Perfusion culture microfluidic device and system

Abstract

The invention belongs to the field of perfusion culture chips, and provides a perfusion culture microfluidic device and system. A perfusion culture microfluidic device comprises a main body, a culture support and a sealing structure, the main body is divided into an upper layer structure, a middle layer structure and a bottom layer structure, the upper layer is provided with a liquid inlet and a liquid outlet which are respectively connected with a channel between the upper layer and the middle layer, and a channel between the middle layer and the bottom layer; the channel penetrates through the culture bracket in the middle layer, and the culture bracket is provided with an upper circular ring and a lower circular ring which are separated by a porous membrane; the sealing structures are respectively arranged on the upper layer and the middle layer of the main body and on the bottom layer of the main body, and seal the upper-layer circular ring, the porous membrane and the lower-layer circular ring of the culture bracket into two culture chambers. According to the perfusion culture micro-fluidic device and system and the double-channel perfusion micro-fluidic device based on the porous membrane culture support, the growth requirements of various tissue or organ simulants can be met, the sealing structure enables the device to be disassembled and assembled without the help of auxiliary equipment, inoculation and characterization of cells are facilitated, and the operation is simple and convenient. The whole device can be repeatedly used.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of perfusion culture chip, in particular to a perfusion culture microfluidic device and system. BACKGROUND

[0002] With the continuous development of cell biology, organ-on-chip and organoid technology, three-dimensional cell culture technology has gradually become a research hotspot and is gradually replacing traditional two-dimensional cell culture technology. Various types of cells, such as pluripotent stem cells, tumor cells and patient-derived primary cells, etc., exhibit strong self-assembly ability and can be used to construct three-dimensional physiological and pathological models. This three-dimensional structure is closer to the real morphology of in vivo tissue cells, which is conducive to in-depth study of its functional mechanism and is widely used in developmental biology, pathology, pharmacology, cancer treatment and other fields. However, traditional three-dimensional cell culture methods, such as hanging drop method, suspension culture, droplet method, etc., have the problem of poor controllability and repeatability, which is difficult to meet the needs of high-throughput and precise control. In contrast, perfusion culture chips based on microfluidic technology have emerged as the times require, providing a more efficient and accurate solution for three-dimensional cell culture. Microfluidic technology can achieve precise control of the cell culture microenvironment, simulate in vivo physiological conditions, and provide an ideal platform for cell growth, differentiation and function research. The core of perfusion culture technology is to continuously update the culture medium to provide sustained supply of nutrients and oxygen to cells, while timely removing metabolic waste to maintain the healthy growth and stable state of cells. This dynamic culture method not only improves cell viability and functional expression, but also better simulates the physiological metabolic process and mechanical environment of in vivo tissues, prolonging the culture and detection time of the model.

[0003] The human body is a complex multi-organ system with close material exchange and signal transmission between organs. In order to more comprehensively simulate the physiological and pathological processes of the human body, constructing a multi-organ system interconnected biomimetic microphysiological platform has become a research hotspot. Perfusion culture chip technology combines the advantages of microfluidic technology, organ-on-chip technology and organoid culture technology, providing new tools and methods for the study of multi-organ systems. Microfluidic technology can precisely manipulate fluids at the microscale, simulating in vivo physiological environments and providing stable nutrient supply and metabolic waste removal for cell and tissue culture. Organ-on-chip and organoid technology can construct three-dimensional cell structures with specific functions from in vitro, which are closer to in vivo tissues and organs. The combination of the two can achieve dynamic culture and interconnection of multiple organ models, simulating the interaction and synergistic function between multiple organs in vivo. This multi-organ system interconnected perfusion culture chip not only can be used to study the communication mechanism and disease transmission pathway between organs, but also can provide a more accurate in vitro model for drug development to evaluate the metabolism, distribution and toxicity of drugs in different organs.

[0004] There are documents in the prior art that introduce organoid perfusion culture technology and commercial product solutions.

[0005] Patent documents are as follows:

[0006] Chinese patent document CN218321440U discloses an organoid perfusion culture chip: the organoid perfusion culture chip of the utility model comprises a placing shell, a liquid storage shell, a mounting plate, a culture shell, a partition assembly and the like. The culture medium is injected into the placing shell through the first liquid inlet pipe, the shunt pipe and the connecting pipe, and then enters the corresponding culture shell through the plurality of second liquid inlet pipes. The cell suspension is added to the partition ring for culture, and the culture medium enters the partition ring through the through hole to provide nutrients and oxygen for the cells. When the liquid level in the culture shell exceeds the upper end of the cannula, the culture medium flows into the liquid storage shell through the cannula and the first liquid outlet pipe, and finally is discharged through the second liquid outlet pipe. This solution realizes unified perfusion of all culture medium storage pools under the condition of reducing the sample interference in the culture shell, and improves the efficiency and stability of the culture process.

[0007] Chinese patent document CN111548939A discloses an organoid perfusion culture chip and a method for using the same: the organoid perfusion culture chip of the invention adopts a two-layer structure of micropores and microcolumns. The micropores in the micropore layer are arranged in an array structure to form cell spheres; the microcolumn structures in the microcolumn layer form relatively independent spaces to accommodate the cell spheres formed in the micropores, and realize the exchange of nutrients and the discharge of metabolic waste during perfusion culture through the gaps between the microcolumns. The method for using the chip includes steps of cell inoculation, culture, chip overturning to make the cell spheres enter the spaces of the microcolumn layer, connecting a fluid control unit to perform perfusion culture, and the like. This solution simplifies the operation process of organoid experiments, meets the demand for high throughput, and is suitable for research in the fields of biomedicine, new drug development, toxicology and the like.

