CN117844636A

CN117844636A - Microfluidic cell perfusion culture device and system based on standard porous plate

Info

Publication number: CN117844636A
Application number: CN202311625976.3A
Authority: CN
Inventors: 胡敏; 王韫芳; 柳娟; 刘锦润
Original assignee: Beijing Tsinghua Changgeng Hospital
Current assignee: Beijing Tsinghua Changgeng Hospital
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2024-04-09

Abstract

The invention relates to the technical field of cell culture appliances, in particular to a microfluidic cell perfusion culture device and system based on a standard porous plate. The microfluidic cell perfusion culture device based on the standard porous plate comprises: and the cover plate, the middle layer plate and the bottom plate assembly are sequentially stacked. The bottom plate subassembly includes: the first plate body is formed with multiunit first micro-fluid channel structure on the first plate body, and every group of first micro-fluid channel structure includes: a parallel structure formed by a plurality of microfluidic channels; a plurality of first through hole structures are formed on the middle layer plate, and each microfluidic channel is connected with the plurality of first through hole structures; the cover plate is provided with a fluid inlet pipeline and a fluid outlet pipeline which are used for communicating with the first through hole structure. The microfluidic cell perfusion culture device and system based on the standard porous plate can realize the automation of cell static culture, the dynamic culture of cells under laboratory conditions, the simulation of in-vivo environment, excellent compatibility, the adoption of multi-channel culture and high screening efficiency.

Description

Microfluidic cell perfusion culture device and system based on standard porous plate

Technical Field

The invention relates to the technical field of cell culture appliances, in particular to a microfluidic cell perfusion culture device and system based on a standard porous plate.

Background

Multiwell plates, also commonly referred to as microwell plates or microplates, play a very important role in cell culture and detection, and are mainly used for: cell culture, high throughput screening, cell counting and cell activity detection, enzyme-linked immunosorbent assay (ELISA for short), cell migration and invasion experiments, apoptosis and cell cycle analysis, cell co-culture, microorganism culture and detection, and the like. The multi-well plate provides a convenient, efficient and standardized platform for cell biology research, so that researchers can perform experiments on multiple samples or conditions at the same time. Cell perfusion culture is another cell culture technique in which a medium is circulated continuously or periodically through a cell culture system to provide the cells with the necessary nutrients and remove metabolic waste. This culture is different from conventional static culture, which generally involves placing cells in a fixed culture environment until the cells are changed or harvested. The characteristics and effects of cell perfusion culture include: continuously supplying nutrition and oxygen, effectively removing metabolic waste, improving product yield, simulating in vivo environment, reducing use of culture medium, and being suitable for mass production. Thus, cell perfusion culture provides a more dynamic and in vivo environment-mimicking culture condition for cells, supporting higher cell growth and product yield.

As a traditional cell static culture device, the cell culture and detection technology of porous plates in a biological laboratory is relatively mature and convenient. But the culture medium in the multiwell plate cannot be circulated continuously or periodically in order to provide the cells with the desired nutrients and remove metabolic waste in time. Therefore, a series of modern biological experiments under laboratory conditions cannot be effectively satisfied, such as:

1) Biopharmaceutical experiments to produce therapeutic proteins, antibodies and other biological products;

2) Cell therapy experiments, expanding cells for cell therapy, such as T cells or stem cells;

3) Tissue engineering experiment, culturing tissue engineering component

To simulate blood flow and nutrient supply in the body;

4) Drug screening experiments, cytotoxicity testing and high throughput screening of drugs.

Therefore, there is a need for a standard well plate compatible and microfluidic technology based cell biological testing device and perfusion platform that solves the above problems and is suitable for high throughput drug screening and in vivo environmental simulation applications based on the existing well plate technology.

Disclosure of Invention

The invention provides a microfluidic cell perfusion culture device and a microfluidic cell perfusion culture system based on a standard porous plate, which are used for solving the defects that a culture medium in the porous plate cannot continuously or periodically circulate in the prior art, is not suitable for high-throughput drug screening, in-vivo environment simulation and the like, and has poor compatibility.

