2mm; the diameters of the venous channel inlet through hole 11 and the venous channel outlet through hole 12 are set to 3-4mm; the characteristic depths in the lower chip are all set to 0.4-0.6mm; the diameters of the venous channel inlet shallow groove 31 and the venous channel outlet shallow groove 32 are set to 3-4mm; the length and width of the venous unit 33 are set to 4mm and 5mm respectively; the widths of the venous inlet connecting channel 34 and the venous outlet connecting channel 35 are set to 1-2mm; the pore size of the porous film 2 is 3-8μm.

[0064] Taking into account the overall experimental design and subsequent application requirements, the chip can use PDMS material with good air permeability, high transparency and easy processing, or it can choose materials with good biocompatibility and thermoplastic mechanical processing, including PMMA, PC, COC and other materials according to the experimental situation.

[0065] During the implementation process, the lower layer of the porous film 2 is inoculated with brain microvascular endothelial cells. The brain tissue culture unit is filled with a porous microfluidic medium embedded with neurons, astrocytes and microglia, which is set to the brain parenchyma and capillaries distributed in the brain parenchyma. The arterial unit is inoculated with brain microvascular endothelial cells. As an arterial structure, basement membrane extract (BME) hydrogel, fibrinogen, gelatin, laminin, collagen, etc. can be selected as the material for the porous microfluidic medium. In practical applications, BME hydrogel is preferred for porous microfluidic media. On the right side of the upper structure is the ventricular structure. The ventricular structure and the brain tissue culture unit liquid interact bidirectionally, but the liquid does not overflow into the artery, that is, the liquid in the artery is set to flow unidirectionally.

[0066] This microfluidic brain organ chip can simulate the overall structure of the brain, including the blood-brain barrier, the cerebrospinal fluid-brain barrier, and the cerebral blood circulation system that passes through the brain and collects into the veins after the blood-brain barrier. The simulation of the blood-brain barrier includes the physical and metabolic barriers that regulate the molecular exchange between the blood and the brain. The simulation of the cerebrospinal fluid-brain barrier includes the selective exchange of substances between the cerebrospinal fluid and the extracellular fluid of brain cells. Ultimately, through the simulation of the cerebral blood circulation system, the organic connection between the blood, blood-brain barrier, brain tissue and cerebrospinal fluid in the simulated cerebral circulation is achieved. At the same time, it has the feasibility of expanding the structural and functional characteristics of the simulated blood-cerebrospinal fluid and cerebrospinal fluid-blood barrier.

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

[0068] Solidworks software is used to draw the design of the chip. The chip can be made of PDMS and thermoplastics. For chips made of thermoplastics, the chip module can be obtained by injection molding or CNC processing, and then the chip is assembled by hot pressing bonding. For PDMS chip production, PMMA is processed by CNC processing to obtain a mold, or the mold can be obtained by 3D printing. The PDMS chip production process is as follows:

[0069] The PDMS chip production process includes:

[0070] S1 Pre-processing

[0071] The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1, and stirred using an existing stirring device (such as a stirring rod or a fork) or the toothed rolling roller device of the present invention until fine bubbles are generated to allow for full mixing (the PDMS prepolymer and the curing agent must be fully mixed, otherwise the chip quality will be affected); because bubbles will naturally be generated when the PDMS prepolymer and the curing agent are stirred, the use of existing stirring devices such as a stirring rod will not only cause excessive air to be stirred into the liquid, but also the stirring and mixing efficiency is low (the PDMS prepolymer is a viscous liquid); in order to reduce the amount of air mixed into the stirring process, improve the efficiency of the subsequent vacuum degassing operation, and improve the stirring and mixing efficiency, in this embodiment, the toothed rolling roller device of the present invention may be preferably used to achieve the above-mentioned technical effects. The first embodiment of the toothed rolling roller device 4, such as Figures 3-4 As shown, it includes a left bracket and a right bracket 41, and the left bracket and the right bracket are connected by a cross bar 49; more than three rolling rollers are arranged between the left bracket and the right bracket 41, and the outer walls of the rolling rollers are provided with tooth marks; the three rolling rollers are arranged in a triangle to form a group, wherein the first rolling roller 42 and the second rolling roller 48 are arranged at the same horizontal line at the lower part, and the third rolling roller 43 is arranged between the first rolling roller 42 and the second rolling roller 48, and the third rolling roller 43 is arranged above the first rolling roller and the second rolling roller, and the tooth marks of the third rolling roller 43 are respectively meshed with the tooth marks of the first rolling roller 42 and the tooth marks of the second rolling roller 48; the effect of such arrangement is: when using, by pushing the first rolling roller 42 and the second rolling roller 48 to roll, the third rolling roller 43 is driven, and the PDMS prepolymer and the curing agent are stirred and mixed through the meshing and rotation of the tooth marks of the three rolling rollers, and at the same time as Figure 4 As shown, when in use, the three rollers are immersed below the liquid surface to achieve mixing, effectively preventing excessive air from mixing into the liquid, and stirring is achieved by relying on the tooth marks of the three rotating rollers, which significantly improves 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 side rod and a right connecting side rod 45, which are rotatably connected to the left bracket and the right bracket 41 respectively through a rotating shaft 44, and the upper parts of the left connecting side rod and the right connecting side rod 45 are connected by a connecting rod, and an operating rod 46 is connected to the middle part of the connecting rod, and 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, the second embodiment of the toothed rolling roller device 4 can adjust the height of the three rolling rollers according to the depth of the liquid, so as to ensure that the height of the three rolling rollers is adjusted along with the depth of the target liquid, and ensure that the three rolling rollers are immersed in the liquid. The only difference between the second embodiment and the first embodiment is that the first adjustment long hole 413, the second adjustment long hole 416 and the third adjustment long hole 411 are respectively provided on the left bracket and the right bracket 41; the first adjustment long hole 413 and the second adjustment long hole 416 are provided in the horizontal direction, and the third adjustment long hole 411 is provided in the vertical direction; as shown in FIG. Figure 6 As shown, taking the first adjustment slot 413 as an example, a T-shaped sleeve 415 is movably provided in the first adjustment slot 413, and then a thread is provided on the outside of the T-shaped sleeve 415, and the T-shaped sleeve 415 is locked and fixed by providing an upward nut 414 on the outside, and the rolling shaft of the first rolling roller 42 can be rotatably inserted into the T-shaped sleeve 415, that is, the position of the first rolling roller 42 is adjusted by adjusting the positions of the T-shaped sleeves 415 on both sides; in some embodiments, the first adjustment slot 413 is provided with a T-shaped sleeve 415, and the T-shaped sleeve 415 is provided with a thread on the outside ... provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 4 3. The second adjustment long hole 416 and the third adjustment long hole 411 are also respectively provided with scales 412 to increase the accuracy of the adjustment; when 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 is engaged with the first rolling roller 42 and the second rolling roller 48 respectively, so as to reduce the height of the three rolling rollers, thereby ensuring that the three rolling rollers can be always immersed below the liquid surface for stirring operation.

[0073] The mixed PDMS prepolymer can be placed in a vacuum drying dish to eliminate bubbles, but using this traditional method, it generally takes more than 1 hour to eliminate bubbles (the bubbles must be fully eliminated, otherwise there will be defects such as bubbles and holes after curing, resulting in unqualified chips); in this embodiment, the double negative pressure degassing device of the present invention can be preferably used, which can significantly improve the bubble elimination speed and elimination effect, and greatly improve the work efficiency; Figures 7 to 9As shown, the double negative pressure defoaming device 6 includes a bottom plate 63, a separation plate 62 and an upper cover 61; the separation plate 62 is detachably arranged between the bottom plate 63 and the upper cover 61, and its contact surface can be provided with a silicone ring to improve the sealing performance; the bottom surface 621 of the separation plate 62 is provided with a plurality of squeezing holes 622; the squeezing holes 622 are in a conical structure, and the aperture d1 of the upper opening is larger than the aperture d2 of the lower opening. The purpose of this design is to promote the mixed PDMS prepolymer to flow downward by relying on the squeezing holes The structure of 622 squeezes small bubbles uniformly mixed in the liquid upward, significantly improving the efficiency of eliminating small bubbles in the liquid; the first exhaust pipe 67 and the second exhaust pipe 66 are respectively provided on one side of the bottom plate 63 and the separation plate 62, and the first exhaust pipe 67 and the second exhaust pipe 66 are respectively provided with a first valve 65 and a second valve 64; a first pressure gauge 611 and 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 exhaust pipe 67. When in use, pour the mixed liquid from which bubbles are to be eliminated onto the bottom surface 621, and then use the air pump 7 to evacuate the top of the separation plate 62 and the bottom plate 63. Gravity can be used to cause the mixed liquid to flow downward from the bubble squeezing hole 622, or the following control method can be selected: the pressures on the upper and lower sides of the liquid can be checked by the first pressure gauge 611 and the second pressure gauge 651, and then the upper and lower pressure differences can be 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 speed at which the liquid flows downward, and the gas in the liquid is squeezed upward and then drawn above the liquid surface, and the liquid flowing downward is further subjected to the vacuum degree of the bottom plate 63 to continue to eliminate bubbles. Through the above principle, the bubble elimination speed and elimination effect in this step can be significantly improved, thereby greatly improving the quality and work efficiency of chip preparation, and having good repeatability.