[0008] Chinese patent document CN112481077A discloses a microfluidic perfusion culture device and a perfusion method thereof: the microfluidic perfusion culture device of the invention comprises a fluid control unit, a culture chamber unit and a base liquid path unit. The fluid control unit connects the culture medium to the base liquid path unit through a pump body, and the base liquid path unit controls the flow of the culture medium through the on-off control of the electromagnetic valve, to realize accurate perfusion culture of the cells in the culture chamber. This device can flexibly control the flow rate, flow volume and replacement frequency of the culture medium, to meet different cell culture and experimental requirements.

[0009] The single perfusion channel chip in the commercial product solution is as follows:

[0010] CN Bio's PhysioMimix Organ-on-Chip (OOC) series microphysiological systems (MPS): Support the culture of multiple organs, as well as the co-culture of different organs. The main difference of the chip they use is mainly reflected in the structural design of the culture chamber, the mechanism and the way of fluid control of different chips are the same. The chip is mainly divided into three layers, from top to bottom, the culture chamber, the membrane layer, and the gas path layer. The overall structure of the culture layer is similar to the traditional cell culture well plate, which is open to the outside world, convenient for cell implantation, drug addition and replacement, and consistent with the outside culture environment. The middle layer uses COC film, which is more suitable for bonding with plastic base than the conventional PDMS film layer, and its mechanical strength is also higher. The bottom layer is the gas path layer, which connects the external gas source to control the deformation of the diaphragm and complete the liquid circulation flow.

[0011] Hesperos' Human-On-A-Chip organ-on-chip is a highly integrated and flexible microphysiological system. It can build multiple interconnected organs such as heart, liver, lung, brain, etc. on a chip, and realize material exchange and signal transmission between organs through microfluidic system. The chip uses a pump-free gravity flow system to maintain cell activity using serum-free medium circulation. Cells are cultured in three dimensions to form microtissue structures similar to in vivo organs, which can better simulate physiological and pathological characteristics. The chip is equipped with advanced non-invasive sensors that can monitor organ activity in real time and generate a large number of data points. Through customized data analysis system, it can quickly analyze the trend of organ function changes. In addition, the chip adopts a modular design, and the organ modules and barrier tissues can be flexibly replaced and adjusted, and the number of organ modules can also be increased or decreased according to needs, with good scalability, which can meet different research needs and help build disease models and drug research.

[0012] Dual-channel perfusion chip with porous membrane structure in commercial product scheme:

[0013] TissUse's HUMIMIC Multi-Organ-Chip (MOC) supports the culture of multiple organ models and can realize the serial culture of multiple organs for evaluating the systemic effects and toxicity of drugs. Its chip structure is a multi-layer structure, including an upper layer with seeding wells, a PDMS middle layer integrating microfluidic channels and pneumatic membranes, and a bottom glass sheet. The series of chips use pneumatic method to control the sequential deformation of PDMS to complete liquid circulation flow. HUMIMIC Chip2 / 3 can simulate dual-organ and triple-organ systems, and culture multiple organs in series. HUMIMIC Chip4 chip integrates a porous membrane to connect two independent microfluidic channels, which can simulate the transport and exchange of substances between different organ models, such as simulating the excretion and reabsorption process of substances in the kidney model.

[0014] BE-Transflow chip is a multifunctional microfluidic chip for cell culture under biomimetic conditions. It contains two independent culture wells with the same volume as a single well of a 96-well plate, allowing 2D or 3D cell culture. The culture wells are connected to microfluidic channels through a porous membrane, allowing exchange of nutrients and signaling molecules, and the channel size can be customized. BE-Doubleflow chip is a multifunctional microfluidic device similar to the use of a glass slide. The chip has a double-channel, with two independent perfusable channels connected by a porous membrane with a selectable pore size, allowing exchange of nutrients and signaling molecules to achieve complex culture configurations. The chip series also has an anti-evaporation reservoir and a spiral reservoir, which facilitates the connection of a microfluidic flow control system. In terms of material, the chip is made of biocompatible plastic, with good optical performance and no non-specific adsorption characteristics, suitable for a variety of detection methods.

[0015] The Dutch Micronit multilayer chip is composed of three layers: the bottom sheet, the cover sheet, and the middle film. The bottom sheet and the cover sheet are glass slides, and the middle film is made of borosilicate glass. The bottom sheet and the cover sheet have rubber sealing rings, which are sealed by mold clamping. After the experiment, it can be easily disassembled for subsequent operation. The middle film layer is both a cell culture support framework and a selective permeable medium for cell culture, forming two independent chambers. This multilayer structure can simulate part of the organ culture environment.

[0016] Emulate's "human simulation system" core component Chip-S1® chip reproduces complex biology through two different culture channels, while enabling cell-to-cell interactions through a porous membrane that is elastic and can be stretched by air chambers on both sides. The entire chip system can reproduce the natural physiological and mechanical forces experienced by human cells, such as fluid shear stress, cyclic strain, etc., thus more realistically simulating human responses.