The invention provides a microfluidic cell perfusion culture device based on a standard porous plate, which comprises: the cover plate, the middle layer plate and the bottom plate component are sequentially stacked;

wherein the floor assembly comprises:

a first plate body, on which a plurality of groups of first microfluidic channel structures are formed, each group of first microfluidic channel structures comprising: a parallel structure formed by a plurality of microfluidic channels;

a plurality of first through hole structures are formed on the middle layer plate, and each microfluidic channel is connected with the plurality of first through hole structures;

the cover plate is provided with a fluid inlet pipeline and a fluid outlet pipeline, and at least one of the plurality of first through hole structures corresponding to each group of first micro-fluid channel structures is communicated with the fluid inlet pipeline and at least one of the plurality of first through hole structures is communicated with the fluid outlet pipeline.

According to the microfluidic cell perfusion culture device based on the standard porous plate provided by the invention, a plurality of groups of second microfluidic channel structures are further formed on the first plate body, and each group of second microfluidic channel structures comprises: and the end parts of each group of second microfluidic channel structures are respectively communicated with one of the corresponding first through hole structures and are used for cell static culture comparison research.

According to the invention, a microfluidic cell perfusion culture device based on a standard porous plate is provided, and the bottom plate assembly further comprises:

the second plate body is provided with a plurality of second through hole structures, and the second through hole structures are arranged corresponding to the first through hole structures;

the first plate body is arranged between the second plate body and the third plate body.

According to the microfluidic cell perfusion culture device based on the standard porous plate, a plurality of channels are formed on the cover plate, and the channels are used for arranging the fluid inlet pipeline and the fluid outlet pipeline.

According to the microfluidic cell perfusion culture device based on the standard porous plate, the cover plate is provided with the transparent hole cover and the transparent observation window which are alternately arranged, the transparent hole cover and the transparent observation window are respectively arranged corresponding to the first through hole structure, and the channel penetrates through the transparent hole cover.

According to the invention, the microfluidic cell perfusion culture device based on the standard porous plate further comprises:

the first side plate is arranged at the outer edge of the cover plate;

the second side plate is of a step structure, the second side plate is arranged on the outer edge of the middle layer plate, and the first side plate is correspondingly attached to the outer wall of the second side plate, so that the cover plate is buckled on the upper surface of the middle layer plate.

According to the microfluidic cell perfusion culture device based on the standard porous plate, the bottom plate component is arranged on the inner side of the second side plate and is fixedly connected with the middle layer plate through bonding or welding to form a composite porous plate structure with a microfluidic channel at the bottom.

According to the present invention, there is provided a microfluidic cell perfusion culture device based on standard multi-well plates, each set of the first microfluidic channel structures comprising: a parallel structure formed by three micro-fluid channels.

According to the microfluidic cell perfusion culture device based on the standard porous plate, each microfluidic channel is connected with three first through hole structures.

The invention also provides a microfluidic cell perfusion culture system based on a standard porous plate, which comprises: the programmable pumping device, the liquid filtering tank, the liquid storage tank, the air release valve and the microfluidic cell perfusion culture device based on the standard porous plate in the embodiment of the invention are sequentially connected through the flexible pipe to form a group of independent fluid perfusion control channels, and under the control of the programmable pumping device, the culture medium in each microfluidic channel automatically and continuously circularly flows or periodically at the same time, and the culture medium cannot overflow from the culture hole.

The invention provides a microfluidic cell perfusion culture device based on a standard porous plate, which comprises: a cover plate, a middle layer plate and a bottom plate assembly; the cover plate is provided with a fluid inlet pipeline and a fluid outlet pipeline and is communicated with the first through hole structure of the middle layer plate; the bottom plate assembly is provided with a plurality of groups of first micro-fluid channel structures, each group of first micro-fluid channel structures is formed by a plurality of micro-fluid channels to form a parallel structure, and the micro-fluid channels are communicated with the fluid inlet pipeline and the fluid outlet pipeline through corresponding first through hole structures. The microfluidic cell perfusion culture device based on the standard porous plate provided by the invention has the following beneficial effects:

1. can realize the automation of the static cell culture. The culture medium can simulate conventional cell static culture, and can be periodically and automatically replaced by a fluid inlet pipeline and a fluid outlet pipeline by virtue of programming, so that the labor and time are saved.