[0074] Pour the debubbled PDMS prepolymer onto the mold and perform vacuum debubbling again until there are no bubbles in the PDMS prepolymer; transfer it to an 80°C oven and heat-cure it for 1 hour; finally, carefully peel off the cured PDMS from the mold and cut it into PDMS blocks of appropriate sizes for subsequent use.

[0075] S2 Punch Hole

[0076] Use a puncher of corresponding size to punch holes at a vertical angle at the corresponding positions on the PDMS upper chip.

[0077] S3 chip package

[0078] Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, the PET film needs to be aminosilanized to achieve bonding. In the aminosilanization process, first prepare a 5% 3-aminopropyltriethoxysilane (APTES) solution with water, preheat it to 80°C with a hot plate, clean the PDMS chip with isopropanol and blow dry it with nitrogen or compressed air, and clean both sides of the PDMS chip with Scotch tape to remove any obvious debris. Treat the PET film with oxygen plasma for 30 seconds (80W) and transfer it to the preheated 5% APTES solution for 30 minutes. Then, wash the PET film with deionized water and dry it at room temperature. The aminosilanized PET film and PDMS chip are simultaneously treated with oxygen plasma for 30 seconds (80W) and bonded. Finally, the assembled PDMS chip is placed in an oven and heated at 60°C for 24 hours.

[0079] Brain Organ Chip Construction

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

[0081] To construct neural tissue on the microfluidic chip, 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 (NEM) supplemented with 2mM glutamine and 0.02μg / ml fibroblast growth factor. To obtain astrocytes and microglia in a resting state, they were cultured in serum-free astrocyte culture medium and astrocyte conditioned medium (ACM) for 1 day before injection. The density of each cell suspension was about 8×10 6 / mL. 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 in a ratio of 4:1 (v / v) of BME hydrogel prepolymer to cell volume. In the cell mixture prepared according to this ratio, the proportion of neural progenitor cells developing into mature neurons exceeded 80%, and finally the cell ratio of neurons, astrocytes and microglia was in the range of 5-6:4-5:1 (n:n:n), which is similar to the proportion of human brain. Before injecting the gel, a pin device consistent with the structure of the ventricular unit was inserted into the ventricular unit to prevent the gel from flowing into the ventricular unit when 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 medium prepared with NEM, AM and ACM in a volume ratio of 8:4:1, v / v / v was injected into the venous unit, arterial unit and ventricular unit, and the medium was changed every day. From day 3, NEM was replaced with neural differentiation medium (NDM). The medium was changed every other day until the BBB was reconstructed.

[0082] After 4 days of co-culture of brain tissue neural cells, brain microvascular endothelial cells and pericytes were seeded. First, a mixture containing 400 μg / mL collagen IV and 100 μg / mL fibronectin was prepared with DPBS to coat the porous film and incubated at 37°C for at least 4 hours. The coating solution was then removed and the film was rinsed with DPBS. Brain microvascular endothelial cells and pericytes were suspended in endothelial cell medium (ECM) and pericyte medium (PM), respectively, with a cell density of approximately 1×10 6 / mL. Brain microvascular endothelial cells and pericytes were mixed in a ratio of 9:1 (n / n), and the cell suspension was then injected into the venous unit of the chip. The chip was gently turned over to allow the brain microvascular endothelial cells and pericytes to adhere to the bottom of the porous film. After 3 hours of culture, unattached cells were removed with fresh mixed culture medium (ECM:PM, 9:1 (v / v)). In the same way, brain microvascular endothelial cells and pericytes were inoculated in the arterial unit. The ECM and PM mixed culture medium in the blood side channel (venous unit and top of the BME gel) was replaced 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.

[0083] For the amount of culture medium added, the liquid level of the ventricular unit culture medium should be lower than that of the arterial unit, and not lower than the liquid level of the culture medium at the inlet and outlet of the venous channel. That is, the liquid level of the arterial unit> the liquid level of the ventricular unit culture medium> the liquid level of the culture medium at the inlet and outlet of the venous channel, so as to generate a pressure difference to drive the flow of the culture medium, so that the culture medium in the arterial unit flows through the porous microfluidic medium to the ventricular unit and the venous unit. The culture medium of the ventricular unit flows to the porous microfluidic medium and finally flows back to the vein without overflowing to the top of the porous microfluidic medium.

