CN114849801A - Microfluidic device for culturing and analyzing cells, tissues and organs in vitro in a quantitative manner - Google Patents
Microfluidic device for culturing and analyzing cells, tissues and organs in vitro in a quantitative manner Download PDFInfo
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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Abstract
The invention belongs to the technical field of in-vitro biological tissue culture and analysis, and particularly relates to a micro-fluidic device for in-vitro cell, tissue and organ culture and analysis. The microfluidic device of the present invention mainly comprises: the standardized pore plate is used for supporting the filter membrane, and the culture of biological tissues or organs such as cells and the like can be realized on the membrane; the culture solution circulation plate is internally connected with a culture solution storage pool in an external connection way and is connected with a peristaltic pump through a hose for constructing a dynamic culture system; the detection liquid circulation plate is provided with channels which are vertically designed with the channels in the culture liquid circulation plate and are used for analyzing and detecting the biological culture; the cover plate avoids the pollution of bacteria and the like to cells and the like in the culture plate. The microfluidic device provided by the invention solves the problem that the conventional cell co-culture equipment cannot realize quantitative and flow culture and analysis, can be used for constructing physiological and pathological models such as biological barrier function structures such as a gas-blood barrier, a blood-brain barrier and an intestinal barrier, multi-organ co-culture and organoid culture, and is used for analysis and detection of model evaluation.
Description
Technical Field
The invention belongs to the technical field of in-vitro biological tissue culture and analysis, and particularly relates to a microfluidic device for in-vitro cell, tissue and organ culture and analysis.
Background
Traditional cell culture techniques, in which a single type of primary cells or proliferating cell lines are used as the cell source for in vitro culture, usually form a monolayer of cells at the solid-liquid interface, and lose many of the biological properties of the cells in their natural growth environment. In order to better reconstruct the organ type structure of the cell in vitro, different types of cells can be co-cultured in a differential matrix constructed by microfluidics in a membrane-supported cell culture mode, and various in vitro tissues and organ cultures are supported by constructing a liquid-liquid or gas-liquid interface through a membrane medium by taking different biological samples as sources. Thereafter, the cultured cells, tissues or organs are subjected to detection in the form of a quantitative analysis, an immunohistochemical analysis or the like. Aims at constructing a specific physiological or pathological model and a disease correlation model for application in aspects of drug screening/activity evaluation, disease mechanism research and the like.
Transwell filter small dish plug-in units are the most widely commercialized cell co-culture system at present, filter membranes are mostly made of translucent or transparent materials (polycarbonate, polyethylene terephthalate and polytetrafluoroethylene), and plug-in units can be embedded into commercially available cell culture plates with different specifications such as 24 wells, 12 wells, 6 wells and the like. Kang et al (Kang YB, Rawat S, Cirillo J, Bouchard M, Noh HM. layred long-term co-culture of liver and endothecial cells on a Transwell membrane: heated engineering the liver disease. Biocontamination 2013, 5(4): 045008.) successfully simulated rat antrum models in vitro, co-cultured rat primary antrum endothelial cells and rat primary hepatocytes on both sides of the Transwell membrane, respectively: the cells can keep the vitality and the normal shape within 57 days; the endothelial cells still have differentiation characteristics after long-term culture, and the liver cells can keep specific functions, so that the endothelial cells can be further used as models for liver disease research, toxicology research and drug screening. Furthermore, this Transwell filter membrane cuvette Insert was also successfully used for The establishment of physiological barriers in vitro (Chang SH, Ko PL, Liao WH, Peng CC, Tung YC. Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alvetor epithelial Barrier Function modules Micromachiens base 2021, 12 (4); Zakharova M, Tie MP, Koch LS, Le-The H, Leferk AM, den Berg A, Transwell-Integrated 2 μ M thin polymeric Membranes with porous particles and Distribution of membrane filter and membrane filter, 2026 technique 2021).
