CN212077076U

CN212077076U - Micro-fluidic experimental board

Info

Publication number: CN212077076U
Application number: CN202020548830.9U
Authority: CN
Inventors: 付伟欣; 何宇涵; 关一民
Original assignee: Shanghai Industrial Utechnology Research Institute
Current assignee: Shanghai Industrial Utechnology Research Institute
Priority date: 2020-04-14
Filing date: 2020-04-14
Publication date: 2020-12-04
Anticipated expiration: 2030-04-14

Abstract

The utility model provides a micro-fluidic experimental plate, this micro-fluidic experimental plate includes upper strata flow path board, the upper seal membrane, lower floor's seal membrane, porous membrane and lower floor's flow path board, wherein, upper strata flow path board is including first, the second liquid import, it is first, the cavity is cultivateed to second liquid export and upper strata, the upper seal membrane is located upper strata flow path board below, lower floor's seal membrane is located upper strata seal membrane below, the porous membrane is located, between the lower floor's seal membrane, lower floor's flow path board is located lower floor's seal membrane below and includes lower floor's cultivation cavity, first liquid import, upper strata cultivation cavity and first liquid export communicate in proper order, the second liquid import, lower floor's cultivation cavity and second liquid export communicate in proper order. The utility model provides a can be used to single face cell culture or the two-sided standard platform of cultivateing altogether of two kinds and above cell contains two-sided can supply liquid in succession, can construct the culture cavity of physiology shearing force microenvironment, can realize higher experimental repeatability to can mass industrial production.

Description

Micro-fluidic experimental board

Technical Field

The utility model belongs to micro-fluidic and bio-pharmaceuticals field relates to a micro-fluidic experiment board.

Background

Conventional cell culture is widely applied to drug screening, new drug testing and biological and pharmaceutical research in universities and colleges as a physiological model. At present, most cell culture modes applied as a drug screening physiological model are limited to single-layer two-dimensional culture of single cells, and a few scientific research institutions in higher schools use two-dimensional or three-dimensional spherical culture of various cells as a physiological model or a pathological model. However, according to the published data in various documents in recent years, the physiological indexes of the two-dimensional culture of only a single cell are different from the biological performances of the cell in the real animal body. These documents demonstrate that new drug screening is responsible for the high failure rate into clinical human trials after passing conventional cell culture drug testing. It should be noted that even animal experimental results (e.g. mouse, rat experiments) often show a high rate of dissimilarity with human clinical experimental results. This phenomenon indicates that neither conventional two-dimensional single-species cell culture nor animal experiments are the most suitable methods and approaches for drug screening and related research physiological models.

The organ chip is used as a means for constructing a physiological model which is closer to human tissues, has the capability of simulating the physical structure of the physiological model, has the possibility of building a physiological microenvironment, is a platform which is more efficient and referential than conventional cell culture and animal experiments, can effectively shorten the research and development period of medicines and evaluate individual differences so as to be beneficial to accurate treatment in the aspect of application of medicine testing and new medicine screening, and can be used as a more advanced tool method for researching physiological and pathological models by scientific research institutions of higher colleges and universities. However, most organ chips in academia are designed and processed temporarily according to the research needs of specific organs, and have no universality and stability. And due to many artificial operation differences, such as the number, distribution position, experiment operation methods and other artificial factors of each cell inoculation, the organ chip physiological models are different in batches, so that the repeatability of the experiment results is poor.

The bio-printing technology is a means which can help to establish a physiological model manually, is standardized and quantifiable, can establish production in batches on the premise of keeping stability, can eliminate a lot of randomness and deviation which cannot be eliminated in manual operation, and can help to establish a better experimental platform and tools for organ chip technology and in-vitro tissue engineering.

SUMMERY OF THE UTILITY MODEL

In view of the above-mentioned shortcomings of the prior art, it is an object of the present invention to provide a microfluidic assay plate for solving the problem of the prior art that lacks a standardized tool for multi-cell co-culture.