[0017] However, the aforementioned existing technologies have the following drawbacks: a. They can only perform single-channel perfusion, and connecting multiple organ mimics in series poses a serious challenge to developing culture media compatible with the growth of multiple types of organ mimics (organoids, etc.); moreover, these chips have significant limitations in constructing complex physiological models such as vascularized ones. For example, single-perfusion channel chips in commercial product solutions, TissUse's HUMIMIC Chip2 / 3, CN218321440U (an organoid perfusion culture chip with a complex structure), and CN111548939A (an organoid perfusion culture chip and its usage method with a complex structure). b. Some chips are closed chips, which are not conducive to sample inoculation and characterization, such as CN112481077A (a microfluidic perfusion culture device and its perfusion method), Emulate's Chip-S1® chip (a core component of the "human simulation system"), and single-perfusion channel chips in commercial product solutions. c. Some chips require the use of special fixtures or auxiliary modules, increasing the cost of use. For example: Micronit multilayer chips from the Netherlands, CN Bio's PhysioMimix organ-on-a-chip (OOC) series microphysiological systems (MPS) and Emulate series chips. d. Non-reusable, such as the Emulate series chips, BE-Doubleflow chips, HUMIMIC Chip4 chips, etc.

[0018] Therefore, there is an urgent need for a microfluidic device and system for perfusion culture. Summary of the Invention

[0019] The purpose of this invention is to provide a perfusion culture microfluidic device and system, which is a dual-channel perfusion microfluidic device based on a porous membrane culture scaffold. It can be compatible with the growth requirements of various organ mimics, and the sealed structure can be disassembled at any time, which is conducive to cell inoculation and characterization. It can be disassembled manually and can be reused.

[0020] To solve the above-mentioned technical problems, the perfusion culture microfluidic device and system provided by the present invention are implemented as follows:

[0021] A perfusion culture microfluidic device includes a main body, a culture support, and a sealing structure. The main body has a three-layer structure: an upper layer with an inlet and an outlet, which are respectively connected to a channel between the upper and middle layers and a channel between the middle and bottom layers. The channel passes through the culture support located in the middle layer. The culture support has two rings, an upper ring and a lower ring, separated by a porous membrane. The sealing structure is located in the upper and middle layers of the main body and in the bottom layer of the main body, sealing the upper ring, the porous membrane, and the lower ring of the culture support to form two culture chambers.

[0022] This invention features a three-layer structure design, integrating two independent perfusion channels (one between the upper and middle layers, and the other between the middle and bottom layers) with the culture chamber. Structurally, it comprises a main structure, a culture scaffold, and a sealing structure. The main structure consists of an upper, middle, and bottom layer, forming two independent perfusion channels. The culture scaffold has a porous membrane in the middle to isolate the upper and lower channels, creating independent culture chambers. The sealing structure ensures the airtightness of the culture chambers, eliminating the need for external clamps. This chip can be used to construct tissue interfaces, biological barriers, or dual-organ models, as well as to build microphysiological systems through the series and parallel connection of multiple chips. It supports various perfusion culture modes, such as unidirectional perfusion, parallel perfusion, and cross-perfusion, and can simulate complex physiological and pathological models, making it suitable for basic theoretical research, drug toxicity testing, and pharmacokinetic studies.

[0023] Optionally, the upper layer has two liquid inlets and two liquid outlets: a first liquid inlet and a second liquid inlet, a first liquid outlet and a second liquid outlet; the first liquid inlet is connected to a first channel between the upper layer and the middle layer, and liquid flows out from the first liquid outlet; the second liquid inlet is connected to a second channel between the middle layer and the bottom layer, and liquid flows out from the second liquid outlet.

[0024] Optionally, the culture scaffold is a disc-shaped disk with an upper ring on the top layer and a lower ring on the bottom layer, with a porous membrane between the upper and lower rings. The upper and lower rings each have slits, and the slits have corresponding flow windows on the middle layer of the main body. When the two are aligned and assembled, the first channel passes through the flow window and the slit of the upper ring to the first culture chamber formed by the upper ring and the porous membrane, and the second channel passes through the flow window and the slit of the lower ring to the second culture chamber formed by the porous membrane and the lower ring of the culture scaffold.

[0025] The culture support is placed on a circular hole in the middle layer of the main body. The diameter of the hole is the same as the diameter of the upper and lower circular rings. A disc supports the culture support on the middle layer of the main body. The culture support is detachable, and the upper and lower circular rings on the culture support can be set as needed. This invention uses two rings arranged side by side.

[0026] Optionally, the lower layer of the culture scaffold is provided with a channel orientation structure, which is a micro-cylinder or parallel rib, and the height of the micro-cylinder or parallel rib is greater than or equal to the height of the second channel.

[0027] Optionally, the channel orientation structure is located on both sides of the radial connection line of the circular cut in the lower layer of the culture scaffold, and there are at least two or more channel orientation structures with corresponding grooves in the corresponding positions of the main body.

[0028] Optionally, the sealing structure has three parts: the first sealing structure and the inner side of the upper circular hole of the main body are provided with matching threads; the first sealing structure passes through the circular hole on the upper part of the main body; the second sealing structure is outside the upper circular ring of the culture support; the first sealing structure, the second sealing structure and the upper circular ring of the culture support seal to form the first culture chamber; the third sealing structure is sleeved on the bottom of the lower circular ring and seals with the lower circular ring and the base on the bottom layer of the main body to form the second culture chamber.

