Mechanical compression and / or co-culture microfluid equipment and application thereof

Abstract

The invention relates to a mechanical compression and / or co-culture microfluid device and application thereof, and belongs to the technical field of biomedical science or pharmacology, the microfluid device comprises a culture chamber and a nutrition supply chamber, the culture chamber comprises at least two compartments for accommodating cells / tissues and a porous limiting plate shared with the nutrition supply chamber, the compartments at least comprise a compression compartment for accommodating cells / tissues subjected to mechanical compression and a non-compression compartment which is not subjected to compression strength or is subjected to compression strength far lower than the compression strength of the compression compartment, and the actuating chamber drives the flexible partition plate on the bottom surface of the culture chamber to periodically deform and restore to the original shape after being in contact with the limiting plate; and at least one compartment of the nutrition supply room corresponds to the culture room. The device is free of complex alignment operation, low in manufacturing difficulty, low in cost and particularly suitable for simulating the microenvironment of cartilage tissue, the thin face shape formed by the compression compartment simulates the shape and nutrition supply mode of cartilage, and meanwhile the non-compression compartment can achieve co-culture of multiple kinds of non-compression tissue and compressed cartilage tissue.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical science or pharmacology technology, and specifically relates to a mechanical compression and / or co-culture microfluidic device and its application. Background Art

[0002] With the advent of an aging population, related aging diseases have imposed a significant economic burden on society and patients, and have seriously affected the quality of life of patients. There are still a large number of aging diseases that have not been conquered, which has put forward an urgent need for the causes of diseases and the development of new drugs.

[0003] In the field of drug research and development, there is a clear market demand for improving the drug development process. It takes an average of 13.5 years and costs $2.5 billion to develop a drug, but 92% of drugs fail in clinical trials. There is an urgent need for human cell-based system models that better reflect the physiological functions of the human body.

[0004] Before clinical testing, drugs need to be tested in experimental models of disease in in vitro models (e.g., cell culture) and in vivo models (i.e., on animals). However, both models have certain problems: in vitro cell culture systems do not reflect the physiological functions of humans or animals very well, and cells are very sensitive to their microenvironment, which is rich in molecular signals from the extracellular matrix, other cells, and mechanical stimulation caused by flow, concentration gradients, and movement. Therefore, in vitro testing does not well represent the physiological / pathological behavior of drugs in vivo. In vitro testing of the activity (toxicity or efficacy) of chemical compounds needs to accurately predict what will happen in the clinic. Problems arise if the test results are false positive (the compound is actually safe, but it has toxic effects) or false negative (the compound is toxic, but no adverse effects are detected). Since many compounds are safe at low doses but toxic at high doses, the sensitivity of the test is crucial.

[0005] On the other hand, the controversy surrounding animal models in the scientific community stems from the recognition of biological differences between humans and animals, even primates. As a result, findings from animal testing cannot be translated into clinical practice. Years of wasted research also exact an economic toll. Many animals are raised under sterile conditions, and they neither produce the immune response required for disease models nor possess a balanced gut microbiota that influences drug metabolism. In addition, ethical debates are associated with the use of animals, and large numbers of animals are sacrificed in experiments. Animal experiments incur huge economic costs in terms of breeding, feeding, and disposal.

[0006] However, currently available models suffer from the same limitations as discussed above for in vitro models. Several improvements to in vitro methods have been identified to obtain more physiological results. These include a shift to three-dimensional culture, the use of primary human cells, the introduction of flow and mechanical stimulation, and the co-culture of multiple cell types. Currently, three-dimensional in vitro culture methods have become more widely adopted and have been shown to be more effective for toxicity screening than simple two-dimensional cultures. However, methods that are more in line with the human physiological environment are still needed for drug testing, exploration of disease mechanisms, and the development of personalized medical programs.

