CN116410863A

CN116410863A - Microfluidic experimental plate and cell culture method

Info

Publication number: CN116410863A
Application number: CN202111670123.2A
Authority: CN
Inventors: 付伟欣; 汪磊; 胡斌; 关一民
Original assignee: Shanghai Industrial Utechnology Research Institute
Current assignee: Shanghai Industrial Utechnology Research Institute
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-07-11

Abstract

The invention provides a microfluidic experimental plate and a cell culture method, wherein the experimental plate comprises an upper substrate, a lower substrate and a porous membrane positioned between the upper substrate and the lower substrate, the upper substrate comprises an upper liquid inlet and an upper liquid outlet positioned on the side surfaces, an upper liquid inlet runner positioned on the lower surface, an upper liquid outlet runner and a plurality of upper culture chamber runners connected in parallel, the upper culture chamber runners comprise a plurality of upper culture chambers connected in parallel and penetrating the upper substrate in the vertical direction, the upper culture chamber runners comprise a first upper culture chamber runner and a second upper culture chamber runner which are distributed on two opposite sides of the culture chamber in the horizontal direction, and the top of the upper culture chamber is sealed by an upper ventilation layer; the lower substrate is similar in structure to the upper substrate. The invention can improve the flux of organ chips, avoid the problem of mutual influence among different culture chambers, and each culture chamber can realize nearly continuous and uniform shearing force culture microenvironment, and the culture chamber is breathable and impermeable. In the aspect of product processing, the invention can realize the industrialized production of stable processing.

Description

Microfluidic experimental plate and cell culture method

Technical Field

The invention belongs to the field of microfluidics and biotechnology, and relates to a microfluidic experimental plate and a cell culture method.

Background

At present, most of cell culture modes used as physiological models for drug screening 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 balling culture of multiple cells as a physiological model or a research model of a pathological model. However, according to the data published in various relevant documents in recent years, only two-dimensional culture of single cells shows physiological indexes and biological performances of cells in real animals are not the same. These documents demonstrate that the new drug screening is responsible for the high failure rate of entering clinical human trials after drug testing by conventional cell culture. It should be noted that even animal test results (e.g., mice, rats) often exhibit a high rate of violation of human clinical test results. This phenomenon shows that conventional two-dimensional single-species cell culture and animal experiments are not 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 tissue, has the capability of simulating the physical structure of the physiological model, has the possibility of constructing a physiological microenvironment, is a more efficient and reference platform than the conventional cell culture and animal experiments, can effectively shorten the drug development period and evaluate individual differences in the application aspect of drug testing and new drug screening, is beneficial to accurate treatment, and can also be used as a more advanced tool method of physiological and pathological models in research of scientific research institutions of higher institutions. However, most of the academic organ chips are designed and processed temporarily according to the specific organ research requirements, and have no versatility and stability. And the organ chip of the existing single-channel culture chamber can not screen the markers related to the diseases in a large batch and rapidly, evaluate the curative effect of the medicines, and the practical application of the organ chip is greatly limited.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a microfluidic experimental plate and a cell culture method, which are used for solving the problems that the failure rate of the existing cell culture method as a drug screening and related research physiological model is high, the existing organ chip has no versatility and stability, can not rapidly screen markers related to diseases in a large scale, evaluate the curative effect of the drug, and limit the practical application of the organ chip.

To achieve the above and other related objects, the present invention provides a microfluidic experimental plate comprising:

the upper substrate comprises an upper liquid inlet and an upper liquid outlet which are positioned on the side surface of the upper substrate, an upper liquid inlet runner, an upper liquid outlet runner and a plurality of upper culture chamber runners which are connected in parallel and penetrate through the upper substrate in the vertical direction, wherein the upper culture chamber runner comprises a first upper culture chamber runner and a second upper culture chamber runner which are distributed on two opposite sides of the upper culture chamber in the horizontal direction, the upper liquid inlet runner, the first upper culture chamber runner, the second upper culture chamber runner, the upper liquid outlet runner and the upper liquid outlet runner are sequentially communicated, and the top of the upper culture chamber is sealed by an upper ventilation layer;