2. Cell dynamic culture under laboratory conditions can be realized. By matching with the perfusion system, under the action of pressure difference, the culture medium can continuously or periodically circulate, so that nutrition required by cells can be provided in time, metabolic waste can be removed, and dynamic culture of the cells under laboratory conditions can be realized.

3. The in vivo environment can be simulated. Microfluidic perfusion is closer to the microcirculation environment in the body, and can better simulate the natural state of cells in the body.

4. The compatibility is excellent. Can be used as a cell static culture device independently, and has the action and effect which are not different from those of the conventional porous plate; it is also fully compatible with the cell biology examination, detection and analysis instruments based on multi-well plates commonly used in laboratories. Therefore, the device can be widely applied to biopharmaceutical experiments, cell therapy experiments, tissue engineering experiments and drug screening experiments under laboratory conditions.

5. The multi-channel culture/screening efficiency is high. The device is provided with a plurality of first micro-fluid channel structures, each first micro-fluid channel structure is connected in parallel by a plurality of micro-fluid channels, and can simultaneously realize the operations such as drug resistance screening test of cells to different drug concentrations under the same environmental condition, and the like, and is suitable for the operations such as high-flux drug screening, comparative tissue engineering experiments and the like.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a microfluidic cell perfusion culture device based on standard multi-well plates according to a first view angle in one embodiment of the present invention;

FIG. 2 is a schematic diagram of a microfluidic cell perfusion culture device based on standard multi-well plates according to a second view angle according to one embodiment of the present invention;

FIG. 3 is a schematic illustration of the construction of one embodiment of the present invention with the cover plate provided with the fluid inlet and outlet conduits removed;

FIG. 4 is a schematic view of the structure of a middle laminate provided in one embodiment of the present invention;

FIG. 5 is a schematic illustration of a microfluidic cell perfusion culture device based on standard multiwell plates according to one embodiment of the present invention with the cover plate removed;

FIG. 6 is a schematic structural view of a floor assembly provided in one embodiment of the invention;

FIG. 7 is a schematic view of a floor assembly provided in one embodiment of the invention with a second panel removed;

FIG. 8 is a top view of a first plate provided in one embodiment of the present invention;

FIG. 9 is a schematic view of a third plate provided in one embodiment of the present invention;

fig. 10 is a schematic diagram of a microfluidic cell perfusion culture system based on standard multi-well plates provided in one embodiment of the present invention.

Reference numerals:

10: a cover plate; 20: a middle layer plate; 30: a base plate assembly;

31: a first plate body; 311: a first microfluidic channel structure; 312: a second microfluidic channel structure; 313: a microfluidic channel; 32: a second plate body; 321: a second via structure; 33: a third plate body;

21: a first via structure; 22: a second side plate; 23: a positioning piece;

11: a fluid inlet conduit; 12: a fluid outlet conduit; 13: a transparent hole cover; 14: a transparent viewing window; 15: a channel; 16: first side plate

100: a pumping device; 200: a liquid filtering pool; 300: a liquid storage tank; 400: microfluidic cell perfusion culture devices based on standard multiwell plates; 500: and a release valve.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. 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.

In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the apparatus or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the embodiments of the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In describing embodiments of the present invention, it should be noted that, unless explicitly stated and limited otherwise, the terms "coupled," "coupled," and "connected" should be construed broadly, and may be either a fixed connection, a removable connection, or an integral connection, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in embodiments of the present invention will be understood in detail by those of ordinary skill in the art.