In order to achieve the above objects and other related objects, the present invention provides a microfluidic circuit board, including:

the upper layer of the channel plate comprises a first liquid inlet, a second liquid inlet, a first liquid outlet, a second liquid outlet and an upper layer of culture chamber, the upper layer of culture chamber vertically penetrates through the upper layer of channel plate, the first liquid inlet, the second liquid inlet, the first liquid outlet and the second liquid outlet are all opened from the surface of the upper layer of channel plate, and the first liquid inlet, the upper layer of culture chamber and the first liquid outlet are sequentially communicated;

the upper-layer sealing film is positioned below the upper-layer flow channel plate, a first flow channel hole, a second flow channel hole and a first through hole are formed in the upper-layer sealing film, the first flow channel hole is communicated with the second liquid inlet, the second flow channel hole is communicated with the second liquid outlet, and at least one part of the upper-layer culture chamber is exposed out of the first through hole;

the lower sealing film is positioned below the upper sealing film, a third flow channel hole, a fourth flow channel hole and a second through hole are formed in the lower sealing film, the third flow channel hole is communicated with the first flow channel hole, the fourth flow channel hole is communicated with the second flow channel hole, and the second through hole is aligned to the first through hole;

the porous membrane is positioned between the upper sealing membrane and the lower sealing membrane and covers the first through hole and the second through hole;

lower floor's runner plate is located lower floor's sealed membrane below, lower floor's runner plate includes the lower floor and cultivates the cavity, the lower floor is cultivateed the cavity and is run through from top to bottom the lower floor's runner plate, the second via hole exposes the lower floor cultivates at least part of cavity, the third runner hole the lower floor cultivates the cavity and the fourth runner hole communicates in proper order.

Optionally, the microfluidic experimental plate further comprises an upper layer breathable film and a lower layer breathable film, wherein the upper layer breathable film is used for sealing the opening of the upper layer culture chamber located on the front surface of the upper layer flow channel plate, and the lower layer breathable film is used for sealing the opening of the lower layer culture chamber located on the back surface of the lower layer flow channel plate.

Optionally, the front of the upper layer runner plate is provided with a cavity, the upper layer culture chamber is located at the front opening of the upper layer runner plate is located at the bottom surface of the cavity, and the upper layer breathable film is attached to the bottom surface of the cavity.

Optionally, the upper strata is cultivateed the cavity and is included upper main culture chamber and upper runner of upper and lower intercommunication, the upper runner is located the upper runner board back, the upper main culture chamber is located the upper runner board is positive, the opening area in upper main culture chamber is less than the opening area of upper runner, just the opening in upper main culture chamber is located the opening range of upper runner, first liquid inlet the upper runner reaches first liquid outlet communicates in proper order.

Optionally, the upper layer flow channel is shuttle-shaped.

Optionally, the cavity is cultivateed to lower floor includes lower floor's main culture chamber and lower floor's runner of upper and lower intercommunication, the runner of lower floor is located the runner board of lower floor is positive, the main culture chamber of lower floor is located the runner board back of lower floor, the opening area in lower floor's main culture chamber is less than the opening area of lower floor's runner, just the main opening of cultivateing the chamber of lower floor is located the opening range of lower floor's runner, the third runner hole lower floor's runner reaches the fourth runner hole communicates in proper order.

Optionally, the lower layer flow channel is shuttle shaped.

Optionally, the microfluidic experimental plate further comprises an upper sealing plug and a lower sealing plug, an upper cavity matched with the upper sealing plug is arranged on the front surface of the upper flow channel plate, a lower cavity matched with the lower sealing plug is arranged on the back surface of the lower flow channel plate, the upper cavity is communicated with the upper culture chamber, and the lower cavity is communicated with the lower culture chamber.

Optionally, the upper layer culture chamber is in the shape of a shuttle and the lower layer culture chamber is in the shape of a shuttle.

Optionally, at least one of the first liquid inlet, the second liquid inlet, the first liquid outlet, and the second liquid outlet is open from a front surface of the upper flow channel plate, or at least one of the first liquid inlet, the second liquid inlet, the first liquid outlet, and the second liquid outlet is open from a side surface of the upper flow channel plate.