[0029] Optionally, the porous membrane is made of PET, PC, PDMS, COC, or COP. The porous membrane has a uniform or normally distributed pore size, a pore size range of 0.1-20 μm, a porosity of 20-95%, a thickness of 5-100 μm, and an effective inoculation area of ​​not less than 19.83 mm². 2 .

[0030] Optionally, the main body, culture scaffold, and first sealing structure may be made of thermoplastic plastics such as PMMA, PC, COC, or COP.

[0031] Optionally, the second and third sealing structures are made of silicone, rubber, fluoropolymer, or polyurethane.

[0032] The present invention also provides a perfusion culture microfluidic system, comprising the perfusion culture microfluidic device as described in claims 1 to 9, a culture medium storage tank, an antifoamer, and a power source; the power source provides power to control the liquid flow, and the perfusion culture microfluidic device performs static culture, unidirectional perfusion, parallel perfusion, reverse perfusion, unidirectional circulating perfusion, and bidirectional circulating perfusion modes.

[0033] The perfusion culture microfluidic device provided by this invention is reusable and exhibits good light transmittance and biocompatibility. The sealing structure, from the upper to the middle and bottom layers of the main body, is equipped with compression-type sealing structures forming nuts, preventing crosstalk between the upper and lower channels in the middle layer. The device is reusable and has good light transmittance and biocompatibility; the culture scaffold with a porous membrane is detachable, facilitating cell seeding and other operations. The culture scaffold also features a channel orientation structure, facilitating chip assembly, sample seeding, and characterization.

[0034] Because the culture scaffold and sealing structure of this invention are detachable, they are easy to operate and avoid crosstalk; and they support diverse perfusion culture modes. These features give this invention significant advantages in constructing complex physiological models, studying cell-cell interactions, and evaluating drugs.

[0035] This invention addresses the limitations of existing perfusion culture chips. Existing single-channel perfusion chips are significantly insufficient for constructing complex physiological models and struggle to meet the growth requirements of various organ mimics. Furthermore, some closed-loop chips in existing technologies are not conducive to cell seeding and characterization. Additionally, some chips require specialized clamps for disassembly and reuse, increasing usage costs. Attached Figure Description

[0036] Figure 1 This is a perspective view of the perfusion culture microfluidic device provided by the present invention;

[0037] Figure 2 This is a top view of the perfusion culture microfluidic device provided by the present invention;

[0038] Figure 3 yes Figure 2 Sectional view along direction A;

[0039] Figure 4 This is an exploded view of the perfusion culture microfluidic device provided by the present invention;

[0040] Figure 5 This is a structural diagram of the culture support in the perfusion culture microfluidic device provided by the present invention, wherein A is a bottom view, B is a top view, C is a left view, D is a front view, and E is a radial section view of the front view;

[0041] Figure 6 This is a graph showing the TEER values ​​of a small intestinal epithelial model constructed using the perfusion culture microfluidic device of the present invention. In graph A, the TEER values ​​of HIMEC and Caco-2 cells seeded on both sides of the porous membrane, the TEER values ​​of only one type of cell seeded, and the TEER values ​​of the control group without cell seeding are compared. In graph B, the TEER values ​​of a small intestinal epithelial organochip model constructed using different thermoplastic materials such as PMMA, PC, COC, and COP are compared.

[0042] Figure 7 These are physiological index detection diagrams of a liver organ-on-a-chip model constructed using the novel perfusion culture microfluidic device of the present invention. In this diagram, A is the difference in physiological activity of albumin secretion and urea synthesis under different perfusion rates, and B is the difference in physiological activity of CYP1A2 and CYP3A4.

[0043] Figure 8 This is a schematic diagram of an intestinal-liver microphysiological system constructed using the novel perfusion culture microfluidic device of the present invention;

[0044] Wherein: 1-Main body, 11-Upper layer, 12-Middle layer, 13-Bottom layer, 14-First liquid inlet, 15-Second liquid inlet, 16-First liquid outlet, 17-Second liquid outlet, 18-Base;

[0045] 2-Cultivation scaffold, 21-Upper ring, 22-Lower ring, 23-Porous membrane, 26-First culture chamber, 27-Second culture chamber, 28-First channel, 29-Second channel, 30-Channel orientation structure;

[0046] 31-First sealing structure, 32-Second sealing structure, 33-Third sealing structure. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] Example 1: The microfluidic device and system for perfusion culture of the present invention

[0050] like Figures 1 to 5 As shown, a perfusion culture microfluidic device includes a main body 1, a culture support 2, and a sealing structure. The main body 1 has a three-layer structure: upper, middle, and lower. The upper layer 11 has an inlet and an outlet, which are respectively connected to a channel between the upper layer 11 and the middle layer 12, and a channel between the middle layer 12 and the lower layer 13. The channel passes through the culture support 2 located in the middle layer. The culture support 2 has two rings, an upper ring and an lower ring, which are separated by a porous membrane 23. The sealing structure seals the upper ring 21, the porous membrane 23, and the lower ring 22 of the culture support into two culture chambers, located in the upper layer 11 and the middle layer 12 of the main body, and in the lower layer 13 of the main body.