In embodiments of the invention, unless expressly specified and limited otherwise, a first feature "up" or "down" on a second feature may be that the first and second features are in direct contact, or that the first and second features are in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the embodiments of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

A microfluidic cell perfusion culture device 400 based on standard multi-well plates according to the present invention is described below in connection with fig. 1-9. The microfluidic cell perfusion culture device 400 based on standard multi-well plates comprises: the cover plate 10, the middle layer plate 20 and the base plate assembly 30 are sequentially stacked. In the use state, the cover plate 10, the middle layer plate 20 and the bottom plate assembly 30 are sequentially stacked from top to bottom.

Wherein the base plate assembly 30 comprises: the first plate 31, the first plate 31 is formed with a plurality of groups of first microfluidic channel structures 311, and each group of first microfluidic channel structures 311 includes: a parallel structure formed by a plurality of microfluidic channels 313. The middle plate 20 has a plurality of first via structures 21 formed thereon, and each microfluidic channel 313 is connected to the plurality of first via structures 21; the cover plate 10 is provided with a fluid inlet pipeline 11 and a fluid outlet pipeline 12, and at least one of the first through hole structures 21 corresponding to each group of the first micro-fluid channel structures 311 is communicated with the fluid inlet pipeline 11 and at least one of the first through hole structures is communicated with the fluid outlet pipeline 12.

Specifically, the first through-hole structure 21 serves as a culture hole for holding a culture medium to provide nutrition required for cells. By designing the plurality of first micro-fluidic channel structures 311 on the first plate 31 for connecting the first through-hole structures 21 with corresponding functions, the plurality of micro-fluidic channels 313 in the first micro-fluidic channel structures 311 in the same group are mutually communicated through the first through-hole structures 21 above the same, and the specific implementation manner is as follows: the diameter of the first through hole structure 21 is larger than the diameter of the circular hole at the end part of the micro fluid channel 313, so that the first through hole structure 21 is at least partially communicated with the flow channel of the middle part of the micro fluid channel 313, thereby realizing parallel communication of a plurality of micro fluid channels 313. It will be appreciated that the fluid inlet channels 11 and the fluid outlet channels 12 on each set of first microfluidic channel structures 311 should be connected in different first through-hole structures 21, respectively, and that the fluid inlet channels 11 and the fluid outlet channels 12 should extend into the first through-hole structures 21, but should not be inserted to the bottom, at a distance from the bottom of the first through-hole structures 21, so as to avoid cell detachment or damage due to surface tension caused by the gas entering the microfluidic channels from the bottom of the first through-hole structures 21 after the liquid is pumped out.

The microfluidic cell perfusion culture device 400 based on the standard porous plate can be matched with a multichannel microfluidic control system (such as a programmable peristaltic pump and the like) to provide power for a culture medium, so that the culture medium can continuously or periodically circulate in the corresponding first through hole structure 21 and the microfluidic channel 313 under the action of liquid pressure difference, thus timely providing nutrition required by cells and removing metabolic wastes, and the culture medium cannot overflow from the first through hole structure 21. It also simulates conventional static cell culture, utilizes programmable control, and through fluid inlet conduit 11 and fluid outlet conduit 12, to achieve periodic culture fluid replacement, saving labor and time.

Since the first microfluidic channel structure 311 is designed on the bottom plate assembly 30, the first microfluidic channel structure 311 is formed by connecting a plurality of microfluidic channels 313 in parallel, thereby realizing the intercommunication with a plurality of first through-hole structures 21, and the culture medium is infused by the fluid inlet pipeline 11 and discharged by the fluid outlet pipeline 12, and the form of micro-fluid infusion is closer to the micro-circulation environment in the body, therefore, the micro-fluid infusion can better simulate the natural state of cells in the body, including the characteristics of biological fluid dynamics.

In the corresponding first through-hole structures 21 and microfluidic channels 313, it is possible to culture and amplify cells or microorganisms and to perform cell counting and/or cell activity detection by staining under a fluorescence microscope. The microfluidic cell perfusion culture device 400 based on the standard porous plate in the embodiment of the invention can be used for conventional cell static culture and dynamic perfusion culture of cells; and is compatible with existing multi-well plate based cytometry instrumentation.