the lower substrate is adhered below the upper substrate, the lower substrate comprises a lower liquid inlet and a lower liquid outlet which are positioned on the side surface of the lower substrate, a lower liquid inlet runner, a lower liquid outlet runner and a plurality of parallel lower culture chamber runners which are positioned on the upper surface of the lower substrate, and a plurality of parallel lower culture chambers which penetrate through the lower substrate in the vertical direction, the lower culture chamber runners comprise a first lower culture chamber runner and a second lower culture chamber runner which are distributed on two opposite sides of the lower culture chamber in the horizontal direction, the lower liquid inlet runner, the first lower culture chamber runner, the second lower culture chamber runner, the lower liquid outlet runner and the lower liquid outlet are sequentially communicated, the bottom of the lower culture chamber is sealed by a lower ventilation layer, and the upper culture chamber is vertically opposite to the lower culture chamber;

and the porous membrane is positioned between the upper substrate and the lower substrate and covers the upper culture chamber, the lower culture chamber, the upper culture chamber runner and the lower culture chamber runner.

Optionally, the lower surface of the upper substrate, the porous membrane and the upper surface of the lower substrate are bonded by applying an adhesive.

Optionally, a first double-sided adhesive film is arranged between the upper substrate and the porous film, a plurality of first through holes are arranged in the first double-sided adhesive film and cover the upper liquid inlet flow channel, the upper liquid outlet flow channel and the upper culture chamber flow channel, and the plurality of first through holes are vertically opposite to the plurality of upper culture chambers respectively; a second double-sided adhesive film is arranged between the lower substrate and the porous film, a plurality of second through holes are arranged in the second double-sided adhesive film and cover the lower liquid inlet flow passage, the lower liquid outlet flow passage and the lower culture chamber flow passage, and the second through holes are vertically opposite to the lower culture chambers respectively.

Optionally, a first double-sided adhesive film is arranged between the upper substrate and the porous film, a plurality of first through holes are arranged in the first double-sided adhesive film and cover the upper liquid inlet flow channel and the upper liquid outlet flow channel, and the first through holes are vertically opposite to the upper culture chambers and the upper culture chamber flow channels respectively; a second double-sided adhesive film is arranged between the lower substrate and the porous film, a plurality of second through holes are arranged in the second double-sided adhesive film and cover the lower liquid inlet flow channel and the lower liquid outlet flow channel, and the second through holes are vertically opposite to the lower culture chambers and the lower culture chamber flow channels respectively.

Optionally, the upper culture chamber flow channel and the lower culture chamber flow channel are both fusiform.

Optionally, the upper ventilation layer comprises a ventilation membrane or a ventilation sealing plug, and the lower ventilation layer comprises a ventilation membrane or a ventilation sealing plug.

Optionally, the materials of the upper layer ventilation layer and the lower layer ventilation layer comprise polydimethylsiloxane, and the upper layer substrate and the lower layer substrate comprise transparent hard materials.

Optionally, one upper layer culture chamber and the corresponding lower layer culture chamber form a culture unit, and all the culture units adopt the same kind of porous membranes, or at least two culture units adopt different kinds of porous membranes.

Optionally, the upper layer culture chamber and the lower layer culture chamber form a culture unit, the culture units are arranged in at least one row or at least one column, the upper layer liquid inlet flow channel and the lower layer liquid inlet flow channel respectively comprise a first-stage flow channel, a second-stage flow channel and a third-stage flow channel, and the upper layer liquid outlet flow channel and the lower layer liquid outlet flow channel respectively comprise a first-stage flow channel, a second-stage flow channel and a third-stage flow channel, wherein the flow resistance of the first-stage flow channel, the second-stage flow channel and the third-stage flow channel is sequentially increased.

The invention also provides a cell culture method, which comprises the following steps:

providing the microfluidic experimental plate according to any one of the above, inoculating a first cell on the upper surface of the porous membrane through the upper liquid inlet, and inoculating a second cell on the lower surface of the porous membrane through the lower liquid inlet;

a culture medium or a drug administration sample is fed through the upper layer liquid inlet and the lower layer liquid inlet to perform cell culture on both sides of the porous membrane.