[0051] This invention features a three-layer structure design, integrating two independent perfusion channels (one between the upper and middle layers, and the other between the middle and bottom layers) with the culture chamber. Structurally, it comprises a main structure, a culture scaffold, and a sealing structure. The main structure consists of an upper, middle, and bottom layer, forming two independent perfusion channels. The culture scaffold has a porous membrane in the middle to isolate the upper and lower channels, creating independent culture chambers. The sealing structure ensures the airtightness of the culture chambers, eliminating the need for external clamps. This chip can be used to construct tissue interfaces, biological barriers, or dual-organ models, as well as to build microphysiological systems through the series and parallel connection of multiple chips. It supports various perfusion culture modes, such as unidirectional perfusion, parallel perfusion, and cross-perfusion, and can simulate complex physiological and pathological models, making it suitable for basic theoretical research, drug toxicity testing, and pharmacokinetic studies.

[0052] The upper layer has two inlets and two outlets, forming two channels: a first inlet 14, a second inlet 15, a first outlet 16, and a second outlet 17. The first inlet 14 connects to the first channel 28 between the upper and middle layers, and the liquid flows out from the first outlet. The second inlet 15 connects to the second channel 29 between the middle and bottom layers, and the liquid flows out from the second outlet.

[0053] The middle layer 12 of the main body continues the tubular structure of the second liquid inlet and the second liquid outlet extending from the upper layer, making it a vertical channel that runs through the upper and middle layers.

[0054] like Figures 1 to 5 As shown, the culture scaffold 2 is a disc-shaped disk with an upper ring 21 on the upper layer and a lower ring 22 on the lower layer. A porous membrane 23 is located between the upper ring 21 and the lower ring 22. The upper ring 21 and the lower ring 22 each have two symmetrical cuts. The liquid in the first channel 28 flows through the cuts into the first culture chamber 26 formed by the upper ring 21 and the porous membrane 23. The liquid in the second channel 29 flows through the cuts into the second culture chamber 27 formed by the porous membrane 23 and the lower ring 22 of the culture scaffold 2.

[0055] The culture support is placed on a circular hole in the middle layer of the main body. The diameter of the hole is the same as the diameter of the upper and lower circular rings. A disc supports the culture support on the middle layer of the main body. The culture support is detachable, and the upper and lower circular rings on the culture support can be set as needed. This invention uses two rings arranged side by side.

[0056] The main middle layer 2 is a flat plate structure with a certain thickness. Its outline matches the overall shape of the device. A large through hole or cavity is opened in the center. The bottom surface of the upper ring of the culture support is attached to the upper surface of the middle layer, and the bottom surface of the lower ring is attached to the base 18 of the bottom layer 3.

[0057] Symmetrical notches are machined on the circumference of the upper and lower rings of the culture scaffold. Corresponding flow windows are provided at the corresponding positions of these notches on the middle layer. When the two are aligned and assembled, they together form a transverse flow channel.

[0058] In other words, corresponding to the positions of the upper and lower circular cuts on the culture scaffold, the middle layer has symmetrical rectangular or approximately rectangular windows, with the windows corresponding to the cut positions, to facilitate the passage of the first and second channels into the culture chamber.

[0059] The lower layer of the culture scaffold is provided with a channel orientation structure 30, which is a micro-cylinder or parallel rib. The height of the micro-cylinder or parallel rib is greater than or equal to the height of the second channel. There are at least two micro-cylinders or parallel ribs, corresponding to each other on the left and right sides of the culture scaffold.

[0060] The channel orientation structure 30 is located on both sides of the radial connection line of the circular cut in the lower layer of the culture scaffold, and there is at least one or more channel orientation structures with corresponding grooves in the corresponding positions of the main body.

[0061] like Figures 1 to 4 As shown, there are three sealing structures. The first sealing structure and the inner side of the upper circular hole of the main body are provided with matching threads. The first sealing structure 31 passes through the circular hole on the upper layer 11 of the main body. The second sealing structure 32 is outside the upper ring 21 of the culture support 2. The first sealing structure 31, the second sealing structure 32 and the upper ring 31 of the culture support seal to form the first culture chamber 26. The third sealing structure 33 is fitted on the bottom of the lower ring and seals with the lower ring 22 and the bottom layer of the main body to form the second culture chamber 27.

[0062] On the upper surface of the middle layer, around the central cavity and the flow window, there is an annular groove for installing the first sealing structure 31 and the second sealing structure 32. On the lower surface of the middle layer, in the annular area that contacts the bottom surface of the lower ring of the culture support, there is an annular groove for installing the third sealing structure 33.

[0063] The porous membrane of this invention is made of PET, PC, PDMS, COC, or COP. The pore size of the porous membrane is uniform or normally distributed. The pore size range is 0.1-20 μm, the porosity is 20-95%, and the thickness is 5-100 μm. The effective inoculation area of ​​the porous membrane is not less than 19.83 mm². 2 .

[0064] The main body, culture scaffold, and first sealing structure of this invention are made of thermoplastic plastics such as PMMA, PC, COC, or COP.

[0065] The second and third sealing structures of this invention are made of silicone, rubber, fluoropolymer, or polyurethane.

[0066] The present invention also provides a perfusion culture microfluidic system, including a perfusion culture microfluidic device, a culture medium storage tank, an antifoamer, and a power source; the power source provides power to control the liquid flow, and the perfusion culture microfluidic device can perform static culture, unidirectional perfusion, parallel perfusion, reverse perfusion, unidirectional circulating perfusion, and bidirectional circulating perfusion modes.