However, it should be noted that commercial Transwell culture systems are usually stationary. Studies have shown that physical factors such as fluid shear are critical to cell/tissue/organ growth (Choe A, Ha SK, Choi I, Choi N, Sun JH. microfluidics Gut-lift chip for reproducing the first pass enzymes metabolism, Biomed Microdevices 2017, 19(1): 4.). Donald et al (Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE. Reconstituting organ-level perfusion functions on a chip, Science 2010, 328(5986): 1662-1668.) established a first microfluidic chip that mimics human alveolar-vascular function, co-culturing human alveolar epithelial cells and human microvascular endothelial cells on both sides of a porous membrane of PDMS (polydimethylsiloxane), wherein the epithelial cell channel is exposed to a gas environment and the medium in the endothelial cell channel is in a flowing state, which was used for inflammatory response studies and nano-toxicology studies. Wu Xintong et al (Wu Xintong, Dingdong, Hanzhuang et al, a double-layered cell culture apparatus for differential co-culture of two cells [ P ]. CN 113234593A, 2021-03-26.) invented a cell co-culture apparatus, which utilizes shear force formed by a flow shear chamber to load stress stimulation on cells to promote in vitro culture of the cells. Wang et al (Wang Y, Wang H, Deng P, Tao T, Liu H, Wu S, et al, Modeling Human Nonalcoholic Fatty Liver Disease (NAFLD) with an organic-on-a-Chip System. Acs Biomaterials Science & Engineering 2020, 6(10): 4. sup. 5743. 573) completed the culture of Human Liver Organoids on a self-made PDMS microfluidic Chip and established a model of Nonalcoholic Fatty Liver Disease, which was subjected to morphological observation, genetic, functional and metabonomic analysis by researchers and explored as a Disease model for the possibility of Disease mechanism research and drug screening. It can be found that the cell/tissue/organ culture device based on the chip design has a flowing culture system, restores respective growth states as much as possible, is closer to an actual physiological state compared with a static culture system, and is more suitable for establishing a bionic model. However, it is worth noting that the design and manufacture of the chip also have problems of time and cost, and it is difficult to achieve the goal of mass and standardized culture and analysis, and there are certain disadvantages. The present invention is therefore directed to the creation of a microfluidic device that enables a throughput of cell/tissue/organ culture and analysis.
Disclosure of Invention
In view of the above analysis, the present invention aims to provide a microfluidic device for in vitro cell, tissue and organ culture and analysis, which is used to solve the problem that the existing cell co-culture equipment cannot realize the quantitative and flow type culture and analysis.
The invention provides a micro-fluidic device for in vitro quantitative cell, tissue and organ culture and analysis, which comprises a cover plate, a standardized pore plate, a filter membrane, a sealing gasket, a culture fluid circulation plate or a detection fluid circulation plate which are arranged from top to bottom; wherein:
the standardized pore plate is used for supporting a filter membrane, and the filter membrane is used for culturing biological tissues or organs such as cells;
the culture solution circulating plate is internally provided with a plurality of rows of channels, each channel is externally connected with a culture solution storage pool and is connected with a peristaltic pump through a hose for constructing a dynamic culture system; each row of channels is provided with an inlet pipeline and an outlet pipeline so as to form a liquid flow path which enables the culture liquid to enter the culture channels from the inlet and flow out from the outlet and to be circulated by the peristaltic pump;
the detection liquid circulation plate is internally provided with a plurality of rows of channels which are vertically designed with the channels in the culture liquid circulation plate and are used for analyzing and detecting the immunohistochemical staining of cells after biological culture and the like; the channels are provided with inlet and outlet conduits to enable the test fluid to be retained from the inlet into the channel or expelled or aspirated from the outlet.
The sealing gasket is arranged between the standardized pore plate and the culture solution circulating plate or the detection solution circulating plate;
the cover plate can avoid the pollution of bacteria and the like to cells and the like in the culture plate.
The standardized pore plate, the filter membrane, the sealing gasket and the culture fluid circulating plate are combined together and used for circulating and quantifying the culture of in vitro cells, tissues and organs;
the standard pore plate, the filter membrane, the sealing gasket and the detection liquid flow plate are combined together and used for analyzing and detecting in vitro cells, tissues and organs.