[0084] Applications of Microfluidic Brain Organ-on-Chips

[0085] The brain organ chip proposed in this proposal has a wide range of applications. Since the chip simulates the arterial-brain-venous cerebral blood flow system and integrates the blood-brain barrier and cerebrospinal fluid-brain barrier, it can relatively completely reconstruct the physiological structure of the brain in vitro, and has the potential feasibility of expanding to complex brain tissue, blood-cerebrospinal fluid barrier, and cerebrospinal fluid-venous barrier function. The chip is used in the study of brain physiological functions, in the study of the effects of substances (food, toxins, drugs) absorbed into the blood on the blood-brain barrier and the brain, and in the study of the brain's response to these substances; or in the study of the effects of intrathecal (subarachnoid space, cerebrospinal fluid) administration on the cerebrospinal fluid-brain barrier and the brain; or in the study of the effects of changes in the internal and external environment on the brain, such as the existence of the cerebral blood circulation system, making it possible to study the effects of environmental factors in the body on the brain, such as the effects of hormones (glucocorticoids produced during chronic stress) or other bioactive substances (lipopolysaccharides) in the circulatory system on the barrier function of brain structures; or in the study of the pathogenesis of various brain diseases and the effects of the brain on the periphery; and in the replacement of the use of experimental animals in drug screening.

Claims

1. A microfluidic brain organ chip, characterized in that: The chip body comprises an upper chip, a porous film and a lower chip; the upper chip is provided with a venous channel inlet through hole, a venous channel outlet through hole, 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 is provided with 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 arranged below the brain tissue culture unit, and the porous film is arranged between the venous unit and the brain tissue culture unit; the position of the venous channel inlet through hole corresponds to the position of the venous channel inlet shallow groove in the vertical direction; the position of the venous channel outlet through hole corresponds to the position of the venous channel outlet shallow groove in the vertical direction; 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 arranged above the brain tissue culture unit; the ventricle unit is arranged on the right side of the brain tissue culture unit.

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

3. The microfluidic brain organ chip according to claim 1, characterized in that: The material used for the chip body is PDMS or PMMA or PC or COC.

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

5. The microfluidic brain organ chip according to claim 1, characterized in that: The brain tissue culture unit is filled with a porous microfluidic medium embedded with neurons, astrocytes, and microglia.

6. A microfluidic brain organ chip according to claim 5, characterized in that: The material of the porous microfluidic medium is basement membrane extract hydrogel or fibrinogen or gelatin or laminin or collagen; the arterial unit is inoculated with brain microvascular endothelial cells and pericytes.

7. The microfluidic brain organ chip according to claim 1, characterized in that: The liquid between the ventricular unit and the brain tissue culture unit is set to bidirectionally exchange, and the liquid does not overflow into the artery thereof, that is, the liquid in the artery is set to flow unidirectionally.