[0067] Example 2: Application of the microfluidic device for perfusion culture provided by the present invention

[0068] The porous membrane of the culture scaffold of this invention can be seeded with the same or different types of organ or tissue mimics on both its upper and lower sides. A single device can be used to construct tissue interfaces, biological barriers, dual-organ or vascularized organ models, etc.; multiple chips connected in series and parallel can be used to construct biomimetic human microphysiological systems. Assembling multiple devices involves linking multiple models through sterile silicone tubing: identical models can be scaled up and cultured through series and parallel connections, while different models can form upstream and downstream connections through series connection. This can be applied to scenarios including, but not limited to, the following: physiological information transmission, sensing, and response between tissues and organs; pharmacokinetic studies; drug toxicity studies; cell migration and metastasis, etc.

[0069] A single device or a complete working system comprises a perfusion culture microfluidic device, a culture medium storage tank, an defoamer, and a power source. The power system controls the liquid flow, and the device can perform various culture modes, including but not limited to static culture, unidirectional perfusion, parallel perfusion, reverse perfusion, unidirectional circulating perfusion, and bidirectional circulating perfusion.

[0070] Culture medium storage tanks and defoamers can be used in combination, with a variety of commercially available models to choose from. Power sources can be selected from syringe pumps, pressure pumps, peristaltic pumps, flow meters, etc., with pumping capacities ranging from 1 μL to 1 mL / min. A variety of commercially available models are also available.

[0071] Organ or tissue mimics can be immortalized cell lines, primary cells, organ or tissue-specific cells induced by various stem cells from animals or humans, as well as multicellular spheroids, organoids, primary tissues, etc. derived therefrom.

[0072] Example 3: Method of using the perfusion culture microfluidic device provided by the present invention

[0073] 3.1 Operation procedure for a single chip:

[0074] 1. All instruments can be sterilized with ethylene oxide;

[0075] 2. Inoculate organ or tissue analog A onto the underside of the porous membrane of the culture scaffold;

[0076] Steps 3-6 are the difference steps. After selecting operation procedure A or B according to the characteristics of the model, proceed with the subsequent steps.

[0077] Operation Procedure A:

[0078] 3. Seed organ or tissue simulator B on the upper side of the porous membrane of the culture scaffold;

[0079] 4. Assemble the culture scaffold with the main body according to the channel positioning structure, paying attention to the correspondence between the cut of the culture scaffold and the middle layer window of the main body;

[0080] 5. Inject the medium (Medium, M) AM into the lower channel through the second inlet;

[0081] 6. Inject culture medium BM (AM and BM can be the same culture medium or different culture media with minor differences) into the upper channel through the first inlet.

[0082] Operation Procedure B:

[0083] 3. Assemble the culture scaffold and the main body of the device according to the channel positioning structure, but do not add the first sealing structure;

[0084] 4. After seeding organ or tissue simulant B on the upper side of the porous membrane of the culture scaffold, add a first sealing structure;

[0085] 5. Inject the medium (Medium, M) AM into the lower channel through the second inlet;

[0086] 6. Inject culture medium BM (AM and BM are significantly different culture media) into the upper channel through the first inlet.

[0087] 7. Static culture requires no power source, culture medium storage tank, or defoamer; only periodic culture medium replacement is needed.

[0088] 8. The upper and lower channels are connected to the power source, the integrated culture medium storage tank, and the defoaming device respectively via hoses, and the corresponding culture medium AM or BM is added to the storage tank.

[0089] 9. Once the power source is turned on and the liquid delivery parameters are set, perfusion culture can be performed. Place the perfusion culture microfluidic system in a carbon dioxide incubator.

[0090] 10. During the culture process or after the application of treatment conditions, the device can be placed under an upright or inverted microscope to monitor the cell growth status; when a fluorescence signal is present, the distribution and intensity changes of the fluorescence signal can be monitored using a fluorescence microscope.

[0091] 11. After the culture is complete, use tweezers to remove the culture scaffold for subsequent characterization.

[0092] 3.2 Operational procedure for linking multiple sets of identical models:

[0093] 1. Inoculate organ or tissue simulants in multiple sets of devices in the same manner as described in 1-6 above;

[0094] 2. Connect the first inlet and second outlet of each device through sterile silicone tubing and connect them to the power source, the integrated culture medium storage tank, and the defoaming device, and add the corresponding culture medium BM to the storage tank;

[0095] 3. Connect the second inlet and second outlet of each device through sterile silicone tubing and connect them to the power source and the integrated culture medium storage tank and defoaming device, and add the corresponding culture medium AM to the storage tank;

[0096] 4. Turn on the power source and set the liquid delivery parameters to start perfusion culture. Place the device system in a carbon dioxide incubator.

[0097] 5. During the culture process or after the application of treatment conditions, the device can be placed under an upright or inverted microscope to monitor the cell growth status; when a fluorescence signal is present, the distribution and intensity changes of the fluorescence signal can be monitored using a fluorescence microscope;

[0098] 6. After the culture is complete, remove the culture support with tweezers and proceed with subsequent characterization.