[0007] Occhetta et al. (2019) described a microfluidic device for simulating the physiological and pathological compression microenvironment of cartilage in vivo. The device simulated physiological compression by 10% compression and pathological compression by 30% and induced an osteoarthritis phenotype. However, on the one hand, the supraphysiological compression is not consistent with the pathogenesis of human osteoarthritis, and the co-culture of cartilage with other cells, such as subchondral bone, macrophages and other tissues, was not achieved. Paggi et al. (2020) described a microfluidic device for simulating cartilage tissue compression, which simulated shear force while achieving compression, but it also lacked co-culture and its compression range could not be precisely controlled.

[0008] Therefore, there is a need in the above-mentioned field to provide an effective and reliable cell tissue model for in vitro testing that overcomes the shortcomings of the known techniques. More specifically, there is a need to provide a method and apparatus for drug screening and development at the preclinical level that is effective and economical while overcoming the shortcomings of the known techniques. Summary of the invention

[0009] The main purpose of the present invention is to overcome the deficiencies in the prior art. The present invention provides a microfluidic device for mechanical compression and / or co-culture and its application.

[0010] The present invention is implemented through the following technical scheme: A microfluidic device for mechanical compression and / or co-culture comprises a housing, a limiting plate and a flexible partition, wherein: A plurality of support columns are arranged on the bottom surface of the shell; A limit plate is arranged horizontally at an upper middle position of the inner cavity of the shell, a plurality of through holes are arranged on the limit plate, and at least one nutrient supply chamber is arranged in the inner cavity of the shell above the limit plate; A flexible partition is arranged horizontally at a lower middle position of the shell cavity, at least two culture chambers are arranged in the shell cavity between the flexible partition and the limiting plate, the culture chambers include a compression compartment and a non-compression compartment, the culture chambers are connected with the nutrient supply chamber through a through hole, and the shell cavity below the flexible partition is arranged as an actuation chamber; the lower surface of the flexible partition is placed on top of the support column, an annular side wall is arranged at the edge position of the upper surface of the flexible partition, a cylindrical central micro-column is arranged at the central part of the upper surface of the flexible partition, a plurality of enclosing micro-columns are evenly distributed along the circumferential direction on the outside of the central micro-column, the height of the central micro-column and the enclosing micro-column is not higher than the height of the annular side wall, the cross-section of the enclosing micro-columns is arranged as a trapezoid, and gaps are arranged between adjacent enclosing micro-columns; when the actuation chamber causes When the actuation chamber is actuated, the flexible partition is driven by the actuation chamber to bulge and deform upward, and the flexible partition drives the surrounding microcolumns and the central microcolumns to push up and contact with the limiting plate. The annular closed cavity between the surrounding microcolumns and the central microcolumns forms a compression compartment, which is used to accommodate three-dimensional cells and / or tissue constructs subjected to mechanical compression. The annular cavity between the surrounding microcolumns and the annular side wall forms a non-compression compartment, which is used to accommodate three-dimensional cells and / or tissue constructs that are not subjected to or are subjected to a compression strength far lower than that in the compression compartment. When the actuation chamber stops actuating, the flexible partition is naturally restored to a horizontal state under the action of gravity, and the central microcolumn and the surrounding microcolumn are separated from the limiting plate. The actuation chamber is periodically actuated to drive the flexible partition to deform periodically at a set deformation rate.

[0011] Furthermore, the limit plate is made of a single chemically inert material, or a composite of chemically inert materials, and the chemically inert material is one or more of silicone rubber, fluorinated rubber, polystyrene (PS), polymethyl methacrylate (PMMA), polycarbonate, ester (PC), glass, silicon or polyethylene glycol (PEG), and the most preferred material is a composite of silicone rubber and PMMA.

[0012] Furthermore, the flexible partition is made of a flexible and deformable chemically inert material, and the flexible and deformable chemically inert material is one or more of silicone rubber, fluorinated rubber, or polyethylene glycol (PEG), and silicone rubber is most preferred.

[0013] Furthermore, a block or a membrane containing nutrients is placed in the nutrient supply chamber as a culture medium, and the culture medium enters the culture chamber through a through hole provided on the limiting plate.