Optionally, the cell culture method is used to simulate a blood brain barrier model, an alveolar barrier model, or an intestinal barrier model.

As described above, the microfluidic experimental plate and the cell culture method of the invention have the following beneficial effects: (1) The cell culture chamber which comprises double-sided continuous liquid supply and can construct physiological shear force microenvironment can be used as a standardized platform for co-culture of two or more cells; (2) Is an organ chip product which can be used for industrial production and mass production; (3) The simulation tool and the simulation method can be used for simulating tissue structures such as blood brain barriers, alveolus barriers, intestinal barriers and the like in physiological models, and can be used as simulation tools and means for conducting research on related physiological model substances; (4) The multistage flow channels ensure that the culture chambers are independent of each other, do not affect each other, and provide approximately equal flow rates and shear forces; (5) The culture chamber flow channel can generate uniform shearing force which accords with physiological values in the range of porous membranes for all cell culture; (6) The bottom of the culture chamber on the substrate is made of a breathable polymer material, so that gas exchange such as oxygen can be provided for the culture chamber; (7) The circulating liquid supply system which can be externally connected with a peristaltic pump is used for continuously generating vascular blood flow rate close to physiological conditions and a proper shearing force microenvironment.

Drawings

Fig. 1 shows a schematic perspective structure of a microfluidic experimental plate according to the present invention.

Fig. 2 shows a top view of a microfluidic experimental plate according to the invention.

Fig. 3 shows a cross-section A-A of fig. 2.

Fig. 4 is a schematic diagram showing an exploded structure of a microfluidic experimental plate according to the present invention in the first embodiment.

Fig. 5 is a schematic perspective view of the upper substrate with its lower surface facing upwards.

FIG. 6 is a schematic top view of the lower culture chamber flow channel and the lower culture chamber.

Fig. 7 shows a cross-sectional view in the direction B-B of fig. 6.

Fig. 8 is a schematic diagram showing an exploded structure of a microfluidic experimental plate according to the present invention in the second embodiment.

FIG. 9 is an enlarged partial schematic view showing the cross-sectional structure of a single culture unit in the second embodiment.

Fig. 10 shows a schematic of the connection of the syringe to the microfluidic assay plate.

Description of element reference numerals

100. Microfluidic experimental plate

1. Upper substrate

101. Upper liquid inlet

102. Upper layer liquid outlet

103. Upper layer liquid inlet channel

1031. Primary flow channel

1032. Two-stage runner

1033. Three-stage runner

104. Upper layer liquid outlet flow channel

105. Flow channel of upper layer culture room

1051. First upper layer culture chamber flow channel

1052. Second upper layer culture chamber flow channel

106. Upper layer culture room

107. Upper ventilation layer

2. Lower substrate

201. Lower liquid inlet

202. Lower layer liquid outlet

203. Lower layer liquid inlet channel

204. Lower layer liquid outlet flow channel

205. Lower layer culture room runner

2051. First lower layer culture chamber runner

2052. Second lower culture chamber flow channel

206. Lower layer culture room

207. Lower ventilation layer

3. Porous membrane

4. First double-sided mucosa

401. First via hole

5. Second double-sided mucosa

501. Second via hole

6. First injector

7. Second injector

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Please refer to fig. 1 to 10. It should be noted that, the illustrations provided in the present embodiment merely illustrate the basic concept of the present invention by way of illustration, and only the components related to the present invention are shown in the drawings and are not drawn according to the number, shape and size of the components in actual implementation, and the form, number and proportion of the components in actual implementation may be arbitrarily changed, and the layout of the components may be more complex.

Example 1

In this embodiment, referring to fig. 1 to 3, fig. 1 is a schematic perspective view of a microfluidic experimental plate 100, fig. 2 is a top view of the microfluidic experimental plate 100, and fig. 3 is a cross-sectional view of fig. 2 from A-A, wherein the microfluidic experimental plate comprises an upper substrate 1, a lower substrate 2, and a porous membrane 3 between the upper substrate 1 and the lower substrate 2.