[0099] 3.3 Multiple chip sets with different models linked together to form a microphysiological system (taking two chip sets as an example)

[0100] 1. As described in 1-6 above, implant corresponding organ or tissue simulators into two sets of chips respectively, with the same or similar organ or tissue simulators implanted on one side of the two sets of chips;

[0101] 2. Connect the inlet and outlet ports of the same or similar organ or tissue simulants inoculated on both devices through silicone tubing and connect them to the power source and the integrated culture medium reservoir and defoaming device, and add the corresponding culture medium to the reservoir;

[0102] 3. Connect the remaining inlets and outlets of devices 1 and 2 respectively through sterile silicone tubing and connect them to the power source, the integrated culture medium storage tank, and the defoaming device respectively, and add the corresponding culture medium to the storage tank respectively;

[0103] 4. Turn on the power source and set the liquid delivery parameters to start perfusion culture. Place the chip system in a carbon dioxide incubator.

[0104] 5. During the culture process or after the application of treatment conditions, the device can be placed under an upright or inverted microscope to monitor the cell growth status; when a fluorescence signal is present, the distribution and intensity changes of the fluorescence signal can be monitored using a fluorescence microscope;

[0105] 6. After the culture is complete, remove the culture support with tweezers and proceed with subsequent characterization.

[0106] 7. The connection and use of multiple devices are not limited to the above methods. Professional operators can perform various assemblies according to actual application needs.

[0107] Example 4: Application of the microfluidic device for perfusion culture of the present invention

[0108] 4.1 Construction of small intestinal epithelial organ-on-a-chip:

[0109] The perfusion culture microfluidic device of this invention is made of PMMA material and CNC machined. The chip body and culture scaffold are bonded together by thermoforming and laser welding. First, human intestinal microvascular endothelial cells (HIMEC) are seeded in the porous membrane sublayer of the culture scaffold in EGM2-MV medium containing VEGF, FGF, EGF, hydrocortisone, and 5% FBS. The volume is 20 μL, and the cell density is 8 × 10⁻⁶ cells / mL. 4 -1×10 5 Between cells / mL, incubate statically for 1 hour to allow cells to adhere to the porous membrane; after assembling the chip components except for sealing structure 1, seed 30 μL (1×10⁻⁶) onto the upper layer of the porous membrane of the culture scaffold. 5 ~7×10 6 Human colon cancer Caco-2 cells (cells / mL) were used; after adding sealing structure 1, the lower channel was filled with EGM2-MV medium containing VEGF, FGF, EGF, hydrocortisone, and 5% FBS. 150 μL of DMEM-HG medium containing 20% ​​FBS + 1% NEAA + 1% P / S was carefully injected into the upper channel. The chip was statically incubated for at least 12 hours. The upper channel was then filled with the appropriate medium. The upper and lower channels were connected to different peristaltic pumps and integrated culture medium reservoirs and defoamers via silicone tubing. Two-thirds of the culture medium volume was added to the corresponding reservoirs. The peristaltic pumps were turned on, and perfusion culture was performed at a flow rate of 60 μL / min, with the medium changed every 3-4 days. Small intestinal epithelial organelle microarrays were obtained after two weeks of culture. TEER analysis showed that, compared to the control group, the values ​​of HIMEC and Caco-2 cells seeded on both sides of the porous membrane were significantly higher than those seeded with only one type of cell and much higher than those of the control group without cell seeding. Figure 6 A). Devices made from different thermoplastic materials such as PMMA, PC, COC, and COP were tested, and no significant difference was found in the TEER values ​​among the constructed small intestinal epithelial organ models. Figure 6 B).

[0110] 3.2 Construction of liver organ-on-a-chip:

[0111] The device was CNC machined from PMMA material, and the main body and culture scaffold were bonded together using thermoforming and laser welding. HepaRG-derived hepatocytes (Chuanglian Biotechnology Co., Ltd., Beijing, China) and human hepatic sinusoidal endothelial cells (HHSEC, Tongpai Biotechnology Co., Ltd., Shanghai, China) served as liver mimics. Liver-derived endothelial cells (HLECP2, Lonza) simulated the endothelial cells of interlobular veins. To induce HepaRG cell differentiation, cells were placed in William's E medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mM glutamine, 0.023 U / mL insulin, and 5 μg / mL hydrocortisone. Cells were then cultured for 2 weeks, followed by another 2 weeks in medium supplemented with 2% dimethyl sulfoxide. Throughout the culture period, cells were maintained in a CO2 incubator (37°C, 5% CO2). HLECP2 cells were cultured in EGM2-MV medium containing VEGF, FGF, EGF, hydrocortisone, and 5% FBS. Throughout the culture, the cells were kept in a CO2 incubator (37°C, 5% CO2). (Du, K., Li, S., Li, C., Li, P., Miao, C., Luo, T., Ding, W. (2021). Modeling nonalcoholic fatty liver disease on a liverlobule chip with dual blood supply. Acta Biomaterialia, 134, 228-239. doi:10.1016 / j.actbio.2021.07.013).