As mentioned above, the utility model discloses a micro-fluidic laboratory panel provides one kind and can be used to the two-sided standard platform of cultivateing of single face cell or two kinds and above cell, contains two-sided can supply liquid in succession, can construct the cell culture cavity of physiology shearing force microenvironment. The utility model discloses a micro-fluidic experiment board can regard as one kind to be convenient for with the standardized platform of cell coculture that cell biological printing technique combines, can inoculate the cell to the chip with standardized, quantized means at the in-process of equipment, realizes higher experimental repeatability, makes the experimental result at every turn have more the referential. The utility model discloses a micro-fluidic experimental board can make in batches in industrial production, is the organ chip product of a stable technology. The utility model discloses can be used to the vascular barrier among the simulation physiological model, the alveolus barrier, organizational structure such as intestines barrier to can regard as simulation tool and means of relevant physiological model material conduction research, the shuttle-type runner can produce even and the shearing force that accords with the physiological value in the porous membrane within range of all cell culture. The utility model discloses a pump bodies such as micro-fluidic experimental board can external peristaltic pump form circulation liquid supply system, can be used to produce the vascular blood velocity of flow that is close physiological value and found high shear force microenvironment.

Drawings

Fig. 1 shows a side view of a microfluidic laboratory plate.

Fig. 2 shows a cross-sectional view of a microfluidic assay plate.

Fig. 3 is a schematic sectional exploded view of the microfluidic test plate.

Fig. 4 is a schematic perspective exploded view of the microfluidic test plate.

Fig. 5 shows a top view of the upper flow field plate.

Fig. 6 shows a bottom view of the upper flow field plate.

Fig. 7 is a top view of the lower flow field plate.

Fig. 8 is a side view of the lower flow field plate.

Fig. 9 shows a top view of a microfluidic assay plate in another embodiment.

Fig. 10 is a schematic perspective exploded view of a microfluidic assay plate in another embodiment.

FIG. 11 is a schematic cross-sectional exploded view of a microfluidic control plate in another embodiment.

Fig. 12 shows the situation that the sealing plug of the microfluidic experimental plate has not blocked the culture chamber opening.

Fig. 13 shows the situation where the sealing plug of the microfluidic assay plate blocks the opening of the culture chamber.

Fig. 14 is a schematic perspective exploded view of the microfluidic test plate.

Description of the element reference numerals

1 upper flow channel plate

101 first liquid inlet

102 second liquid inlet

103 first liquid outlet

104 second liquid outlet

105 upper culture chamber

1051 upper main culture cavity

1052 upper layer flow channel

106 cavity

107 upper cavity

2 Upper sealing film

201 first flow channel hole

202 second flow passage hole

203 first via

3 lower layer sealing film

301 third flow passage hole

302 fourth flow passage hole

303 second via hole

4 porous membranes

5 lower flow channel plate

501 lower layer culture chamber

5011 lower main culture cavity

5012 lower runner

502 cavity

503 lower cavity

6 upper layer breathable film

7 lower layer breathable film

8 external pipeline

9 upper sealing plug

10 lower layer sealing plug

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The present invention can also be implemented or applied through other different specific embodiments, and various details in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Please refer to fig. 1 to 14. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the invention in a schematic manner, and only the components related to the invention are shown in the drawings rather than being drawn according to the number, shape and size of the components in actual implementation, and the form, quantity and proportion of the components in actual implementation may be changed at will, and the layout of the components may be more complicated.

Example one

In this embodiment, please refer to fig. 1 to 4, in which fig. 1 is a side view of the microfluidic circuit board, fig. 2 is a cross-sectional view of the microfluidic circuit board, fig. 3 is a schematic cross-sectional exploded structure diagram of the microfluidic circuit board, and fig. 4 is a schematic three-dimensional exploded structure diagram of the microfluidic circuit board. The micro-fluidic experimental plate comprises an upper flow channel plate 1, an upper sealing film 2, a lower sealing film 3, a porous membrane 4 and a lower flow channel plate 5.

