CN110106083B - Cell co-culture flow chamber device for simulating blood brain barrier in vitro - Google Patents
Cell co-culture flow chamber device for simulating blood brain barrier in vitro Download PDFInfo
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- 238000003501 co-culture Methods 0.000 title claims abstract description 47
- 230000008499 blood brain barrier function Effects 0.000 title claims abstract description 35
- 210000001218 blood-brain barrier Anatomy 0.000 title claims abstract description 35
- 238000000338 in vitro Methods 0.000 title claims abstract description 35
- 239000012528 membrane Substances 0.000 claims abstract description 64
- 239000012530 fluid Substances 0.000 claims description 31
- 238000007789 sealing Methods 0.000 claims description 28
- 230000002093 peripheral effect Effects 0.000 claims description 13
- 238000004088 simulation Methods 0.000 claims 1
- 230000000638 stimulation Effects 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 230000024437 response to mechanical stimulus Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 9
- 239000006143 cell culture medium Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 238000010008 shearing Methods 0.000 description 4
- 238000004891 communication Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 230000003139 buffering effect Effects 0.000 description 2
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- 238000013461 design Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
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- 229920001296 polysiloxane Polymers 0.000 description 2
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- 238000012545 processing Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 230000004938 stress stimulation Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M23/34—Internal compartments or partitions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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Abstract
The invention relates to a cell co-culture flow chamber device for simulating a blood brain barrier in vitro, and belongs to the field of research on cell mechanical stimulus response. The cell co-culture flow chamber device for simulating the blood brain barrier in vitro comprises a first plate, a second plate, a third plate and a fourth plate; a smooth flow cavity, an inlet steady flow cavity communicated with one end of the smooth flow cavity and an outlet steady flow cavity communicated with the other end of the smooth flow cavity are defined between the lower surface of the fourth plate and the upper surface of the third plate; the upper surface of the second plate is provided with a plurality of convex bosses, and the middle part of each boss is concave to form a lower cavity; the third plate is provided with a through hole corresponding to the boss; the upper end face of the boss is covered with a semi-permeable membrane with cells planted on both sides, and the semi-permeable membrane covers the lower chamber. The device and the method can realize experiments which closely imitate the stimulation of cells under shear stress in the environment of biological entities.
Description
Technical Field
The invention relates to the field of in-vitro cell culture and research on mechanical stimulus response of cells, in particular to a cell co-culture flow chamber device for simulating a blood brain barrier in vitro.
Background
Forces (e.g., shear forces) can regulate cellular functions by affecting intracellular gene expression and protein synthesis, thereby playing an important role in physiological and pathological processes of cells.
In the prior art, a fluid device for researching the influence of shear force stimulation on cells exists, however, the shearing environment of the existing device and the biological entity environment have large differences, and the test result cannot well reflect the actual situation.
Disclosure of Invention
The invention aims to provide a cell co-culture flow chamber device for simulating a blood brain barrier in vitro, which aims to solve the problem that the fluid shearing environment and the biological entity environment of the existing fluid device have larger difference, so that the test result can not well reflect the actual situation.
Embodiments of the present invention are implemented as follows:
The embodiment of the invention provides a cell co-culture flow chamber device for simulating a blood brain barrier in vitro, which comprises a first plate, a second plate, a third plate and a fourth plate, wherein the four plates are configured to be sequentially overlapped and connected together from bottom to top along the vertical direction; a smooth flow cavity, an inlet steady flow cavity communicated with one end of the smooth flow cavity and an outlet steady flow cavity communicated with the other end of the smooth flow cavity are defined between the lower surface of the fourth plate and the upper surface of the third plate; an inlet channel communicated with the inlet steady flow cavity and an outlet channel communicated with the outlet steady flow cavity are formed on the cell co-culture flow cavity device for simulating the blood brain barrier in vitro, so that a flow channel which can enable fluid to flow from the inlet channel to the outlet channel after sequentially flowing through the inlet steady flow cavity, the smooth flow cavity and the outlet steady flow cavity is formed; the upper surface of the second plate is provided with a plurality of convex bosses, and the middle part of each boss is concave to form a lower cavity; the third plate is provided with a through hole corresponding to the boss; the boss is matched in the through hole in a state that the third plate and the second plate are overlapped; the upper end surface of the boss is covered with a semi-permeable membrane with cells planted on both sides, and the semi-permeable membrane covers the lower chamber so as to partially and transparently separate the advection chamber from the lower chamber; the upper surface of the semipermeable membrane and the bottom surface of the advection chamber are coplanar.