The present invention provides a microfluidic cell perfusion culture device 400 based on a standard multi-well plate, comprising: a cover plate 10, a middle layer plate 20 and a bottom plate assembly 30; the cover plate 10 is provided with a fluid inlet pipeline 11 and a fluid outlet pipeline 12 and is communicated with a first through hole structure 21 of the middle layer plate 20; the bottom plate assembly 30 is formed with a plurality of groups of first microfluidic channel structures 311, each group of first microfluidic channel structures 311 is formed by a plurality of microfluidic channels 313 to form a parallel structure, and the microfluidic channels 313 are communicated with the fluid inlet pipeline 11 and the fluid outlet pipeline 12 through corresponding first through hole structures 21. The microfluidic cell perfusion culture device 400 based on the standard porous plate provided by the invention has the following beneficial effects:

1. can realize the automation of the static cell culture. It can simulate conventional cell static culture and rely on programming, can regularly automatic change culture solution through fluid inlet line 11 and fluid outlet line 12, uses manpower sparingly and time.

5. The multi-channel culture/screening efficiency is high. The device is provided with a plurality of first micro-fluid channel structures 311, and each first micro-fluid channel structure 311 is connected in parallel by a plurality of micro-fluid channels 313, so that the operations of drug resistance screening test and the like of cells to different drug concentrations under the same environmental condition can be realized simultaneously, and the device is suitable for the operations of high-flux drug screening, comparative tissue engineering experiments and the like.

In one embodiment of the present invention, the first plate 31 further has a plurality of sets of second microfluidic channel structures 312 formed thereon, and each set of second microfluidic channel structures 312 includes: and one micro-fluidic channel 313, and the end part of each group of second micro-fluidic channel structures 312 is respectively communicated with one of the corresponding plurality of first through hole structures for cell static culture comparison research. As shown in fig. 7 and 8, on the first plate body 31, second microfluidic channel structures 312 are disposed outside the first microfluidic channel structures 311, each second microfluidic channel structure 312 has only a single microfluidic channel 313, and both ends of the microfluidic channel 313 are respectively connected to two of the plurality of first through-hole structures 21, so that the device can be used alone as a cell static culture device, and its effect is not different from that of a conventional multi-well plate; it is also fully compatible with the cell biology examination, detection and analysis instruments based on multi-well plates commonly used in laboratories. Therefore, the device can be widely applied to biopharmaceutical experiments, cell therapy experiments, tissue engineering experiments and drug screening experiments under laboratory conditions. In the present embodiment, the first plate 31 has a plurality of groups of first microfluidic channel structures 311 and second microfluidic channel structures 312 formed thereon, which can realize static cell culture and dynamic cell culture, and has high compatibility.

In one embodiment of the present invention, as shown in fig. 6 and 9, the floor assembly 30 further includes: a second plate 32 and a third plate 33. Wherein, the second plate 32 has a plurality of second through hole structures 321 formed thereon, and the second through hole structures 321 are disposed corresponding to the first through hole structures 21; the first plate 31 is disposed between the second plate 32 and the third plate 33. In the present embodiment, the bottom plate assembly 30 is formed by sequentially laminating and fixing the second plate body 32, the first plate body 31 and the third plate body 33 from top to bottom, so as to form a composite porous plate structure (hereinafter, referred to as a composite porous plate for short), the second plate body 32 is formed with a porous structure, that is, a second through hole structure 321, which is disposed corresponding to the first through hole structure 21, and the fluid inlet pipe 11 and the fluid outlet pipe 12 can be connected to the second through hole structure 321 through the first through hole structure 21, respectively. The third plate 33 is a bottom layer structure of the bottom plate assembly 30, which is a non-porous transparent plate structure, as shown in fig. 9, and from the perspective of fig. 2, the structure of the first plate 31 can be directly observed through the transparent third plate 33.