Referring to fig. 5 and 6, fig. 5 is a top view of the upper flow channel plate 1, and fig. 6 is a bottom view of the upper flow channel plate. The upper layer flow channel plate 1 comprises a first liquid inlet 101, a second liquid inlet 102, a first liquid outlet 103, a second liquid outlet 104 and an upper layer culture chamber 105, the upper layer culture chamber 105 penetrates through the upper layer flow channel plate 1 from top to bottom, the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103 and the second liquid outlet 104 are all from the surface opening of the upper layer flow channel plate, and the first liquid inlet 101, the upper layer culture chamber 105 and the first liquid outlet 103 are sequentially communicated.

Referring back to fig. 4, the upper sealing film 2 is disposed under the upper flow channel plate 1, the upper sealing film 2 is provided with a first flow channel hole 201, a second flow channel hole 202 and a first through hole 203, the first flow channel hole 201 is communicated with the second liquid inlet 102, the second flow channel hole 202 is communicated with the second liquid outlet 104, and the first through hole 203 exposes at least a portion of the upper culture chamber 105; the lower sealing film 3 is positioned below the upper sealing film 2, a third flow passage hole 301, a fourth flow passage hole 302 and a second through hole 303 are arranged in the lower sealing film 3, the third flow passage hole 301 is communicated with the first flow passage hole 201, the fourth flow passage hole 302 is communicated with the second flow passage hole 202, and the second through hole 303 is aligned with the first through hole 203; the porous film 4 is positioned between the upper sealing film 2 and the lower sealing film 3 and covers the first via hole 203 and the second via hole 303; the lower runner plate 5 is positioned below the lower sealing film 3.

Referring to fig. 7 and 8, fig. 7 is a top view of the lower flow channel plate 5, and fig. 8 is a side view of the lower flow channel plate 5. The lower flow channel plate 5 comprises a lower culture chamber 501, and the lower culture chamber 501 penetrates through the lower flow channel plate 5 from top to bottom.

Referring back to fig. 4, the second through hole 303 exposes at least a portion of the lower culture chamber 501, and the third flow channel hole 301, the lower culture chamber 501, and the fourth flow channel hole 302 are sequentially communicated with each other.

For example, the upper sealing film 2 and the lower sealing film 3 may include pressure-sensitive films, and the upper flow channel plate 1 and the lower flow channel plate 5 may be made of polymer plastic. Of course, the upper sealing film 2 and the lower sealing film 3 may also be made of other films with good biocompatibility, as long as the upper flow channel plate and the lower flow channel plate 5 can be bonded and sealed well.

As an example, referring back to fig. 2 to 4, the microfluidic experimental plate further includes an upper gas permeable membrane 6 and a lower gas permeable membrane 7, wherein the upper gas permeable membrane 6 is used to close the opening of the upper culture chamber 105 on the front surface of the upper flow channel plate 1, and the lower gas permeable membrane 7 is used to close the opening of the lower culture chamber 501 on the back surface of the lower flow channel plate 5.

For example, as shown in fig. 2 and 3, the front surface of the upper flow channel plate 1 is provided with a cavity 106, the opening of the upper culture chamber 105 on the front surface of the upper flow channel plate 1 is located on the bottom surface of the cavity 106, the upper air permeable membrane 6 can be attached to the bottom surface of the cavity 106 by adhesion to close the opening, and the lower air permeable membrane 7 can also be attached to the predetermined position on the back surface of the lower flow channel plate 5 by adhesion.

It should be noted that a cavity 502 (as shown in fig. 8) may be formed at a predetermined position on the back of the lower flow channel plate 5, the lower breathable film 7 is attached to the bottom surface of the cavity 502, the cavity may not be formed at the predetermined position on the back of the lower flow channel plate 5 (as shown in fig. 2 and 3), and the lower breathable film 7 is protruded from the back of the lower flow channel plate 5.