Further: the cell co-culture flow chamber device for simulating the blood brain barrier in vitro further comprises a locking ring, wherein the height of the locking ring is smaller than that of the boss; the lock ring is in contact with the periphery of the boss; after the semipermeable membrane is covered on each boss, the locking ring locks the semipermeable membrane on the boss from top to bottom; the lower surface of the third plate is provided with a ring groove for accommodating the locking ring, and in the superposed state of the second plate and the third plate, the locking ring is matched in a ring cavity defined by the upper surface of the second plate and the ring groove. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the semipermeable membrane is conveniently fixed on the boss through the matching between the locking ring and the boss, and can be ensured to be in a tensioning state; the ring groove is arranged at the lower end of the third plate, so that the locking ring can be conveniently accommodated.
Further: the bosses are sequentially arranged along the flow direction of the advection cavity. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the bosses are sequentially arranged along the flow direction of the advection cavity, the lower cavities defined by the bosses are sequentially distributed along the flow direction of the advection cavity, so that metabolites of cells on two sides of the semipermeable membrane corresponding to different lower cavities can be exchanged under the driving of fluid flow, and a co-culture environment which is more in line with the biological entity environment is formed.
Further: the middle part of the boss is recessed to penetrate through the second plate, so that the upper surface of the first plate and the recess enclose a lower chamber. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the boss sets up to link up, makes things convenient for the processing of structure to cavity inner wall under convenient clearance after experimental completion. Also, in this embodiment, it may be further configured that: and an elastic gasket is also overlapped between the first plate and the second plate, is of an annular structure and is enclosed outside each lower cavity. The elastic pad is elastically deformable in the vertical direction to elastically change its thickness. The elastic pad is arranged so that the device also has an additional use mode: under the state that each plate of device sealing connection together, suitably reduce locking force for the second plate is jacked to the elasticity gasket elasticity on the ground, thereby makes the annular inboard at the elasticity gasket form the gap, and the lower terminal surface of each lower cavity of this gap intercommunication makes can realize the material exchange between each lower cavity, thereby forms another type and cultivates the environment altogether, adapts to different experimental demands.
Further: and the connecting screws penetrate through the plates and lock and connect the four plates together. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the connecting screws penetrate through the plates and connect the plates together, so that assembly and disassembly of the plates can be conveniently realized. In this embodiment, in order to avoid leakage from gaps between adjacent plates, the contact between the four plates after the connection of the connecting screws should be tight enough to ensure tightness.
Further: the first plate and/or the fourth plate are/is arranged to be transparent in the area corresponding to the advection cavity. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the first plate and/or the fourth plate are/is arranged to be transparent in the area corresponding to the advection cavity, so that the device can observe the test process in real time.
Further: the upper surface of the first plate is vertically provided with an inlet connecting pipe and an outlet connecting pipe, the inlet connecting pipe is correspondingly communicated with the inlet steady flow cavity, and the outlet connecting pipe is correspondingly communicated with the outlet steady flow cavity. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the inlet connecting pipe and the outlet connecting pipe can facilitate the communication between an externally connected cell culture liquid pipeline and the device. And the inlet connecting pipe and the outlet connecting pipe are vertically arranged, so that the flow velocity of fluid flowing vertically can be converted into horizontal flow through the advection cavity after being fully absorbed and buffered by 90 degrees, and the flow of liquid flowing through the advection cavity is ensured to meet the requirements.