Further:
the standardized well plate is consistent with the pore size and relative position of a commercially available 6-well, 12-well, 24-well, 48-well, 96-well, 384-well or other standard culture plate; correspondingly, the size and the relative position of the channel of each row in the culture fluid circulating plate are basically consistent with the size of the standardized pore plate; the size and relative position of each row of channels in the detection liquid flow plate are basically consistent with the size of the standardized pore plate, and the detection liquid flow plate is vertically designed with the channels in the culture liquid channel plate.
The plate size and relative position of the plate is consistent with the size and relative position of a commercially available 6-well, 12-well, 24-well, 48-well, 96-well, 384-well or other format plate.
The diameter of each channel on the culture fluid flow plate is in the range of 0.1-5.0 cm, preferably 0.1-2.0 cm.
On the detection liquid flow plate, the diameter of each channel ranges from 0.1 cm to 5.0 cm, and preferably ranges from 0.1 cm to 2.0 cm.
The pore size range of the filter membrane is 0.2-100.0 mu m, preferably 0.2-20 mu m.
When the filter membrane is used for cell co-culture, a track etching filter membrane of 0.4-3.0 mu m is preferred.
The filter membrane is made of polycarbonate, polyethylene terephthalate or polytetrafluoroethylene filter membrane.
The pores of the standardized pore plate are filled with nontoxic and elastic materials, and the materials can be cured Polydimethylsiloxane (PDMS), rubber, silica gel and the like.
The same positions on the periphery of the standardized pore plate, the culture solution circulating plate and the detection solution circulating plate are all subjected to perforation design; when the standard pore plate is used for subsequent detection and analysis, the standard pore plate is separated from the culture fluid circulating plate, is superposed with the detection fluid circulating plate, and is penetrated through the holes on the periphery by screws for fixation and sealing.
The cover plate, the standardized pore plate, the culture fluid circulating plate and the detection fluid circulating plate are all made of non-toxic materials (biocompatible resin, polystyrene, polymethyl methacrylate and the like) and can be manufactured by a 3D printing technology or a mold casting mode and the like.
Under the condition of culture use, the standardized pore plate, the filter membrane, the sealing gasket and the culture fluid circulation plate are combined together, screws penetrate through the standardized pore plate and the holes on the periphery of the culture fluid circulation plate and are used for fixing and sealing, extracellular matrix, gelatin and the like can be selectively coated on the filter membrane according to different culture requirements, after a culture is inoculated on the membrane, a container filled with the culture fluid is connected with the inlet of the culture fluid circulation plate through a peristaltic pump or an injection pump by a hose, and the outlet is connected back to the culture fluid by the hose, so that the dynamic circulation of the culture fluid in a channel is realized.
After culturing, under the detection use state, separating the whole of the standard pore plate, the filter membrane and the sealing gasket from the culture fluid circulating plate, then superposing the standard pore plate, the filter membrane and the sealing gasket with the detection fluid circulating plate, penetrating the holes around by screws for fixing and sealing, respectively adding detection or marking reagents for detecting different cell functions into the holes on the upper layer or leading the detection or marking reagents into the channels on the lower layer, dyeing or marking according to the operation rules of the detection reagents, and then placing the detection or marking reagents in a general quantitative analysis instrument such as an enzyme labeling instrument for determination.
The micro-fluidic device for in vitro cell, tissue and organ culture and analysis can be used for constructing physiological and pathological models of biological barrier function structures such as a gas-blood barrier, a blood-brain barrier, an intestinal barrier and the like, multi-organ co-culture, organoid culture and the like, and can be used for analysis and detection of model evaluation.