[0014] Furthermore, the three-dimensional cells and / or tissue constructs introduced into the compression compartment and the non-compression compartment are different. The three-dimensional cells and / or tissue constructs introduced into the compression compartment and the non-compression compartment are periodically squeezed by the changes in the gap formed between the enclosing microcolumns, the central microcolumns, the annular side walls and the limiting plates.

[0015] Further, the culture medium placed in the nutrient supply chamber corresponds to the three-dimensional cells and / or tissue constructs introduced into the compression compartment and the non-compression compartment, respectively.

[0016] Furthermore, the three-dimensional cell and / or tissue construct comprises stem cells and / or somatic cells, wherein the stem cells are at least one of ex vivo embryonic or ex vivo adult stem cells; and the somatic cells are at least one of ex vivo endothelial cells, bone cells, chondrocytes, nerve cells, fat cells, epithelial cells, fibroblasts, myofibroblasts, mesenchymal cells, hepatocytes, pancreatic cells, blood cells or muscle cells.

[0017] Further, the actuation of the actuation chamber is achieved pneumatically, hydraulically, mechanically, electrically, magnetically or thermally.

[0018] An application of a microfluidic device as described above: for generating in vitro tissue models; or for pharmaceutical screening of active ingredients or drugs; or for evaluating the effects of molecules and / or nanoparticles; or for disease modeling; or for drug discovery.

[0019] Further, the application of the microfluidic device as described above comprises the following steps: S1. Install the microfluidic equipment; S2, seeding cells in the compressed compartment and / or the non-compressed compartment; S3, introducing the culture medium corresponding to the inoculated cells into at least one nutrient supply chamber S4, the actuation chamber is actuated, the actuation chamber drives the flexible partition to bulge upward and deform, the flexible partition drives the surrounding micro-column and the central micro-column to push up and contact the limit plate, the annular closed cavity between the surrounding micro-column and the central micro-column forms a compression compartment, and the annular cavity between the surrounding micro-column and the annular side wall forms a non-compression compartment; S5, the actuation chamber stops actuating, the flexible partition naturally returns to a horizontal state under the action of gravity, and the central micro-column and the surrounding micro-column are separated from the limit plate; S6. Repeat the above steps S4 to S5, the actuation chamber is actuated periodically, and then the flexible partition is driven to deform periodically at a set deformation rate, and the gap formed between the enclosing micro-column, the central micro-column, the annular side wall and the limiting plate changes periodically, so that the three-dimensional cells and / or tissue constructs introduced into the compression compartment and the non-compression compartment are periodically squeezed.

[0020] The beneficial effects of the present invention are: 1. The device of the present invention does not require complicated alignment operations and is easy to manufacture; 2. The present invention is made by CNC and laser engraving, without the need for traditional photolithography templates, and has low cost; 3. The nutrient supply chamber of the present invention provides nutrient supply to the tissue in the culture chamber on one side, thus avoiding insufficient nutrient supply due to the increase of tissue volume; 4. The present invention is particularly suitable for simulating the microenvironment of cartilage tissue. The thin surface shape formed by the compressed compartment simulates the shape and nutrient supply method of cartilage. At the same time, its non-compressed compartment can realize the co-culture of various non-compressed tissues and compressed cartilage tissue, simulating the complex microenvironment of cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of a culture chamber of a device according to a preferred embodiment of the present invention before deformation is shown; Figure 2 A schematic diagram showing a culture chamber of a device implemented according to a preferred embodiment of the present invention after deformation is shown; Figure 3 A schematic top view of a culture chamber of a device implemented according to a preferred embodiment of the present invention is shown.