Further: the middle part of the upper surface of the third plate is provided with a concave rectangular groove, and the two long ends of the rectangular groove are concave downwards to form a buffer groove; the lower surface of the fourth plate is a plane, and the rectangular groove and the buffer groove are closed under the state that the fourth plate is connected with the third plate so as to form a advection cavity, an inlet steady flow cavity and an outlet steady flow cavity. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the bottom surface of the buffer tank is positioned below the bottom surface of the advection cavity, so that after fluid enters the buffer tank, the bottom surface and the side wall of the buffer tank are firstly pressed downwards due to inertia to absorb most of vertical speed and then flow upwards to the inlet of the advection cavity, and therefore, due to continuous flow of the fluid, a downward flowing part and an upward flowing part are formed in the buffer tank, the vertical speed component of the fluid is further reduced due to mutual friction and adhesion between the downward flowing part and the upward flowing part, and the excessive vertical speed component affecting the flow stability of the fluid entering the advection cavity is avoided.
Further: the semipermeable membrane is a circular flaky base membrane and an annular peripheral membrane connected with the edge of the base membrane; the semipermeable membrane is reversely buckled on the boss, and the peripheral membrane is tightly pressed between the outer cylindrical surface of the boss and the inner peripheral surface of the through hole of the third plate; the periphery of boss is equipped with elastic sealing ring layer, and elastic sealing ring layer's external diameter is greater than the diameter of through-hole under unstressed condition to form interference fit between messenger's elastic sealing ring layer and the through-hole. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: due to the interference fit, the peripheral membrane of the semipermeable membrane is elastically sealed and pressed between the inner surface of the through hole and the elastic sealing ring layer by the elastic sealing ring layer, so that the semipermeable membrane is prevented from being mechanically damaged on one hand, and the tightness between the through hole and the boss can be ensured on the other hand.
Further: and sealing gaskets are arranged between any two adjacent plates of the four plates. The cell co-culture flow chamber device simulating the blood brain barrier in vitro after the addition of the further content has the additional technical effect that: the sealing gasket can ensure the tightness of the contact surface between the adjacent plates and avoid liquid leakage. Alternatively, the sealing gasket may be a silicone gasket or a gasket of other suitable material. The sealing gasket is of an annular structure corresponding to the peripheral position of each plate, and the advection cavity, the lower cavity, the inlet steady flow cavity and the outlet steady flow cavity are all positioned on the inner side of the sealing gasket, so that liquid leakage on the outer surface of the device is avoided.
The beneficial effects are that:
when the cell co-culture flow cavity device is used, cell culture medium can be introduced from the inlet channel, after the inlet steady flow cavity is used for buffering and stabilizing flow, the cell co-culture flow cavity device passes through the advection cavity in a advection manner, and then is buffered by the outlet steady flow cavity and is introduced from the outlet channel. The semipermeable membrane with two sides for cell planting is fixed on the boss and partially separates the advection cavity above and the lower cavity below. Due to the selective permeation characteristic of the semipermeable membrane, part of substances in the advection cavity can permeate the semipermeable membrane to enter each lower cavity, so that a cell co-culture environment is formed, and when fluid passes through the advection cavity in an advection mode, cells planted on the semipermeable membrane can be applied to be sheared, and therefore shear force stimulation is carried out on co-cultured cells. Because one side of the semi-permeable membrane with cells planted on both sides contacts the fluid in the advection cavity, and the other side contacts the fluid in the lower cavity, the whole semi-permeable membrane is under the infiltration of the fluid, and the semi-permeable membrane is more in line with the biological entity environment compared with the culture device with the existing structure.