In one embodiment of the present invention, the third plate 33 at the bottom layer is a sheet of material with a thickness of about 200 μm, such as a colorless transparent amorphous thermoplastic material such as PET, PP, and PC, and is cut to form a bottom of the fluid channel; the first plate 31 of the middle layer is a double-sided adhesive layer (ARcare 8939) with the thickness of about 450 micrometers and cut with fluid channels and structures, and is used as a structural surface of the fluid channels; the second plate 32 of the uppermost layer is a PCR plastic film designed with fluid through holes as the top of the fluid channels. The three-layer structure may also be made of other materials that are cell biocompatible. Finally, the three-layer mechanism of the entire bottom plate assembly 30 is bonded or otherwise connected to the bottomless porous plate (i.e., the middle plate 20) in a leak-free manner to form a composite porous plate with microfluidic channels at the bottom.

In one embodiment of the present invention, the cover plate 10 is formed with a plurality of channels 15, the channels 15 being used to provide the fluid inlet conduit 11 and the fluid outlet conduit 12. In this embodiment, as shown in fig. 3, by providing a plurality of channels 15 on the cover plate 10 for accommodating the fluid inlet pipe 11 and the fluid outlet pipe 12, when the cover plate 10 is buckled with the middle layer plate 20, one ends of the fluid inlet pipe 11 and the fluid outlet pipe 12 extend into the corresponding first through hole structures 21, and the other ends are respectively connected with the male luer connector and the female luer connector, and can be used for sealing self connection for static cell culture (or optical detection), or connected with a multichannel peristaltic pump for dynamic cell perfusion.

In one embodiment of the present invention, the cover plate 10 is formed with transparent hole covers 13 and transparent observation windows 14 alternately arranged, the transparent hole covers 13 and the transparent observation windows 14 are respectively arranged corresponding to the first through hole structures 21, and the channels 15 are penetrated through the transparent hole covers 13, so that the fluid inlet pipes 11 and the fluid outlet pipes 12 are communicated with the first through hole structures 21 from below the transparent hole covers 13. In this embodiment, a portion of the first via structure 21 is covered by the transparent hole cover 13 on the cover plate 10 to avoid contamination of the medium in the hole. Due to the parallel connection structure of the plurality of micro-fluid channels 313 of the first micro-fluid channel structure 311, at least one of the first through hole structures 21 in each group of the first micro-fluid channel structures 311 is communicated with the fluid inlet pipeline 11, and at least one of the first through hole structures is communicated with the fluid outlet pipeline 12, so that the intercommunication of culture mediums can be satisfied.

In one embodiment of the present invention, the standard multi-well plate based microfluidic cell perfusion culture device 400 further comprises: a first side panel 16 and a second side panel 22. Wherein, the first side plate 16 is arranged at the outer edge of the cover plate 10; the second side plate 22 is of a step structure, the second side plate 22 is arranged at the outer edge of the middle layer plate 20, and the first side plate 16 is correspondingly attached to the outer wall of the second side plate 22, so that the cover plate 10 is buckled on the upper surface of the middle layer plate 20. In this embodiment, the first side plate 16 is fastened to the step structure of the second side plate 22, so that the cover plate 10 can be easily assembled to the upper layer of the middle layer plate 20, and a limit is formed between the first side plate 16 and the step structure, and after the cover plate 10 is fastened to the upper side of the middle layer plate 20, the fluid inlet pipe 11 and the fluid outlet pipe 12 can be correspondingly inserted into the first through hole structure 21, so that the assembly and positioning are facilitated.

In one embodiment of the present invention, the bottom plate assembly 30 is disposed inside the second side plate 22 and fixedly connected to the middle plate by bonding or welding to form a composite porous plate structure with microfluidic channels at the bottom. In this embodiment, the bottom plate assembly 30 and the middle layer plate are fixed together by bonding or welding, and the positioning piece 23 is disposed on the inner side of the second side plate 22 to assist in accurately positioning the bottom plate assembly 30 and the middle layer plate 20, so as to ensure that the through hole structures (i.e. the second through hole structure 321 and the first through hole structure 21) between the two are aligned one by one.