As an example, the upper substrate 1 and the lower substrate 2 comprise transparent hard materials, such as hard polymer materials including polycarbonate and polymethyl methacrylate, and the hard substrate materials have low elasticity and are not easy to deform, so that the microfluidic experimental plate can be standardized in industry and produced in large quantities.

As an example, the porous membrane 3 has many pores therein, which allow liquid to pass through, but do not allow cells to pass through. The porous membrane 3 may be made of PET (polyester) or other suitable material.

As an example, the lower surface of the upper substrate 1, the porous membrane 3 and the upper surface of the lower substrate 2 are bonded by applying an adhesive, and the bonding material can realize bonding seal between the components. In this embodiment, the adhesive material is a biocompatible material.

As an example, referring to fig. 4 and 5, fig. 4 is a schematic exploded view of the microfluidic experimental plate, fig. 5 is a schematic three-dimensional view of the upper substrate 1 when the lower surface is upward, wherein the upper substrate 1 includes an upper liquid inlet 101 and an upper liquid outlet 102 on the sides of the upper substrate 1, an upper liquid inlet channel 103, an upper liquid outlet channel 104 and a plurality of parallel upper culture chamber channels 105 on the lower surface of the upper substrate 1, and includes a plurality of parallel upper culture chambers 106 penetrating the upper substrate 1 in a vertical direction, the upper culture chamber channels 105 include a first upper culture chamber channel 1051 and a second upper culture chamber channel 1052 distributed on opposite sides of the upper culture chamber 106 in a horizontal direction, the upper liquid inlet 101, the upper liquid inlet channel 103, the first upper culture chamber channel 1051, the upper culture chamber 106, the second upper culture chamber channel 1052, the upper culture chamber channel 104 and the upper liquid outlet channel 102 are sequentially connected to each other via an upper liquid outlet port 107; the lower substrate 2 is adhered below the upper substrate 1, the lower substrate 2 comprises a lower liquid inlet 201 and a lower liquid outlet 202 positioned on the side surfaces of the lower substrate 2, a lower liquid inlet runner 203, a lower liquid outlet runner 204 and a plurality of parallel lower culture chamber runners 205 positioned on the upper surface of the lower substrate 2, and a plurality of parallel lower culture chambers 206 penetrating through the lower substrate 2 in the vertical direction, the lower culture chamber runners 205 comprise a first lower culture chamber runner 2051 and a second lower culture chamber runner 2052 distributed on two opposite sides of the lower culture chamber 206 in the horizontal direction, the lower liquid inlet 201, the lower liquid inlet runner 203, the first lower culture chamber runner 2051, the lower culture chamber 206, the second lower culture chamber runner 2052, the lower liquid outlet runner 204 and the lower liquid outlet 202 are sequentially communicated, and the bottom of the lower culture chamber 206 is vertically opposite to the lower culture chamber 106 through a lower ventilation layer 207; the porous membrane 3 is located between the upper substrate 1 and the lower substrate 2, and covers the upper culture chamber 106, the lower culture chamber 206, the upper culture chamber flow path 105, and the lower culture chamber flow path 205.

Specifically, the porous membrane 3 forms a cell culture chamber which is closed at the upper and lower layers and can be used for flowing medium liquid, and a culture unit is composed of the upper culture chamber 106 and the lower culture chamber 206 opposite to the upper culture chamber.

As an example, the upper ventilation layer 107 employs a ventilation film or a ventilation sealing plug, and the lower ventilation layer 207 employs a ventilation film or a ventilation sealing plug. In this embodiment, the ventilation layer takes a ventilation sealing plug as an example. In this embodiment, the upper ventilation layer 107 and the lower ventilation layer 207 are made of a breathable polymer material, such as polydimethylsiloxane, which not only can provide gas exchange such as oxygen for the culture chamber, but also is made of a transparent material to facilitate real-time observation of cells.

As an example, a plurality of the culture units are arranged in at least one row or at least one column. In this embodiment, a parallel structure of 9 culture units of 3×3 is constructed on one microfluidic experimental plate. In other embodiments, the number and specific arrangement of the culture units may be adjusted as desired.