The cell co-culture flow chamber device in the scheme adopts a combined design of four plate overlapping, thereby skillfully forming experiments which are convenient for the semi-permeable membrane to fix and realize the stimulation of the cells under the shearing stress very closely imitating the biological entity environment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
A schematic structural diagram (cross-sectional illustration) of a first embodiment of a cell co-culture flow chamber device that mimics the blood-brain barrier in vitro is shown in fig. 1;
a top view of the second plate and locking ring mated is shown in fig. 2;
A front view of the second plate and locking ring in cooperation is shown in fig. 3;
A top view of the third plate is shown in fig. 4;
FIG. 5 is a proportional view of the cross-sectional view of FIG. 4 taken along line A-A;
A top view of the gasket seal is shown in fig. 6;
a top view of the fourth plate is shown in fig. 7;
A top view of the first plate is shown in fig. 8;
A schematic of the structure of a second embodiment of a cell co-culture flow chamber device that mimics the blood brain barrier in vitro (cross-sectional display) is shown in fig. 9.
Icon: 100-cell co-culture flow chamber device simulating blood brain barrier in vitro; 1-a first plate; 2-a second plate; 3-a third plate; 4-fourth plate members; q1-advection cavity; q2-an inlet steady flow cavity; q3-an outlet steady flow cavity; q4-inlet channel; q5-outlet channel; 8-a boss; q6-lower chamber; k1-through holes; 30-a semipermeable membrane; 5-connecting screws; 19-a sealing gasket; 15-inlet connection; 16-outlet connection; a C1-rectangular groove; c2-a buffer tank; 9-locking ring; c4-ring grooves; 35-a carrier film; 36-peri-membrane; 38-an elastic sealing ring layer; 31-elastic pad; f 1-a gap; k5-connecting holes; 17-transparent structure.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the present invention, it should be noted that, if the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate an azimuth or a positional relationship based on that shown in the drawings, or an azimuth or a positional relationship in which the inventive product is conventionally put in use, it is merely for convenience of describing the present invention and simplifying the description, and it is not indicated or implied that the apparatus or element referred to must have a specific azimuth, be constructed and operated in a specific azimuth, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like in the description of the present invention, if any, are used for distinguishing between the descriptions and not necessarily for indicating or implying a relative importance.
Furthermore, the terms "horizontal," "vertical," and the like in the description of the present invention, if any, do not denote a requirement that the component be absolutely horizontal or overhang, but rather may be slightly inclined. As "horizontal" merely means that its direction is more horizontal than "vertical", and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.
In the description of the present invention, it should also be noted that, unless explicitly stated and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" should be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
Example 1
Referring to fig. 1, the present embodiment provides a cell co-culture flow chamber device 100 simulating blood brain barrier in vitro, which comprises four plates, namely a first plate 1, a second plate 2, a third plate 3 and a fourth plate 4, and the four plates are configured to be sequentially connected together in a vertically overlapping manner from bottom to top. A advection cavity Q1, an inlet steady flow cavity Q2 communicated with one end of the advection cavity Q1 and an outlet steady flow cavity Q3 communicated with the other end of the advection cavity Q1 are defined between the lower surface of the fourth plate 4 and the upper surface of the third plate 3. The cell co-culture flow chamber device 100 for simulating the blood brain barrier in vitro is provided with an inlet channel Q4 communicated with the inlet steady flow chamber Q2 and an outlet channel Q5 communicated with the outlet steady flow chamber Q3, so as to form a flow channel (the direction of the flow channel is shown by an arrow in the figure) which can enable fluid to flow from the inlet channel Q4 to the inlet steady flow chamber Q2, the advection chamber Q1 and the outlet steady flow chamber Q3 in sequence and then flow out of the outlet channel Q5. Referring to fig. 2 and 3, a plurality of convex bosses 8 are distributed on the upper surface of the second plate 2, and the middle part of the boss 8 is concave to form a lower chamber Q6. Referring to fig. 4 and 5 in cooperation, the third plate 3 has a through hole K1 corresponding to the boss 8. In a state where the third plate 3 and the second plate 2 are overlapped, the boss 8 is fitted in the through hole K1. The upper end surface of the boss 8 is covered with a semi-permeable membrane 30 with cells planted on both sides, and the semi-permeable membrane 30 covers the lower chamber Q6 to partially and transparently separate the advection chamber Q1 from the lower chamber Q6. The upper surface of the semipermeable membrane 30 and the bottom surface of the advection chamber Q1 are coplanar.