It should be pointed out, compare in the micro-fluidic chip of common laboratory self-control PDMS or PDMS glass piece bonding confined, the utility model discloses open cavity design after the equipment is accomplished can let the user more conveniently carry out stability better, and the less cell inoculation step of operating error, and be convenient for combine with the biological printing technique, make on the chip make cell print after the resealing become possible, can do further propulsion in repeatability and the stability of organ chip experiment.

As an example, as shown in FIG. 6, upper culture chamber 105 includes upper main culture chamber 1051 and upper flow channel 1052 which are vertically connected, upper flow channel 1052 is located on the back surface of upper flow channel plate 1, upper main culture chamber 1051 is located on the front surface of upper flow channel plate 1, the opening area of upper main culture chamber 1051 is smaller than the opening area of upper flow channel 1052, the opening of upper main culture chamber 1051 is located within the opening range of upper flow channel 1052, and first liquid inlet 101, upper flow channel 1052 and first liquid outlet 103 are sequentially connected.

As shown in FIG. 7, the lower culture chamber 501 includes a lower main culture chamber 5011 and a lower runner 5012 which are vertically connected, the lower runner 5012 is located on the front surface of the lower runner plate 5, the lower main culture chamber 5011 is located on the rear surface of the lower runner plate 5, the opening area of the lower main culture chamber 5011 is smaller than the opening area of the lower runner 5012, and the opening of the lower main culture chamber 5011 is located within the opening area of the lower runner 5012. As shown in fig. 4, the third flow passage hole 301, the lower flow passage 5012, and the fourth flow passage hole 302 are sequentially communicated with each other.

By way of example, the upper flow path 1052 and the lower flow path 5012 are in the shape of a shuttle, which facilitates the generation of uniform and physiological shear forces across all porous membranes for cell culture.

As an example, at least one of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103, and the second liquid outlet 104 is opened from the front surface of the upper flow path plate 1, or at least one of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103, and the second liquid outlet 104 is opened from the side surface of the upper flow path plate 1.

As shown in fig. 5, the openings of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103, and the second liquid outlet 104 are all opened on the front surface of the upper flow channel plate 1, and this arrangement can facilitate the reduction of the thickness of the microfluidic experimental plate as much as possible, and has lower requirements for the focal length configuration of the microscope.

As shown in fig. 9 and 10, openings of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103 and the second liquid outlet 104 are all opened on a side surface of the upper flow channel plate 1 in another embodiment, wherein fig. 9 is a top view of a microfluidic experimental plate, and fig. 10 is a schematic perspective exploded structure view of the microfluidic experimental plate. Specifically, the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103, and the second liquid outlet 104 are all bent to penetrate through the back surface of the upper flow channel plate 1. The openings of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103 and the second liquid outlet 104 are all arranged on the side surface of the upper flow channel plate 1, so that the real-time observation of double-sided cell culture is facilitated, and two sides of the microfluidic experiment plate can be horizontally placed on a microscope for focusing.

As an example, the first liquid inlet and the second liquid inlet may be externally connected to a peristaltic pump or other pump body through an external connection pipe 8, so as to achieve a double-sided microfluidic continuous liquid supply function, and achieve continuous shear force stimulation on cells on both sides of the porous membrane, which is close to a physiological microenvironment value.

As shown in fig. 11 to 14, it is shown that in another embodiment, the culture chamber is closed by a sealing plug instead of a gas permeable membrane, wherein fig. 11 is a schematic cross-sectional exploded structure of the microfluidic circuit board, fig. 12 is a schematic cross-sectional exploded structure of the microfluidic circuit board, fig. 13 is a schematic cross-sectional exploded structure of the microfluidic circuit board, and fig. 14 is a schematic three-dimensional exploded structure of the microfluidic circuit board.

Specifically, the sealing plug divide into upper sealing plug 9 and lower floor's sealing plug 10, upper strata flow path board 1 openly be equipped with upper sealing plug 9 matched with upper cavity 107, lower floor flow path board 5 back be equipped with lower floor's sealing plug 10 matched with lower cavity 503, upper cavity 107 with upper cultivation cavity 105 communicates, lower floor's cavity and 503 lower floor cultivates cavity 501 and communicates.