When the cell co-culture medium is used for cell co-culture, the pores of the standardized pore plate adhered with the filter membrane are filled with nontoxic elastic materials, then the standardized pore plate and the culture solution circulating plate are overlapped, and screws penetrate through the holes on the periphery for fixing and sealing. And turning the whole body, injecting a certain amount of lower-layer cell culture solution into each row of channels, covering a cover plate, placing the whole body in a cell culture box, standing until the lower-layer cells are coated with a membrane, taking out the whole body, turning the whole body, inoculating a certain amount of upper-layer cell culture solution into each hole, covering the cover plate, placing the whole body in the cell culture box, standing until the upper-layer cells are coated with the membrane, and taking out the whole body. And (3) sucking the culture solution to remove the non-membrane-attached cells, adding fresh culture medium into each hole to nourish the upper layer cells, and optionally, when the culture solution is used for gas-liquid interface culture, not adding fresh culture solution to the upper layer cells. Each row of channels of the culture solution circulation plate are communicated with the culture solution storage pool through hoses, dynamic circulation of the culture solution in the channels is realized by adopting a peristaltic pump, a dynamic culture environment is provided for the layer of cells, and the flow speed range is as follows: 0.0025-1 mL/min. Optionally, the filter membrane can be selectively coated with extracellular matrix, gelatin and the like according to different culture requirements.
After the cell culture is finished, when the cell culture plate is used for subsequent detection and analysis, the peristaltic pump is stopped, residual culture solution in the plate is sucked, the standard pore plate is separated from the culture solution circulating plate, then the standard pore plate is superposed with the detection solution circulating plate, and screws penetrate through the holes on the periphery of the standard pore plate for fixing and sealing. Optionally, adding detection reagents for detecting different cell functions into the upper-layer hole or the lower-layer channel respectively, and completing the analysis and detection steps according to the operation rules of the detection reagents; the detection liquid flow plate may be designed in a communication mode or an independent detection hole mode in order to prevent crosstalk and contamination of the detection liquid depending on the purpose of detection.
When the micro-fluidic device is used for culturing or analyzing biological tissues or organs, the material of the filter membrane, the size of the filter membrane, the flow rate range, the detection test solution and the like can be adjusted according to the purpose of an experiment, and the working mode of the micro-fluidic device is basically consistent with that of the micro-fluidic device.
The microfluidic device provided by the invention solves the problem that the conventional cell co-culture equipment cannot realize quantitative and flow culture and analysis, can be used for constructing physiological and pathological models such as biological barrier function structures such as a gas-blood barrier, a blood-brain barrier and an intestinal barrier, multi-organ co-culture and organoid culture, and is used for analysis and detection of model evaluation.
Drawings
FIG. 1 shows the components and operation of the microfluidic device according to the present invention for cell, tissue and organ culture. Wherein (A) is the composition of the culture device; (B) is a simple drawing of the working mode of the device.
FIG. 2 shows the components and operation of the microfluidic device of the present invention for post-incubation analysis and detection. Wherein, (a) the composition of the analysis device; (B) simple and easy drawing of the working mode of the device.
FIG. 3 is a graph showing the establishment of a pneumonia model of the present invention in which the microfluidic device is used for the air-blood barrier and the co-infection of bacteria and viruses.
Fig. 4 shows the use of a microfluidic device of the invention for the establishment of a blood-brain barrier.
Fig. 5 is a schematic diagram of the microfluidic device of the present invention used for establishing an intestinal barrier and bacterial-host symbiosis model.
Fig. 6 shows the establishment of the microfluidic device of the present invention for multi-organ co-culture model.
Fig. 7 shows the use of the microfluidic device of the present invention for the establishment of a lung organoid model.
Reference numbers in the figures: 1 is a cover plate, 2 is a standardized pore plate, 3 is a filter membrane, 4 is a sealing gasket, 5 is a culture fluid circulating plate, and 6 is a detection fluid circulating plate.
Detailed Description
The present invention is described in detail below with reference to the attached drawing figures, which form a part of this application and together with the embodiments of the invention serve to explain the principles of the invention and not to limit the scope of the invention.
First, the basic structure and operation of the apparatus of the present invention will be described with reference to the drawings.