As an example, all of the culture units employ the same kind of the porous membrane, or at least two culture units employ different kinds of porous membranes, thereby satisfying diversified demands.

As an example, the upper liquid inlet channel 103 and the lower liquid inlet channel 203 each include a primary channel, a secondary channel and a tertiary channel, so as to construct a plurality of channel structures for supplying liquid to different culture units, and the upper liquid outlet channel 104 and the lower liquid outlet channel 204 each include a primary channel, a secondary channel and a tertiary channel that are sequentially connected, so as to construct a plurality of channel structures for discharging liquid to different culture units, wherein flow resistance of the primary channel, the secondary channel and the tertiary channel is sequentially increased. The primary runner 1031, the secondary runner 1032, and the tertiary runner 1033 of the upper liquid inlet runner 103 are identified in fig. 5. In other embodiments, the layout of the multi-stage flow channels may be adjusted as desired, and is not limited to the case shown in fig. 5. The multistage flow channel realizes parallelization of a plurality of culture units, can ensure that culture chambers are independent of each other, cannot affect each other, and provides approximately equal flow velocity and shearing force.

Specifically, the dimensions of the primary flow channel, the secondary flow channel and the tertiary flow channel of the substrate follow the hydrodynamic relation, the cross-sectional area, the length and the like of the flow channels are adjusted to achieve the proportional relation between the flow resistance of the secondary flow channel and the primary flow channel and the flow resistance of the tertiary flow channel and the secondary flow channel, and the optimal situation is that the proportional relation is more than 5 times.

Specifically, the upper culture chamber and the lower culture chamber of the microfluidic experimental plate can be respectively filled with the same or different culture mediums or administration samples through the upper flow channel and the lower flow channel. The flowing culture medium can generate corresponding shearing force in each culture chamber so as to provide cell culture conditions close to physiological microenvironment, namely a certain shearing force environment; the shear force applied to the cells in each culture chamber of the upper layer and the lower layer can be controlled by controlling the inflow speed of the culture medium, so that a good physiological model and a good physiological microenvironment can be constructed.

As an example, please refer to fig. 6 and 7, wherein fig. 6 is a schematic top view of the lower culture chamber flow channel 205 and the lower culture chamber 206, fig. 7 is a cross-sectional view of fig. 6 from the B-B direction, and in this embodiment, both the upper culture chamber flow channel 105 and the lower culture chamber flow channel 205 are shuttle-shaped. The shuttle-type flow channel is beneficial to generating uniform shearing force which accords with physiological values in the range of the porous membrane for all cell culture.

As an example, the microfluidic experimental plate may be externally connected with a circulating liquid supply system of a peristaltic pump for continuously generating a vascular blood flow rate close to a physiological condition and a suitable shearing force microenvironment.

The microfluidic experimental plate can be used for a standardized platform for co-culturing two or more cells, comprises a cell culture chamber with two surfaces capable of continuously supplying liquid and constructing a physiological shearing force microenvironment, and is used for constructing an organ chip, wherein a plurality of upper culture chambers 106 and corresponding lower culture chambers 206 form a plurality of culture units connected in parallel, so that the flux of the organ chip can be improved, and the problem of mutual influence between the culture chambers on the organ chip can be avoided. Each culture chamber can achieve a near continuous uniform shear culture microenvironment and is air permeable and water impermeable. The chip processing aspect of the organ chip can realize the industrialized production of stable processing. In addition, the microfluidic experimental plate can improve the integration level of the polar chip, for example, the product appearance size of the 3×3 organ chip can be designed to be 37mm×45mm×4.3mm, and the integration level is higher.

Example two

The embodiment adopts substantially the same technical scheme as the first embodiment, except that the bonding manner between the upper substrate 1 and the lower substrate 2 is different.

Referring to fig. 8 and 9, fig. 8 is a schematic exploded view of the microfluidic experimental plate according to the present embodiment, and fig. 9 is a schematic enlarged view of a section of a single culture unit.