When the cell co-culture flow chamber device in the scheme is used, cell culture medium can be introduced from the inlet channel Q4, after the inlet steady flow chamber Q2 is used for buffering and stabilizing, the cell culture medium passes through the advection chamber Q1 in a advection manner, and then is buffered through the outlet steady flow chamber Q3 and is introduced from the outlet channel Q5. The semipermeable membrane 30 on both sides of which cells are planted is fixed on the boss 8 and partially penetratingly separates the upper advection chamber Q1 and the lower chamber Q6 therebelow. Due to the selective permeation characteristic of the semipermeable membrane 30, part of substances in the advection cavity Q1 can permeate the semipermeable membrane 30 to enter each lower cavity Q6, so that a cell co-culture environment is formed, and cells planted on the semipermeable membrane 30 can be applied to shear force when fluid passes through the advection cavity Q1 in an advection mode, so that shear force stimulation is applied to co-cultured cells. Because one side of the semi-permeable membrane 30 with cells planted on both sides contacts the fluid in the advection chamber Q1 and the other side contacts the fluid in the lower chamber Q6, the whole is under the infiltration of the fluid, which is more in line with the biological entity environment than the culture device with the existing structure.
The cell co-culture flow chamber device in the scheme adopts a combined design of four plate overlapping, thereby skillfully forming experiments which are convenient for the semi-permeable membrane 30 to be fixed and realizing the stimulation of the cells under the shearing stress very closely simulated biological entity environment.
The aforementioned overlapping connection of the four plates can be achieved by passing through the respective plates by a connection screw 5 as shown in fig. 1 and locking the four plates together by threaded connection (the connection screw 5 is shown in the unconnected state). The connecting screws 5 can be distributed at intervals along the circumferential direction of the plates, and specific number and distribution modes can be set according to actual conditions, and only the connection of the plates and the tightness of the contact surfaces of the adjacent plates are required to be realized. For the connection screw 5 to pass through and connect, the four plates are all distributed with a circle of connection holes K5 which are correspondingly communicated with each other. The assembly or disassembly of the plates can be conveniently accomplished by passing through the plates and connecting the plates together by means of the connecting screws 5. In this embodiment, in order to avoid leakage at the contact surface between the adjacent plates, the contact between the four plates after the connection of the connection screw 5 should be tight enough to ensure tightness. In one embodiment, a gasket 19 is interposed between any two adjacent plates of the four plates. The sealing gasket 19 can ensure the tightness of the contact surface between the adjacent plates and avoid liquid leakage. Alternatively, the sealing gasket 19 may be a silicone gasket or a gasket made of other suitable material. Referring to fig. 6, the sealing gasket 19 is of an annular structure corresponding to the peripheral position of each plate, and the advection cavity Q1, the lower cavity Q6, the inlet steady flow cavity Q2 and the outlet steady flow cavity Q3 are all located on the inner sides of the annular structure, so that leakage of the outer surface of the device is avoided. To allow the passage of the connection screw 5, the outer periphery of the sealing gasket 19 is also provided with a corresponding connection hole K5.
Of course, the four plates may be joined together by other means, such as welding, sealant bonding, etc.