1. A microfluidic device for the quantitative culture and analysis of cells, tissues or organs, the construction and operation of which is shown in fig. 1:
the device is constructed as shown in the attached figure 1 (A): comprises a cover plate 1, a standardized pore plate 2, a filter membrane 3, a sealing gasket 4 and a culture fluid circulating plate 5 from top to bottom.
The standardized well plate 2 is sized to conform to the pore size and relative position of commercially available 6-, 12-, 24-, 48-, 96-, 384-well or other format plates, as shown in FIG. 1A for a standard 96-well plate.
The material of the filter membrane 3 can be track etched polycarbonate, polyethylene terepthalate or polytetrafluoroethylene filter membrane, and the aperture range is 0.2-100.0 mu m according to the experimental requirements.
The material of the sealing gasket 4 can be solidified Polydimethylsiloxane (PDMS), rubber, silica gel and the like, and is nontoxic and has certain elasticity.
The cover plate 1, the standardized pore plate 2 and the culture solution circulating plate 5 are all made of non-toxic materials (such as biocompatible resin, polystyrene, polymethyl methacrylate and the like) and can be manufactured by a 3D printing technology, a mold casting method and the like.
The standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulation plate 5 are combined together, and screws are used for fixing and sealing after penetrating through holes at the periphery of the standardized pore plate 2 and the culture fluid circulation plate 5, so that the culture fluid circulation plate can be used for culturing cells/tissues/organs. The basic operation is shown in the attached figure 1B: after the device is assembled, after cell, tissue or organ samples are inoculated on the two sides of the filter membrane, each row of channels of the culture solution circulation plate are respectively communicated with a culture solution storage pool through a hose, the dynamic circulation of the culture solution in the channels is realized by adopting a peristaltic pump, and a dynamic culture environment is provided for the biological sample on one side of the membrane. The cover plate 1 is used for preventing pathogens such as bacteria and the like from polluting cells, tissues or organs, the cover plate 1 can be replaced by a culture solution circulating plate 5, the culture solution circulating plate is reversely buckled and fastened and sealed on the standard pore plate 2, similarly, the culture storage pool is communicated with a hose, the dynamic circulation of the culture solution in the channel is realized by a peristaltic pump, and a dynamic culture environment is provided for a biological sample on the other side of the membrane. One physiological or pathological model can be constructed in each column, and taking a 96-well plate as an example, 12 columns are 12 physiological or pathological models.
2. A micro-fluidic device for the culture and analysis of cells, tissues and organs, which is used for the analysis and detection after the culture of cells, tissues or organs, and has the structure and the working mode as shown in the attached figure 2:
the whole standard pore plate 2, the filter membrane 3 and the sealing gasket 4 are separated from the culture fluid circulating plate 5, then are superposed with the detection fluid circulating plate 6, and pass through the holes at the periphery by screws for fixing and sealing (figure 2A). The detection reagents (1-8, totally 8) for detecting different cell functions are respectively added into the upper layer of holes or the lower layer of channels, and can be placed into a quantitative analysis instrument such as a microplate reader for determination according to the operation rules of the detection reagents, and by taking a 96-well plate as an example, 12 physiological or pathological models respectively obtain data results of 8 different indexes (figure 2B). Depending on the purpose of detection, the detection flow-through plate may be designed in a communication mode or in an independent detection hole mode in order to prevent detection crosstalk or contamination.
Hereinafter, a specific application of the apparatus of the present invention will be described with reference to the following embodiments and accompanying drawings.