Specifically, a first double-sided adhesive film 4 is disposed between the upper substrate 1 and the porous membrane 3, a plurality of first through holes 401 are disposed in the first double-sided adhesive film 4 and cover the upper liquid inlet flow channel 103, the upper liquid outlet flow channel 104 and the upper culture chamber flow channel 105, and the plurality of first through holes 401 are vertically opposite to the plurality of upper culture chambers 106; a second double-sided adhesive film 5 is disposed between the lower substrate 2 and the porous film 3, a plurality of second through holes 501 are disposed in the second double-sided adhesive film 5 and cover the lower liquid inlet channel 203, the lower liquid outlet channel 204 and the lower culture chamber channel 205, and the plurality of second through holes 501 are vertically opposite to the plurality of lower culture chambers 206.

In particular, the first double-sided adhesive film 4 and the second double-sided adhesive film 5 can realize bonding seal between each component. In this embodiment, the first double-sided adhesive film 4 and the second double-sided adhesive film 5 are made of biocompatible materials.

Example III

The embodiment adopts the substantially same technical scheme as that of the second embodiment, and is different in that in the second embodiment, the first double-sided adhesive film 4 covers the upper liquid inlet channel 103, the upper liquid outlet channel 104 and the upper culture chamber channel 105 at the same time, and the first via hole 401 is vertically opposite to the upper culture chamber 106 only; the second double-sided adhesive film 5 covers the lower liquid inlet channel 203, the lower liquid outlet channel 204 and the lower culture chamber channel 205, and the second via 501 is vertically opposite to the lower culture chamber 206 only. In this application, the first double-sided adhesive film 4 covers the upper liquid inlet channel 103 and the upper liquid outlet channel 104, but does not cover the upper culture chamber channel 105, and the first via 401 is vertically opposite to the upper culture chamber 106 and is also vertically opposite to the upper culture chamber channel 105; the second double-sided adhesive film 5 covers the lower liquid inlet channel 203 and the lower liquid outlet channel 204, but does not cover the lower culture chamber channel 205, and the second via hole is vertically opposite to the lower culture chamber 206 and the lower culture chamber channel 205.

Example IV

In this embodiment, a cell culture method is provided, which includes the following steps:

s1: providing the microfluidic experimental plate according to the first, second or third embodiments, wherein a first cell is inoculated on the upper surface of the porous membrane 3 through the upper liquid inlet 101, and a second cell is inoculated on the lower surface of the porous membrane 3 through the lower liquid inlet 101;

s2: culture medium or a drug administration sample is fed through the upper layer liquid inlet 101 and the lower layer liquid inlet 201 to perform cell culture on both sides of the porous membrane 3.

Specifically, the cell culture method can be used for simulating tissue structures such as blood brain barrier, alveolar barrier, intestinal barrier and the like in a physiological model, and can be used as a simulation tool and means for conducting research on related physiological model substances. The following will specifically describe an example of a model for simulating the blood brain barrier.

Referring to fig. 10, a schematic diagram of connection of an injector and a microfluidic experimental plate 100 is shown, when a blood brain barrier model is simulated, brain endothelial cells are first infused from the inlet of the upper substrate of the microfluidic experimental plate 100 by a first injector 6, after the cells are grown on the porous membrane of each culture chamber of the upper substrate, brain astrocyte and brain pericyte are infused from the inlet of the lower substrate by a second injector 7, and after the cells are grown on the porous membrane of each culture chamber of the lower substrate, the inlets of the upper and lower substrates can be continuously infused with culture medium to perform cell culture conforming to physiological microenvironment. Meanwhile, the bottom of the culture chamber of the upper and lower substrates is filled with transparent polymer materials, so that the cells can be conveniently observed in real time. After the whole cell culture period is finished, the subsequent fixing, fluorescent staining and other operations can be performed on the cell experimental sample inside.