In one embodiment of the present invention, each set of first microfluidic channel structures 311 comprises: a parallel structure formed by three microfluidic channels 313; each microfluidic channel 313 is connected to three first via structures 21. Specifically, as shown in fig. 8, 8 groups of first microfluidic channel structures 311 and 8 groups of second microfluidic channel structures 312 are disposed on the first plate 31, each group of first microfluidic channel structures 311 is formed by connecting three microfluidic channels 313 in parallel, each group of second microfluidic channel structures 312 is composed of only one microfluidic channel 313, each microfluidic channel 313 is correspondingly connected with three first through-hole structures 21 (i.e., culture holes), one of which is connected with the fluid inlet pipeline 11, and the other of which is connected with the fluid outlet pipeline 12.

In one embodiment of the present invention, the standard multi-well plate based microfluidic cell perfusion culture device 400 is a disposable consumable.

The invention also provides a microfluidic cell perfusion culture system based on the standard porous plate. The microfluidic cell perfusion culture system based on the standard porous plate comprises: the programmable peristaltic pumping device 100, the liquid filtering tank 200, the liquid storage tank 300, the air release valve 500 and the microfluidic cell perfusion culture device 400 based on the standard porous plate in the embodiment of the invention are sequentially connected through the flexible pipe to form a group of independent fluid perfusion control channels, and under the control of the programmable pumping device, the culture medium in each microfluidic channel automatically and continuously circulates or periodically at the same time, and the culture medium cannot overflow from the culture hole. I.e. the system can be set by programmable pumping means, the flow rate and flow rate of the fluid flow in the pipeline, the start-up time, the continuous or periodic circulation mode.

As shown in fig. 10, a pumping device 100, a fluid reservoir 200, a fluid reservoir 300 and a microfluidic cell perfusion culture device 400 based on a standard porous plate according to an embodiment of the present invention are sequentially connected and assembled through a fluid pipe, a connector, etc., and a gas release valve 500 is provided at an inlet end of the fluid reservoir 300, wherein the fluid reservoir 300 is used for storing fresh culture medium, the fluid reservoir 200 is used for collecting the culture medium containing metabolic wastes, and the gas release valve 500 is used for assisting in controlling the gas pressure of the system. Each of the above-described sets of components are connected by flexible tubing to form a separate fluid infusion control channel and allow continuous or periodic circulation of the culture medium in each microfluidic channel 313 without spilling the culture medium from the culture wells under the control of pumping device 100 (e.g., a programmable peristaltic pump).

A standard multi-well plate based microfluidic cell perfusion culture device 400 according to embodiments of the present invention is in a fast attachable and detachable relationship with its adaptive perfusion control system (i.e., including programmable peristaltic pumping device 100, reservoir 200, reservoir 300, etc.). When in a disassembled state, the microfluidic channel 313 of the microfluidic cell perfusion culture device 400 based on the standard porous plate and the fluid channel of the adaptive perfusion control system can be respectively connected by self-closing of the male luer connector and the female luer connector so as to prevent the flow channel from being polluted, and the sterilization operation of the device and the maintenance of the sterile state of the device are facilitated.

The working principle of the microfluidic cell perfusion culture system based on the standard porous plate provided by the embodiment of the invention is shown in fig. 10, and the purpose is to inject fresh culture medium from the liquid storage tank 300 into the corresponding microfluidic channel 313 on the composite porous plate through pipeline connection and driving of the peristaltic pump so as to dynamically culture cells in the culture system, and remove metabolic waste liquid from the cell culture microfluidic channel 313 and collect the metabolic waste liquid in the independent filtrate tank 200, so that continuous or periodic circulating flow of the culture medium is realized, and nutrition and metabolic waste are removed, which are required by the cells.