[0112] First, HLECP2 liver-derived endothelial cells were seeded in EGM2-MV medium containing VEGF, FGF, EGF, hydrocortisone, and 5% FBS in the lower layer of the porous membrane of the culture scaffold. The volume was 20 μL, and the cell density was 5 × 10⁶ cells / mL. 4 -1×10 5 Cells were cultured at a concentration between [number] cells / mL for 5 hours to allow them to adhere to the porous membrane. After assembling the chip components except for sealing structure 1, 30 μL of a mixture of hepatocytes derived from HHSEC and HepaRG cells, suspended in 2.5 mg / mL bovine fibrinogen solution, was seeded onto the upper layer of the porous membrane on the culture scaffold. The hepatocyte content in this mixture was 5 × 10⁻⁶ cells / mL. 6cells / mL, HHSEC content is 1×10 6 Cells / mL; crosslinking at room temperature for 15 min. After assembling the sealed structure 1, the lower channel was filled with EGM2-MV medium containing VEGF, FGF, EGF, hydrocortisone, and 5% FBS. The upper channel was filled with William's E medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mM glutamine, 1 nM insulin, and 50 μM hemiacetyl hydrocortisone. The upper and lower channels were connected to different peristaltic pumps and integrated culture medium reservoirs and defoamers via silicone tubing. Two-thirds of the culture medium volume was added to the corresponding reservoirs. The chip was placed in a CO2 incubator (37°C, 5% CO2), and the peristaltic pump was turned on for perfusion culture at a flow rate of 40-70 μL / min. The culture medium was changed every 3-4 days. This model can simulate basic physiological functions of the liver, such as urea synthesis and albumin secretion. Physiological parameters were measured on the seventh day of perfusion culture. The results showed that the model exhibited certain differences in urea synthesis, albumin secretion, and the physiological activities of CYP1A2 and CYP3A4 under different perfusion rates. All values ​​were standardized according to cell number and culture time. Figure 7 ).

[0113] 4.3 Construction of the gut-liver microphysiological system:

[0114] Based on the model construction described in 4.1 and 4.2, two sets of devices are connected in series via sterile silicone tubing, and the corresponding culture medium is pumped through a peristaltic pump to form an intestinal-hepatic microphysiological system for studying drug absorption and metabolism. The assembly of the microphysiological system is as follows: Figure 8 As shown.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic device for perfusion culture, characterized in that, The device includes a main body, a culture support, and a sealing structure. The main body has a three-layer structure: an upper layer with an inlet and an outlet, which connect to channels between the upper and middle layers and between the middle and bottom layers, respectively. The channels pass through the culture support located in the middle layer. The culture support has two rings, an upper ring and a lower ring, separated by a porous membrane. The sealing structure is located in the upper and middle layers of the main body and in the bottom layer of the main body, respectively, sealing the upper ring, the porous membrane, and the lower ring of the culture support to form two culture chambers.

2. The microfluidic device for perfusion culture according to claim 1, characterized in that, The upper layer has two liquid inlets and two liquid outlets: a first liquid inlet and a second liquid inlet, and a first liquid outlet and a second liquid outlet. The first liquid inlet connects to the first channel between the upper layer and the middle layer, and the liquid flows out from the first liquid outlet. The second inlet connects to the second channel between the middle layer and the bottom layer, and the liquid flows out from the second outlet.

3. The microfluidic device for perfusion culture according to claim 2, characterized in that, The culture scaffold is a disc-shaped disk with an upper ring on the top layer and a lower ring on the bottom layer, with a porous membrane between the upper and lower rings. Each of the upper and lower rings has a slit, and corresponding flow windows are provided at the slits on the middle layer of the main body. When the two are aligned and assembled, the first channel connects to the first culture chamber formed by the upper ring and the porous membrane through the flow window and the slit in the upper ring, and the second channel connects to the second culture chamber formed by the porous membrane and the lower ring of the culture scaffold through the flow window and the slit in the lower ring.

4. The microfluidic device for perfusion culture according to claim 3, characterized in that, The lower layer of the culture scaffold is provided with a channel orientation structure, which is a micro-cylinder or parallel rib, and the height of the micro-cylinder or parallel rib is greater than or equal to the height of the second channel.

5. The microfluidic device for perfusion culture according to claim 4, characterized in that, The channel orientation structure is located on both sides of the radial connection line of the circular cut in the lower layer of the culture scaffold, and there are at least two or more channel orientation structures, with corresponding grooves in the corresponding positions of the main body.

6. The perfusion culture microfluidic device according to any one of claims 1 to 5, characterized in that, The sealing structure has three parts. The first sealing structure and the inner side of the upper circular hole of the main body are provided with matching threads. The first sealing structure passes through the circular hole on the upper part of the main body. The second sealing structure is located outside the upper circular ring of the culture support. The first sealing structure, the second sealing structure and the upper circular ring of the culture support seal to form the first culture chamber. The third sealing structure is fitted on the bottom of the lower circular ring and seals with the lower circular ring and the bottom layer of the main body to form the second culture chamber.

7. The microfluidic device for perfusion culture according to claim 6, characterized in that, The porous membrane is made of PET, PC, PDMS, COC, or COP. The pore size of the porous membrane is uniform or normally distributed. The pore size range is 0.1-20 μm, the porosity is 20-95%, the thickness is 5-100 μm, and the effective inoculation area of ​​the porous membrane is not less than 19.83 mm². 2 .

8. The microfluidic device for perfusion culture according to claim 6, characterized in that, The main body, culture scaffold, and first sealing structure are made of thermoplastic plastics such as PMMA, PC, COC, or COP.

9. The microfluidic device for perfusion culture according to claim 6, characterized in that, The second and third sealing structures are made of silicone, rubber, fluoropolymer, or polyurethane.

10. A microfluidic system for perfusion culture, characterized in that, The device includes the perfusion culture microfluidic device as described in claims 1 to 9, a culture medium storage tank, an antifoamer, and a power source; the power source provides power to control the flow of liquid, and the perfusion culture microfluidic device can perform static culture, unidirectional perfusion, parallel perfusion, reverse perfusion, unidirectional circulating perfusion, and bidirectional circulating perfusion modes.

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