By way of example, the openings of the upper cavity 107 and the lower cavity 503 include, but are not limited to, circular shapes, and the sidewalls of the upper cavity 107 and the lower cavity 503 may be stepped to achieve better sealing effect. The material of the upper layer sealing plug 9 and the lower layer sealing plug 10 includes but is not limited to polymer.

In this embodiment, the upper culture chamber and the lower culture chamber are in a spindle shape, which is beneficial to generate uniform shear force in the range of all porous membranes for cell culture and meet physiological values.

It should be noted that fig. 11 to 14 show the case that the openings of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103 and the second liquid outlet 104 are all opened at the side of the upper flow channel plate 1, however, in other embodiments, the positions of the openings of the first liquid inlet 101, the second liquid inlet 102, the first liquid outlet 103 and the second liquid outlet 104 may also be adjusted as required, for example, may be disposed at the front of the upper flow channel plate 1, and herein, the protection scope of the present invention should not be limited too.

It should be noted that the cell migration and invasion test plate has similar functions for culturing cells on both sides of the semipermeable membrane, but the cell migration and invasion test plate has no continuous liquid supply channel and can only provide static culture without shear force, and such cell invasion test plate is mostly difficult to operate when different cells are respectively inoculated on both sides, and a large amount of cell suspension is needed to be soaked, which causes waste. And the utility model can overcome the problem.

In addition, the self-made microfluidic organ chip for general experiments is usually made into a closed flow channel chamber, and cell suspension is not only allowed to stand after flowing in during cell inoculation, so that the cell suspension can be attached to the wall at will. Such unstable mode of examining the operation technique of the experiment easily causes the difference of the experiment result of each batch between each experiment, even if the same experiment condition is changed to be operated by one person, the same result can not be repeated, and the self-made chip for the experiment is made of PDMS (polydimethylsiloxane) which is a polymer material with large tolerance and elasticity, and the defects of the polymer material are that the standardized production can not be realized, the polymer material is easy to deform and adsorb small molecular substances, and a considerable bottleneck exists in the high-flux drug screening and drug testing. And the utility model discloses but current cell bio-printing technique of adaptation is standardized and quantifiable stable cell inoculation and special construction's printing construction can overcome these problems.

The microfluidic experimental plate of the embodiment integrates and standardizes microfluidic liquid supply control and cell culture, solves all the structural and processing problems encountered in the integration, can be used as a general cell co-culture tool capable of simulating different organ tissues, can realize a continuous uniform high-shear force culture microenvironment close to physiological substances on the cell co-culture structure, and can realize the culture of co-culture cells on two sides of a porous membrane on the tool, thereby simulating interstitial bands among different cells, and can realize the continuous shear force stimulation close to physiological microenvironment values on the cells on two sides by double-sided continuous liquid supply when bilateral cell co-culture is carried out. The microfluidic experimental plate can also be used as an organ chip platform and can be adapted to the existing cell bio-printing technology to perform standardized and quantifiable stable cell inoculation and printing construction of a special structure. Meanwhile, the microfluidic experimental plate can realize industrial production of stable processing in the aspect of chip processing.

Example two

In this embodiment, a cell culture method is provided, comprising the steps of:

s1: providing a microfluidic assay plate as described in example one, seeding cells on at least one side of said porous membrane;

s2: culturing of cells on at least one side of the porous membrane is performed by delivering a culture medium or administering a sample through at least one of the first fluid inlet and the second fluid inlet.

For example, the cells are seeded on both sides of the porous membrane, and the culture medium or the drug sample is introduced into the upper layer culture chamber through the first liquid inlet, and the culture medium or the drug sample is introduced into the lower layer culture chamber through the second liquid inlet, thereby co-culturing the cells on both sides of the porous membrane.