8. A method for constructing a microfluidic brain organ chip, characterized in that: Constructing a microfluidic brain organ chip as claimed in any one of claims 1 to 7, wherein the brain organ chip is made of PDMS or thermoplastic plastic; The PDMS chip production process includes: S1 Pre-processing The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1 and stirred until fully mixed; the mixed PDMS prepolymer is vacuumed to eliminate bubbles; the debubbled PDMS prepolymer is poured onto the mold to ensure that there are no bubbles in the PDMS prepolymer; then it is transferred to an oven and heated to cure; S2 Punch Hole Use a puncher of corresponding size to punch holes in the PDMS upper chip at a vertical angle at the corresponding position; S3 chip packaging Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, bonding was achieved by aminosilanization of the PET film; The aminosilanization process is: (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water and preheat it to 80°C. Clean the PDMS chip with isopropanol and blow dry it with nitrogen or compressed air. Clean both sides of the PDMS chip to remove any obvious debris. (2) Treat the PET film with oxygen plasma for 30 seconds and transfer it to a preheated 5% APTES solution for 30 minutes; (3) washing the PET film with deionized water and drying it at room temperature; (4) The aminosilanized PET film and the PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and bonded; (5) Place the assembled PDMS chip in an oven and heat at 60 °C for 24 h; S4 Brain Organ Chip Construction The assembled PDMS chip was sterilized by washing the channels with 70% ethanol and irradiated with UV light in a biosafety cabinet for at least 15 minutes before use; (1) In order to construct neural tissue on a brain organ chip, 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 a neural expansion medium supplemented with 2 mM glutamine and 0.02 μg / ml fibroblast growth factor, and in order to obtain resting astrocytes and microglia, they were cultured in serum-free astrocyte culture medium and astrocyte ACM conditioned medium for 1 day 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 in a ratio of 4:1 (v / v) of BME hydrogel prepolymer to cell volume, and the cell mixture prepared according to this ratio was , the proportion of neural progenitor cells developing into mature neurons exceeds 80%, and finally the cell ratio of neurons, astrocytes and microglia is within the range of 5-6:4-5:1 (n:n:n); before injecting the gel, insert the pin device consistent with the structure of the ventricular unit into the ventricular unit, and then inject the gel cell mixture into the brain tissue culture unit of the chip placed on ice; after injection, transfer the chip to a 37°C cell culture incubator and culture for 30 minutes to gel; after gelation, inject a serum-free mixed culture medium prepared with a ratio of NEM:AM:ACM of 8:4:1 (v / v / v) into the venous unit, arterial unit and ventricular unit, and then replace the culture medium every day; from the third day onwards, replace NEM with neural differentiation medium; in the later stage, the neural differentiation medium is replaced every other day until the BBB is reconstructed; (2) After 4 days of co-culture of brain tissue neural cells, brain microvascular endothelial cells and pericytes were inoculated: First, a mixture containing 400 μg / mL collagen IV and 100 μg / mL fibronectin was prepared with DPBS to coat the porous membrane and incubated at 37°C for at least 4 h; Then remove the coating solution and rinse the film with DPBS; suspend brain microvascular endothelial cells and pericytes in endothelial cell culture medium and pericyte culture medium respectively; mix brain microvascular endothelial cells and pericytes in a ratio of 9:1 (n / n), and then inject the cell suspension into the venous unit of the chip; gently flip the chip to allow brain microvascular endothelial cells and pericytes to attach to the bottom of the porous film; after culturing for 3 hours, remove the unattached cells; and inoculate brain microvascular endothelial cells and pericytes in the arterial unit in the same way; replace the ECM and PM mixed culture medium in the blood side channel, i.e., the venous unit and the top of the BME gel, every other day; inject the ventricular unit with a mixed culture medium of NDM, AM and ACM; the chip is cultured for another 3 days to form a BBB; The culture medium level of the ventricular unit is lower than that of the arterial unit, and is not lower than the culture medium level at the inlet and outlet of the venous channel; that is, the arterial unit level> the ventricular unit level> the venous channel inlet and outlet level, so as to generate a pressure difference to drive the flow of the culture medium, so that the culture medium in the arterial unit flows through the porous microfluidic medium to the ventricular unit and the venous unit; the ventricular unit culture medium flows to the porous microfluidic medium, and finally flows back to the vein, and does not overflow to the top of the porous microfluidic medium.

9. The method for constructing a microfluidic brain organ chip according to claim 8, characterized in that: In the S1 preliminary processing step, The PDMS prepolymer and the curing agent are mixed by a toothed rolling roller device, and the mixed PDMS prepolymer is vacuumed to eliminate bubbles by a double negative pressure degassing device; The toothed rolling roller device comprises a left bracket and a right bracket, and the left bracket and the right bracket are connected by a cross bar; more than three rolling rollers are arranged between the left bracket and the right bracket, and the outer walls of the rolling rollers are provided with 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 same horizontal line at the lower part, and the third rolling roller is arranged between the first rolling roller and the second rolling roller, and 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 are respectively meshed with the tooth marks of the first rolling roller and the tooth marks of the second rolling roller; The double negative pressure defoaming device includes a base plate, a separation plate and an upper cover; the separation plate is detachably arranged between the base plate and the upper cover; a plurality of squeezing holes are arranged on the bottom surface of the separation plate; the squeezing holes are in a conical structure, and the aperture of the upper opening is larger than the aperture of the lower opening; a first exhaust pipe and a second exhaust pipe are respectively arranged on one side of the base plate and the separation plate, and a first valve and a second valve are respectively arranged on the first exhaust pipe and the second exhaust pipe.

10. Application of microfluidic brain organ chips, characterized by: The microfluidic brain organ chip according to any one of claims 1 to 7 is used for: research on the effects of substances absorbed into the blood on the blood-brain barrier and the brain, and research on the brain's response to these substances; Or applied to the study of the effects of intrathecal drug delivery on the cerebrospinal fluid-brain barrier and the brain; Or the study of the effects of changes in the internal and external environment of the body on the brain; It may be used to study the pathogenesis of various brain diseases and the effects of the brain on the periphery; and to replace the use of experimental animals in the drug screening process.

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