Referring to fig. 1, 2 and 3, in this embodiment, a plurality of bosses 8 may be further provided, which are sequentially arranged along the flow direction of the advection chamber Q1. The bosses 8 are sequentially arranged along the flow direction of the advection cavity Q1, the lower cavities Q6 defined by the bosses 8 are sequentially distributed along the flow direction of the advection cavity Q1, so that metabolites of cells on two sides of the semipermeable membrane 30 corresponding to different lower cavities Q6 can be exchanged under the driving of fluid flow, and a co-culture environment which is more in line with the biological entity environment is formed.
In an alternative embodiment, referring to fig. 1 and 7, the upper surface of the first plate 1 is vertically provided with an inlet connection pipe 15 and an outlet connection pipe 16, the inlet connection pipe 15 is correspondingly communicated with the inlet steady flow cavity Q2, and the outlet connection pipe 16 is correspondingly communicated with the outlet steady flow cavity Q3. The inlet connection 15 and the outlet connection 16 can facilitate the communication between an external cell culture fluid pipeline and the device. And the inlet connecting pipe 15 and the outlet connecting pipe 16 are vertically arranged, so that the flow velocity of fluid flowing vertically is fully absorbed and buffered, and then 90-degree angle is converted into horizontal flow through the advection cavity Q1, and the flow of liquid flowing through the advection cavity Q1 is ensured to meet the requirements. In the present embodiment, the middle portion of the upper surface of the third plate 3 has a concave rectangular groove C1, and both long ends of the rectangular groove C1 are concave downward to form a buffer groove C2. The lower surface of the fourth plate 4 is a plane which closes the rectangular groove C1 and the buffer groove C2 in a state where the fourth plate 4 is connected to the third plate 3 to form a advection chamber Q1, an inlet steady flow chamber Q2, and an outlet steady flow chamber Q3. The bottom surface of the buffer tank C2 is located below the bottom surface of the advection cavity Q1, so that after fluid enters the buffer tank C2, the bottom surface and the side wall of the buffer tank C2 are firstly pressed downwards due to inertia to absorb most of the vertical velocity and then flow upwards to the inlet of the advection cavity Q1, so that due to continuous flow of the fluid, a downward flowing part and an upward flowing part are formed in the buffer tank C2, the vertical velocity component of the fluid is further reduced due to mutual friction and adhesion between the downward flowing part and the upward flowing part, and the excessive vertical velocity component affecting the flow stability of the fluid entering the advection cavity Q1 is avoided.
Referring to fig. 7 and 8, for the convenience of observation, the area of the first plate 1 and/or the fourth plate 4 corresponding to the advection chamber Q1 is provided with a transparent structure 17 as an observation window. Alternatively the transparent structure 17 may be quartz glass. The area of the first plate 1 and/or the fourth plate 4 corresponding to the advection cavity Q1 is provided with a transparent structure 17, so that the device can observe the test process in real time.
Referring to fig. 1,2 and 3, in this embodiment, the cell co-culture flow chamber device 100 simulating the blood brain barrier in vitro further comprises a locking ring 9, and the height of the locking ring 9 is smaller than the height of the boss 8. The lock ring 9 is in contact with the outer periphery of the boss 8. After the semipermeable membrane 30 is covered on each boss 8, the locking ring 9 locks the semipermeable membrane 30 on the boss 8 from top to bottom. The lower surface of the third plate 3 is provided with a ring groove C4 for receiving the locking ring 9, and in the folded state of the second plate 2 and the third plate 3, the locking ring 9 is fitted in a ring cavity defined by the upper surface of the second plate 2 and the ring groove C4. The semipermeable membrane 30 is conveniently fixed on the boss 8 by the cooperation between the locking ring 9 and the boss 8, and can be ensured to be in a tensioned state. The lower end of the third plate 3 is provided with a ring groove C4 which can conveniently accommodate the locking ring 9. Further, the semipermeable membrane 30 has a circular sheet-like base membrane 35 and an annular peripheral membrane 36 connected to the edge of the base membrane 35. The semipermeable membrane 30 is inversely buckled on the boss 8, and the peripheral membrane 36 is tightly pressed between the outer cylindrical surface of the boss 8 and the inner peripheral surface of the through hole K1 of the third plate 3. The periphery of boss 8 is equipped with elastic sealing ring layer 38, and elastic sealing ring layer 38 external diameter under the unstressed condition is greater than the diameter of through-hole K1 to form interference fit between elastic sealing ring layer 38 and the through-hole K1. Due to the interference fit, the peripheral membrane 36 of the semipermeable membrane 30 is elastically sealed and pressed between the inner surface of the through hole K1 and the elastic sealing ring layer 38 by the elastic sealing ring layer 38, so that on the one hand, the semipermeable membrane 30 is prevented from being mechanically damaged, and on the other hand, the sealability between the through hole K1 and the boss 8 can be ensured.