In summary, the microfluidic experimental plate of the invention comprises a cell culture chamber with double-sided continuous liquid supply and capable of constructing physiological shear force microenvironment, can be used as a standardized platform for co-culture of two or more cells, and is an organ chip product for industrial production and mass production; the microfluidic experimental plate can be used for simulating tissue structures such as blood brain barrier, alveolus barrier, intestinal barrier and the like in a physiological model, and can be used as a simulation tool and a simulation means for conducting research on related physiological model substances; wherein the multistage flow channels ensure that the culture chambers are independent of each other, do not affect each other, and provide approximately equal flow rates and shear forces; the culture chamber flow channel can generate uniform shearing force which accords with physiological values in the range of porous membranes for all cell culture; the bottom of the culture chamber on the substrate is made of a breathable polymer material, so that gas exchange such as oxygen can be provided for the culture chamber; the microfluidic experimental plate can be externally connected with a circulating liquid supply system of a peristaltic pump and is used for continuously generating vascular blood flow velocity close to physiological conditions and a proper shearing force microenvironment. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A microfluidic assay plate comprising:

2. The microfluidic assay plate of claim 1, wherein: the lower surface of the upper substrate, the porous membrane and the upper surface of the lower substrate are bonded by smearing adhesive.

3. The microfluidic assay plate of claim 1, wherein: a first double-sided adhesive film is arranged between the upper substrate and the porous film, a plurality of first through holes are arranged in the first double-sided adhesive film and cover the upper liquid inlet flow channel, the upper liquid outlet flow channel and the upper culture chamber flow channel, and the first through holes are vertically opposite to the upper culture chambers respectively; a second double-sided adhesive film is arranged between the lower substrate and the porous film, a plurality of second through holes are arranged in the second double-sided adhesive film and cover the lower liquid inlet flow passage, the lower liquid outlet flow passage and the lower culture chamber flow passage, and the second through holes are vertically opposite to the lower culture chambers respectively.

4. The microfluidic assay plate of claim 1, wherein: a first double-sided adhesive film is arranged between the upper substrate and the porous film, a plurality of first through holes are arranged in the first double-sided adhesive film and cover the upper liquid inlet flow channel and the upper liquid outlet flow channel, and the first through holes are vertically opposite to the upper culture chambers and the upper culture chamber flow channels respectively; a second double-sided adhesive film is arranged between the lower substrate and the porous film, a plurality of second through holes are arranged in the second double-sided adhesive film and cover the lower liquid inlet flow channel and the lower liquid outlet flow channel, and the second through holes are vertically opposite to the lower culture chambers and the lower culture chamber flow channels respectively.

5. The microfluidic assay plate of claim 1, wherein: the upper layer culture chamber flow passage and the lower layer culture chamber flow passage are both fusiform.

6. The microfluidic assay plate of claim 1, wherein: the upper breathable layer comprises a breathable film or a breathable sealing plug, and the lower breathable layer comprises a breathable film or a breathable sealing plug.

7. The microfluidic assay plate of claim 1, wherein: the upper layer breathable layer and the lower layer breathable layer are made of polydimethylsiloxane, and the upper layer substrate and the lower layer substrate are made of transparent hard materials.

8. The microfluidic assay plate of claim 1, wherein: one upper layer culture chamber and the corresponding lower layer culture chamber form a culture unit, and all the culture units adopt the same kind of porous membranes or at least two culture units adopt different kinds of porous membranes.

9. The microfluidic assay plate of claim 1, wherein: the upper layer culture chamber and the lower layer culture chamber are correspondingly arranged to form a culture unit, a plurality of culture units are arranged in at least one row or at least one column, the upper layer liquid inlet flow channel and the lower layer liquid inlet flow channel respectively comprise a first-stage flow channel, a second-stage flow channel and a third-stage flow channel, and the upper layer liquid outlet flow channel and the lower layer liquid outlet flow channel respectively comprise a first-stage flow channel, a second-stage flow channel and a third-stage flow channel, wherein flow resistance of the first-stage flow channel, the second-stage flow channel and the third-stage flow channel is sequentially increased.

10. A method of cell culture comprising the steps of:

providing the microfluidic assay plate according to any one of claims 1-9, seeding a first cell on the upper surface of the porous membrane through the upper liquid inlet and a second cell on the lower surface of the porous membrane through the lower liquid inlet;

11. The cell culture method of claim 10, wherein: the cell culture method is used to simulate a blood brain barrier model, an alveolar barrier model, or an intestinal barrier model.