Example 1: establishment of gas-blood barrier and related pathological model and subsequent analysis and detection application
A microfluidic device for the quantification of cell, tissue, organ culture and analysis for the establishment of a gas-blood barrier, as shown in figure 3: the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5 are integrally combined and turned over, a culture fluid containing a certain number of endothelial cells (HUVEC cells, primary pulmonary microvascular endothelial cells and the like) is injected into each row of channels, the cover plate 1 is covered and placed in a cell culture box, the cells are taken out after the endothelial cells are subjected to membrane sticking, then the whole body is turned over, a culture fluid containing a certain number of epithelial cells (A549 cells, NCI-H411 cells, primary alveolar epithelial cells and the like) is inoculated into each hole, the cover plate is covered and placed in the cell culture box, and the cells are taken out after the epithelial cells are subjected to membrane sticking. Sucking up the culture solution of the epithelial cell layer, continuously culturing by adopting a gas-liquid interface mode, replacing the culture solution of the endothelial cell layer, communicating each row of channels with a hose to a culture solution storage pool, realizing dynamic circulation of the culture solution in the channels by adopting a peristaltic pump, providing a dynamic culture environment for endothelial cells, and ensuring the flow rate range to be as follows: 0.0025-1 mL/min, and establishing a gas-blood barrier model.
Based on the gas-blood barrier model, a relevant pathological model can be further constructed, and a pneumonia model constructed by infection of bacteria (staphylococcus aureus and the like) and viruses (influenza virus IV) is taken as an example: and (3) stopping dynamic culture conditions on the basis of the established gas-blood barrier, introducing influenza virus IV and staphylococcus aureus into the epithelial layer in sequence, co-infecting for 0-120 min, replacing fresh culture solution, removing redundant infectious agents, continuously recovering dynamic culture of endothelial cells, and establishing a pneumonia model of bacterial combined virus co-infection.
The effects of the gas-blood barrier model, the pneumonia model and the like are evaluated, and the related indexes can be measured: stopping the dynamic culture conditions of the whole culture device of the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5, absorbing and removing the culture fluid in the channel, washing the culture fluid circulating plate 5 by using preheated Phosphate Buffer Solution (PBS), then taking down the culture fluid circulating plate 5, assembling and sealing and fastening the detection fluid circulating plate 6 with the standardized pore plate 2, the filter membrane 3 and the sealing gasket 4, and adding corresponding detection fluid into each row of upper pores or lower channels according to the detection purpose, for example: and (3) adopting ZO-1, E-cadherin and other immunofluorescence staining reagents (a channel can be designed to be communicated) when evaluating the integrity of the epithelial barrier, adopting FITC-dextran test solution (the channel is designed to be an independent hole) when evaluating the cell permeability, and adopting CCK8, MTT and other test solutions (the channel is designed to be an independent hole) when evaluating the cell activity.
Example 2: establishment of the blood-brain barrier
A microfluidic device for the quantification of cell/tissue/organ culture and analysis for use in the establishment of the blood-brain barrier, as shown in figure 4: the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5 are integrally combined and turned over, a culture fluid containing a certain amount of endothelial cells (HCMEC/D3 cells, primary cerebral microvascular endothelial cells and the like) is injected into each row of channels, the cover plate 1 is covered and placed in a cell culture box, the cells are taken out after the endothelial cells are subjected to membrane sticking after standing, the whole is turned over, a culture fluid containing a certain amount of epithelial cells (astrocytes and the like) is inoculated into each hole, the cover plate is covered and placed in the cell culture box, and the cells are taken out after the epithelial cells are subjected to membrane sticking after standing. Fresh culture solution is replaced for epithelial and hypodermal cell layers, each row of channels are communicated with a culture solution storage pool through a hose, dynamic circulation of the culture solution in the channels is realized by adopting a peristaltic pump, a dynamic culture environment is provided for endothelial cells, and the flow rate range is as follows: 0.0025-1 mL/min, and establishing a blood-brain barrier model.
The pathological model is constructed according to experimental needs, and the subsequent detection is basically consistent with that described in example 1 according to experimental purposes.