Specifically, the cell types seeded on both surfaces of the porous membrane may be the same or different. In this embodiment, the cell types seeded on both surfaces of the porous membrane are at least one, and at least a part of the cells seeded on both surfaces of the porous membrane are different in type, thereby realizing co-culture of a plurality of cells. The microfluidic experiment plate is used for culturing co-cultured cells on two sides of the porous membrane, so that intercellular zones among different cells can be simulated, and the cell interaction through the semipermeable membrane can be researched.

As an example, there are two methods of seeding cells:

1) directly titrating solution at the opening of one culture chamber of an assembled microfluidic experimental plate (a breathable film is not attached yet), sealing the opening of the culture chamber on the surface by the breathable film or a sealing plug after the cells adhere well, filling the culture medium, turning over to the other side of the titration solution, sealing the opening of the culture chamber on the surface by the breathable film or the sealing plug after the cells adhere well, filling the culture medium, and performing closed culture on the cells on the two sides; the titration solution may include cell suspension containing cells, extracellular matrix solution, etc. and may also include other solution without cells, and in the titration operation, more than one solution may be titrated, and the titration amount, order and titration number of these solutions may be regulated as required.

2) The method comprises the steps of directly printing needed patterns and structures on one culture chamber of an assembled microfluidic experiment plate (a breathable film is not attached or a sealing plug is not plugged) by using a cell printing technology, sealing the culture chamber with the breathable film or the sealing plug after the cells adhere to the wall well, turning to the other side to print the needed patterns and structures by using a biological printing technology, sealing the culture chamber with the breathable film or the sealing plug after the cells adhere to the wall well, and printing the two sides with proper cell structures for closed culture.

As an example, after the inoculation of the cells on both sides is completed, at least one of the first liquid inlet and the second liquid inlet can be connected with the pump body through an external pipeline, so as to realize the continuous liquid supply on one side or both sides. The first liquid outlet and the second liquid outlet can also be connected with other external pipelines. The pump body includes, but is not limited to, a peristaltic pump and the external conduit includes, but is not limited to, a hose.

Specifically, different culture mediums or administration samples can be simultaneously introduced into the upper layer culture chamber and the lower layer culture chamber. The flowing culture medium or the administration sample can generate corresponding shearing force in the cell culture chamber to simulate the cell culture condition close to the physiological microenvironment, namely a shearing force environment with a certain numerical value; the shearing force applied to the cells in the upper and lower chambers is controlled by controlling the flowing speed of the culture medium, so that the physiological model and the physiological microenvironment can be well constructed. Therefore, a fast flow rate (for example, a blood flow rate of blood vessels close to physiological value) can be generated, a uniform shear force microenvironment which meets the physiological value can be generated to the maximum extent in the flow channel of the fusiform design and on the porous membrane for cell growth, and continuous shear force stimulation can be carried out on cells on one side or two sides of the porous membrane.

By way of example, the cell culture method can be used for simulating tissue structures such as vascular barriers, alveolar barriers, intestinal barriers, blood brain barriers and the like in a physiological model, and can be used as a simulation tool and means for substance conduction research of the relevant physiological model. For example, when a Blood Brain Barrier (Blood Brain Barrier) model is to be simulated, Brain endothelial cells, Brain astrocyte and Brain pericytes can be respectively subjected to standardized and quantized cell inoculation on two sides of a porous membrane by using a biological printing technology, three different types of cells are accurately printed and inoculated at the opening of a culture chamber of the microfluidic experiment plate according to the proportion of various types of Brain cells of a real physiological model in proportion, the printing inoculation can be carried out according to the arrangement structure of the real physiological model, and finally the cell culture chamber with double-sided printing can be cultured by filling culture medium with a breathable membrane in a sealed manner. After the steps are completed, the culture medium can be introduced to culture the cells in accordance with a physiological microenvironment, and the upper and lower transparent breathable films of the microfluidic experiment plate facilitate the real-time observation of the cells. After the whole cell culture cycle is finished, the breathable film can be easily torn off, and subsequent operations such as fixation, fluorescent staining and the like can be directly performed on the cell experiment sample in the breathable film.