Example two
Referring to fig. 9, the cell co-culture flow chamber device 100 simulating the blood brain barrier in vitro disclosed in this embodiment is a variation from the first embodiment in that, on the basis of recessing the middle portion of the boss 8 through the second plate 2 to form the lower chamber Q6 by the upper surface of the first plate 1 and the recessing, an elastic spacer 31 is further stacked between the first plate 1 and the second plate 2, and the elastic spacer 31 has a ring-shaped structure and is enclosed outside each lower chamber Q6. The elastic pad 31 is elastically deformable in the vertical direction to elastically change its thickness. The provision of the resilient pad 31 allows the device to have an additional mode of use: in the state that the plates of the device are connected together in a sealing way, the locking force is properly reduced, so that the elastic gasket 31 elastically pushes up the second plate 2, a gap f1 is formed on the annular inner side of the elastic gasket 31, the gap f1 is communicated with the lower end face of each lower chamber Q6, and the substances in each lower chamber Q6 can be exchanged, thereby forming another type of co-culture environment, and adapting to different test requirements. In addition, boss 8 sets up to link up, makes things convenient for the processing of structure to cavity Q6 inner wall under convenient clearance after the experiment is accomplished.
In this embodiment, the sealing gasket 19 between the first plate 1 and the second plate 2 may be omitted or may be used.
Example III
Referring to fig. 1-9, the present embodiment discloses a cell fluid shear stress stimulation test method, which is based on the cell co-culture flow chamber device 100 simulating blood brain barrier in vitro according to the first/second embodiment, and comprises the following steps: cell culture medium is introduced from inlet channel Q4. After the cell culture medium is buffered and stabilized in the inlet steady flow cavity Q2, the cell culture medium passes through the advection cavity Q1 in a advection manner, and after being buffered in the outlet steady flow cavity Q3, the cell culture medium is discharged from the outlet channel Q5. The permeable substance including water in the fluid in the cell culture medium permeates the semipermeable membrane 30 and fills each lower chamber Q6 (the co-culture environment in which substance exchange between each lower chamber Q6 is performed through the gap f1 can also be realized in the second embodiment), so that the semipermeable membrane 30 is immersed in the fluid at both sides, and a cell co-culture environment is formed. Cells seeded on the semipermeable membrane 30 are applied with shear force while fluid is advectively passed through the advection chamber Q1, thereby achieving loading shear force stimulation to co-cultured cells.