Example 3: establishment of intestinal barrier and bacterial and host symbiosis model
A microfluidic device for the quantification of cell/tissue/organ culture and analysis for use in the establishment of intestinal barrier, as shown in figure 5: the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5 are integrally combined and turned over, a culture fluid containing a certain amount of endothelial cells (HIMEC cells, primary intestinal microvascular endothelial cells and the like) is injected into each row of channels, the cover plate 1 is covered and placed in a cell culture box, the cells are taken out after the endothelial cells are subjected to membrane sticking, the whole body is turned over, a culture fluid containing a certain amount of epithelial cells (Caco-2 cells and the like) is inoculated into each hole, the cover plate is covered and placed in the cell culture box, and the cells are taken out after the epithelial cells are subjected to membrane sticking. Sucking up the culture solution of the epithelial cell layer, continuously culturing by adopting a gas-liquid interface mode, replacing the culture solution of the endothelial cell layer, communicating each row of channels with a hose to a culture solution storage pool, realizing dynamic circulation of the culture solution in the channels by adopting a peristaltic pump, providing a dynamic culture environment for endothelial cells, and ensuring the flow rate range to be as follows: 0.0025-1 mL/min, and establishing an intestinal barrier model.
Based on the established intestinal barrier model, a model for symbiosis of intestinal flora and hosts can be further constructed: on the basis of the established intestinal barrier, stopping dynamic culture conditions, introducing different types of bacteria into the epithelial layer, continuously recovering dynamic culture of endothelial cells for different days, and establishing a symbiotic model of intestinal flora and hosts.
The subsequent tests were substantially identical to those described in example 1 for experimental purposes.
Example 4: establishment of multi-organ co-culture model
A microfluidic device for the throughput of cell/tissue/organ culture and analysis can be used for the establishment of multi-organ co-culture model, as shown in FIG. 6: the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5 are integrally combined and turned over, a culture solution containing a certain amount of endothelial cells (HUVEC cells, primary endothelial cells and the like) is injected into each row of channels, the cover plate 1 is covered and placed in a cell culture box, the cells are taken out after the endothelial cells are statically pasted with membranes, then the whole body is turned over, a culture solution containing a certain amount of epithelial cells (liver: HepRG cells; intestine: Caco-2 cells; lung: A549 cells or corresponding primary cells) representing different organs is respectively inoculated into different holes of each row (A-L), the cover plate is covered and placed in the cell culture box, and the cells are taken out after the epithelial cells are statically pasted with membranes. Absorbing and drying culture solution in epithelial cells (intestines, lungs and the like) holes cultured in an air-liquid interface mode; replacing fresh culture solution in epithelial cell (liver, etc.) hole without gas-liquid interface culture; the fresh culture solution is changed to the endothelial cell layer, will be every row of passageway with hose intercommunication culture solution reservoir, adopts the peristaltic pump to realize the dynamic circulation of culture solution in the passageway, for endothelial cell provides the dynamic culture environment, the velocity of flow scope is: 0.0025-1 mL/min, and establishing a model for multi-organ co-culture.
The subsequent tests were substantially identical to those described in example 1 for experimental purposes.
Example 5: establishment of lung organoid model
A microfluidic device for the quantification of cell/tissue/organ culture and analysis for lung organoid culture, as shown in figure 7: the standardized pore plate 2, the filter membrane 3, the sealing gasket 4 and the culture fluid circulating plate 5 are integrally combined and turned over, a culture fluid containing a certain amount of endothelial cells (HUVEC cells, primary pulmonary microvascular endothelial cells and the like) is injected into each row of channels, the cover plate 1 is covered, the channels are placed in a cell culture box, the cells are taken out after the endothelial cells are pasted with membranes, then the whole body is turned over, and different holes in each row (A-L) are inoculated: lung organoid sphere solution containing type I collagen or human lung induced multifunctional precursor stem cell solution containing type I collagen; when the organoid sphere is embedded into extracellular matrix and attached to the filter membrane, fresh culture solution is replaced for the upper lung organoid and the lower endothelial cell layer, each row of channels are communicated with a culture solution storage pool through a hose, dynamic circulation of the culture solution in the channels is realized by adopting a peristaltic pump, a dynamic culture environment is provided for endothelial cells, and the flow rate range is as follows: 0.0025-1 mL/min, covering the cover plate, and placing the cover plate in a cell culture box for continuous culture to establish a model for multi-organ co-culture.