To sum up, the utility model discloses a micro-fluidic experiment board provides one kind and can be used to the two-sided standard platform of cultivateing of single face cell culture or two kinds and above cell, contains two-sided can supply liquid in succession, can construct the cell culture cavity of physiology shearing force microenvironment. The utility model discloses a micro-fluidic experiment board can regard as one kind to be convenient for with the standardized platform of cell coculture that cell biological printing technique combines, can inoculate the cell to the chip with standardized, quantized means at the in-process of equipment, realizes higher experimental repeatability, makes the experimental result at every turn have more the referential. The utility model discloses a micro-fluidic experimental board can make in batches in industrial production, is the organ chip product of a stable technology. The utility model discloses can be used to the vascular barrier among the simulation physiological model, the alveolus barrier, organizational structure such as intestines barrier to can regard as simulation tool and means of relevant physiological model material conduction research, the shuttle-type runner can produce even and the shearing force that accords with the physiological value in the porous membrane within range of all cell culture. The utility model discloses a pump bodies such as micro-fluidic experimental board can external peristaltic pump form circulation liquid supply system, can be used to produce the vascular blood velocity of flow that is close physiological value and found high shear force microenvironment. Therefore, the utility model effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A microfluidic assay plate, comprising:

2. The microfluidic assay plate of claim 1, wherein: the micro-fluidic experimental plate further comprises an upper breathable film and a lower breathable film, wherein the upper breathable film is used for sealing the upper culture chamber and the lower culture chamber are located at the front opening of the upper flow channel plate, and the lower breathable film is used for sealing the lower culture chamber and the lower culture chamber are located at the back opening of the lower flow channel plate.

3. The microfluidic assay plate of claim 2, wherein: the upper strata runner plate openly is equipped with the cavity, the upper strata is cultivateed the cavity and is located the positive opening of upper strata runner plate is located the bottom surface of cavity, upper ventilated membrane attach in the bottom surface of cavity.

4. The microfluidic assay plate of claim 1, wherein: the upper culture chamber comprises an upper main culture chamber and an upper flow channel which are communicated from top to bottom, the upper flow channel is located on the back of the upper flow channel plate, the upper main culture chamber is located on the front of the upper flow channel plate, the opening area of the upper main culture chamber is smaller than that of the upper flow channel, the opening of the upper main culture chamber is located in the opening range of the upper flow channel, and the first liquid inlet is communicated with the upper flow channel and the first liquid outlet in sequence.

5. The microfluidic assay plate of claim 4, wherein: the upper layer flow passage is in a shuttle shape.

6. The microfluidic assay plate of claim 1, wherein: the cavity is cultivateed to lower floor includes lower floor's main culture chamber and lower floor's runner of upper and lower intercommunication, the runner of lower floor is located the lower floor runner board is positive, the main culture chamber of lower floor is located the lower floor runner board back, the open area in lower floor's main culture chamber is less than the open area of lower floor's runner, just the main opening of cultivateing the chamber of lower floor is located in the open range of lower floor's runner, third runner hole lower floor's runner reaches the fourth runner hole communicates in proper order.

7. The microfluidic assay plate of claim 6, wherein: the lower runner is in a shuttle shape.

8. The microfluidic assay plate of claim 1, wherein: the microfluidic experiment board further comprises an upper sealing plug and a lower sealing plug, an upper cavity matched with the upper sealing plug is arranged on the front side of the upper flow channel board, a lower cavity matched with the lower sealing plug is arranged on the back side of the lower flow channel board, the upper cavity is communicated with the upper culture chamber, and the lower cavity is communicated with the lower culture chamber.

9. The microfluidic assay plate of claim 8, wherein: the upper layer culture chamber is in a fusiform shape, and the lower layer culture chamber is in a fusiform shape.

10. The microfluidic assay plate of claim 1, wherein: the first liquid inlet, the second liquid inlet, the first liquid outlet and the second liquid outlet have at least one opening from the front surface of the upper flow channel plate, or the first liquid inlet, the second liquid inlet, the first liquid outlet and the second liquid outlet have at least one opening from the side surface of the upper flow channel plate.