The cell fluid shear stress stimulation test method is based on the cell co-culture flow chamber device 100 for simulating the blood brain barrier in vitro, can simulate the biological entity environment well, and has higher use value as a test result.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The utility model provides a cell co-culture flow chamber device of in vitro simulation blood brain barrier which characterized in that:
The device comprises a first plate, a second plate, a third plate and a fourth plate, wherein the four plates are sequentially overlapped and connected together from bottom to top along the vertical direction;
a advection cavity, an inlet steady flow cavity communicated with one end of the advection cavity and an outlet steady flow cavity communicated with the other end of the advection cavity are defined between the lower surface of the fourth plate and the upper surface of the third plate; an inlet channel communicated with the inlet steady flow cavity and an outlet channel communicated with the outlet steady flow cavity are formed on the cell co-culture flow cavity device for simulating the blood brain barrier in vitro, so that a flow channel which can enable fluid to flow through the inlet steady flow cavity, the advection cavity and the outlet steady flow cavity from the inlet channel in sequence and then flow out of the outlet channel is formed;
A plurality of convex bosses are distributed on the upper surface of the second plate, and the middle part of each boss is concave to form a lower cavity;
The third plate is provided with a through hole corresponding to the boss; the boss is matched in the through hole in a state that the third plate and the second plate are overlapped;
the upper end face of the boss is covered with a semi-permeable membrane with cells planted on two sides, and the semi-permeable membrane covers the lower cavity so as to partially and penetratively separate the advection cavity from the lower cavity;
the upper surface of the semi-permeable membrane and the bottom surface of the advection cavity are coplanar;
The middle part of the upper surface of the third plate is provided with a concave rectangular groove, and the long ends of the rectangular groove are concave downwards to form buffer grooves;
the lower surface of the fourth plate is a plane, and the rectangular groove and the buffer groove are sealed under the state that the fourth plate is connected with the third plate so as to form the advection cavity, the inlet steady flow cavity and the outlet steady flow cavity;
The upper surface of the first plate is vertically provided with an inlet connecting pipe and an outlet connecting pipe, the inlet connecting pipe is correspondingly communicated with the inlet steady flow cavity, and the outlet connecting pipe is correspondingly communicated with the outlet steady flow cavity.
2. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein:
The device also comprises a locking ring, wherein the height of the locking ring is smaller than that of the boss; the lock ring is in contact sleeving connection with the periphery of the boss; after the semipermeable membrane is covered on each boss, the locking ring locks the semipermeable membrane on the boss from top to bottom;
The lower surface of the third plate is provided with a ring groove for accommodating the locking ring, and the locking ring is matched in a ring cavity defined by the upper surface of the second plate and the ring groove in the overlapped state of the second plate and the third plate.
3. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein:
The bosses are sequentially arranged along the flow direction of the advection cavity.
4. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein:
and a connecting screw passing through the plurality of plates and locking and connecting the four plates together.
5. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein: and the first plate and/or the fourth plate are/is arranged in a transparent structure in the area corresponding to the advection cavity.
6. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein:
the semi-permeable membrane is a circular flaky bottom membrane and an annular peripheral membrane connected to the edge of the bottom membrane;
The semi-permeable membrane is reversely buckled on the boss, and the peripheral membrane is tightly pressed between the outer cylindrical surface of the boss and the inner peripheral surface of the through hole of the third plate;
The periphery of boss is equipped with the elastic sealing ring layer, the external diameter of elastic sealing ring layer under the unstressed condition is greater than the diameter of through-hole, so that elastic sealing ring layer with form interference fit between the through-hole.
7. The in vitro blood brain barrier mimicking cell co-culture flow chamber device of claim 1, wherein:
And sealing gaskets are arranged between any two adjacent plates of the four plates.
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| CN112986545B (en) * | 2021-02-02 | 2021-09-28 | 四川大学 | Mechanical experiment device for simulating tumor cell invasion in vitro |
| CN113234593A (en) * | 2021-03-26 | 2021-08-10 | 北京航空航天大学 | Double-layer cell culture device for differential co-culture of two cells |
| CN113648287B (en) * | 2021-07-30 | 2023-05-05 | 深圳华源再生医学有限公司 | Cell loading device and preparation method thereof |
| CN114181830B (en) * | 2021-12-09 | 2023-05-30 | 中国人民解放军空军军医大学 | Cell culture device for simulating blood brain barrier in vitro |
| CN115595268A (en) * | 2022-12-14 | 2023-01-13 | 四川大学（Cn） | Cell co-culture flow cavity device for simulating human body microcirculation vortex in vitro |
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