The subsequent tests were substantially identical to those described in example 1 for experimental purposes.
Claims (10)
1. A micro-fluidic device for in vitro cell, tissue and organ culture and analysis is characterized by comprising a cover plate, a standardized pore plate, a filter membrane, a sealing gasket, a culture fluid circulation plate or a detection fluid circulation plate which are arranged from top to bottom; wherein:
the standardized pore plate is used for supporting a filter membrane, and the filter membrane is used for culturing cells, tissues or organs;
the culture solution circulating plate is internally provided with a plurality of rows of channels, the channels are externally connected with a culture solution storage pool and are connected with a peristaltic pump through hoses for constructing a dynamic culture system; each channel is provided with an inlet pipeline and an outlet pipeline so as to form a liquid flow path which enables the culture liquid to enter the culture channel from the inlet and flow out from the outlet and to be circulated by the peristaltic pump;
the detection liquid circulation plate is internally provided with a plurality of rows of channels, and the channels are vertically designed with the channels in the culture liquid circulation plate and are used for analyzing and detecting biological cultures; each channel is provided with an inlet pipeline and an outlet pipeline, so that the detection liquid can be injected into the channel from the inlet and retained in the channel and can be discharged or sucked out from the outlet;
the sealing gasket is arranged between the standardized pore plate and the culture solution circulating plate or the detection solution circulating plate;
the standardized pore plate, the filter membrane, the sealing gasket and the culture fluid circulating plate are combined together and used for circulating and quantifying the culture of in vitro cells, tissues and organs;
the standard pore plate, the filter membrane, the sealing gasket and the detection liquid flow plate are combined together and used for analyzing cells, tissues and organs in vitro.
2. The microfluidic device according to claim 1, wherein the standardized well plate is consistent with the pore size and relative position of a 6-well, 12-well, 24-well, 48-well, 96-well, 384-well or other format culture plate; correspondingly, the size and the relative position of the channel of each row in the culture solution circulating plate are consistent with the size of the standardized pore plate; detecting the size and the relative position of each row of channels in the liquid flow plate to be consistent with the size of the standardized pore plate;
the plate format is consistent with the aperture and relative position of 6-well, 12-well, 24-well, 48-well, 96-well, 384-well or other format plates.
3. The microfluidic device according to claim 1, wherein each channel on the culture fluid flow plate has a diameter in the range of 0.1-5.0 cm; the diameter of each channel on each detection liquid flow plate ranges from 0.1 cm to 5.0 cm.
4. The microfluidic device according to claim 1, wherein the filter membrane has a pore size in the range of 0.2-100.0 μm.
5. The microfluidic device according to claim 1, wherein the filter membrane is etched with a 0.4-3.0 μm track for cell co-culture.
6. The microfluidic device according to claim 4 or 5, wherein the filter membrane material is a polycarbonate, polythyleneterephthalate or polytetrafluoroethylene filter membrane.
7. The microfluidic device according to claim 1, wherein the pores of the standardized well plate are filled with a non-toxic, resilient material.
8. The microfluidic device according to claim 1, wherein the standardized well plate, the culture solution flow through plate, and the detection solution flow through plate are perforated at the same positions around the standardized well plate, the culture solution flow through plate, and the detection solution flow through plate; when the standard pore plate is used for subsequent detection and analysis, the standard pore plate is separated from the culture fluid circulating plate, is superposed with the detection fluid circulating plate, and is penetrated through the holes on the periphery by screws for fixation and sealing.
9. The microfluidic device according to claim 1, wherein the cover plate, the standardized well plate, the culture fluid flow plate, and the detection fluid flow plate are made of non-toxic materials and manufactured by 3D printing technology or mold casting.
10. Use of the microfluidic device according to claims 1-9 for constructing physiological and pathological models of gas-blood barrier, blood-brain barrier, intestinal barrier biological barrier function, multi-organ co-culture, organoid culture.
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