The specific operation comprises the following steps: 1) Cells to be dynamically cultured may be first implanted in suspension via a pipette into the corresponding microfluidic channel 313 on the composite multiwell plate; 2) Adding fresh culture medium or culture solution with different concentration medicines to be screened into corresponding liquid storage tanks 300, and then sequentially and hermetically connecting the culture solution with corresponding fluid channels, peristaltic pumps and corresponding waste liquid filter tanks 200 on a composite porous plate cover plate 10 through pipelines (and connectors); 3) Setting the flow speed and the flow rate of the fluid flow in the pipeline, the starting and stopping time and a continuous or periodic circulating mode through a programmable peristaltic pump; 4) Placing the whole culture perfusion system on a tray, starting a peristaltic pump, checking pipeline connection and ensuring that fluid of each channel is not leaked, and placing the culture perfusion system and the tray into a cell incubator for cell dynamic perfusion culture; 5) After the incubation is completed, the whole incubation perfusion system is removed from the cell incubator along with the tray. Then, the fluid channels on the composite porous plate cover plate 10 are disconnected from the perfusion control system, and the fluid inlet pipeline 11 and the fluid outlet pipeline 12 of each channel are in butt joint through the male luer connector and the female luer connector to form self-closing connection. Alternatively, a set of conventional perforated plates may be taken with their cover plates exchanged with the composite perforated plate cover plate 10; 6) At this time, the whole closed porous plate microfluidic cell perfusion culture device is completely compatible with a conventional porous plate, and can perform subsequent operations such as staining and cell biological testing on cells in the device.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic cell perfusion culture device based on standard multi-well plates, comprising: the cover plate, the middle layer plate and the bottom plate component are sequentially stacked;

wherein the floor assembly comprises:

2. The standard multi-well plate based microfluidic cell perfusion culture device of claim 1, wherein the first plate further has a plurality of sets of second microfluidic channel structures formed thereon, each set of second microfluidic channel structures comprising: and the end parts of each group of second microfluidic channel structures are respectively communicated with one of the corresponding first through hole structures and are used for cell static culture comparison research.

3. The standard multiwell plate based microfluidic cell perfusion culture device according to claim 1, wherein the bottom plate assembly further comprises:

4. The standard multiwell plate based microfluidic cell perfusion culture device according to claim 1, wherein a plurality of channels are formed on the cover plate for providing the fluid inlet and outlet conduits.

5. The microfluidic cell perfusion culture device based on a standard multi-well plate according to claim 4, wherein transparent hole covers and transparent observation windows are alternately formed on the cover plate, the transparent hole covers and the transparent observation windows are respectively arranged corresponding to the first through hole structures, and the channels penetrate through the transparent hole covers.

6. The standard multiwell plate based microfluidic cell perfusion culture device according to claim 1, further comprising:

the first side plate is arranged at the outer edge of the cover plate;

7. The microfluidic cell perfusion culture device based on standard porous plates according to claim 6, wherein the bottom plate component is arranged on the inner side of the second side plate and is fixedly connected with the middle layer plate through bonding or welding to form a composite porous plate structure with a microfluidic channel at the bottom.

8. The standard multi-well plate based microfluidic cell perfusion culture device according to any one of claims 1 to 7, wherein each set of the first microfluidic channel structures comprises: a parallel structure formed by three micro-fluid channels.

9. The microfluidic cell perfusion culture device based on standard multi-well plates according to any one of claims 1 to 7, wherein each microfluidic channel is connected to three of the first through-hole structures.

10. A microfluidic cell perfusion culture system based on standard multi-well plates, comprising: a programmable pumping device, a liquid filtering tank, a liquid storage tank, a gas release valve and the microfluidic cell perfusion culture device based on the standard porous plate according to any one of claims 1 to 9, which are sequentially connected through a hose, so as to form a group of independent fluid perfusion control channels, and under the control of the programmable pumping device, the culture medium in each microfluidic channel automatically and continuously circulates or periodically at the same time, and the culture medium cannot overflow from the culture hole.