[0022] In the figure, 1 is a microfluidic device, 2 is a culture chamber, 3 is a nutrient supply chamber, 4 is a limit plate, 5 is a compression compartment, 6 is a non-compression compartment, 7 is a surrounding microcolumn, 8 is a central microcolumn, 9 is an annular side wall, 10 is an actuation chamber, 11 is a support column, and 12 is a flexible partition. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0024] like Figures 1 to 3 A mechanical compression and / or co-culture microfluidic device is shown, comprising a housing, a limiting plate 4 and a flexible partition 12, wherein: A plurality of support columns 11 are arranged on the bottom surface of the housing; A limit plate 4 is horizontally arranged at the upper middle part of the inner cavity of the shell, a plurality of through holes are arranged on the limit plate 4, and at least one nutrient supply chamber 3 is arranged in the inner cavity of the shell above the limit plate 4; A flexible partition 12 is horizontally arranged at the lower middle part of the shell cavity, at least two culture chambers 2 are arranged in the shell cavity between the flexible partition 12 and the limiting plate 4, the culture chamber 2 includes a compression compartment 5 and a non-compression compartment 6, the culture chamber 2 is connected with the nutrient supply chamber 3 through a through hole, and the shell cavity below the flexible partition 12 is arranged as an actuation chamber 10; the lower surface of the flexible partition 12 is placed on top of the support column 11, an annular side wall 9 is arranged at the edge position of the upper surface of the flexible partition 12, a cylindrical central micro-column 8 is arranged at the central part of the upper surface of the flexible partition 12, and a plurality of enclosing micro-columns 7 are evenly distributed on the outside of the central micro-column 8 along the circumferential direction, the height of the central micro-column 8 and the enclosing micro-column 7 is not higher than the height of the annular side wall 9, the cross-section of the enclosing micro-column 7 is arranged as a trapezoid, and a gap is arranged between adjacent enclosing micro-columns 7; when the actuation chamber When actuated, the actuation chamber 10 drives the flexible partition 12 to bulge and deform upward, and the flexible partition 12 drives the enclosing micro-column 7 and the central micro-column 8 to push up and contact with the limiting plate 4, and the annular closed cavity between the enclosing micro-column 7 and the central micro-column 8 forms a compression compartment 5, and the compression compartment 5 is used to accommodate three-dimensional cells and / or tissue constructs subjected to mechanical compression, and the annular cavity between the enclosing micro-column 7 and the annular side wall 9 forms a non-compression compartment 6, and the non-compression compartment 6 is used to accommodate three-dimensional cells and / or tissue constructs that are not subjected to or are subjected to a compression strength far lower than that in the compression compartment 5; when the actuation chamber 10 stops actuating, the flexible partition 12 naturally returns to a horizontal state under the action of gravity, and the central micro-column 8 and the enclosing micro-column 7 are separated from the limiting plate 4, and the actuation chamber 10 is periodically actuated to drive the flexible partition 12 to deform periodically at a set deformation rate.

[0025] Furthermore, the limit plate 4 is made of a single chemically inert material, or a composite of chemically inert materials, wherein the chemically inert material is one or more of silicone rubber, fluorinated rubber, polystyrene PS, polymethyl methacrylate PMMA, polycarbonate, ester PC, glass, silicon or polyethylene glycol PEG, and the most preferred material is a composite of silicone rubber and PMMA.

[0026] Furthermore, the flexible partition 12 is made of a flexible and deformable chemically inert material, and the flexible and deformable chemically inert material is one or more of silicone rubber, fluorinated rubber, or polyethylene glycol (PEG), and silicone rubber is most preferred.

[0027] Furthermore, a block or a membrane containing nutrients is placed in the nutrient supply chamber 3 as a culture medium, and the culture medium enters the culture chamber 2 through the through holes provided on the limiting plate 4 .

[0028] Furthermore, the three-dimensional cells and / or tissue constructs introduced into the compression compartment 5 and the non-compression compartment 6 are different. The three-dimensional cells and / or tissue constructs introduced into the compression compartment 5 and the non-compression compartment 6 are periodically squeezed by the changes in the gap formed between the enclosing microcolumn 7, the central microcolumn 8, the annular side wall 9 and the limiting plate 4.

[0029] Furthermore, the present invention can achieve the culture of more tissues by adding new enclosing microcolumns outside the enclosing microcolumns 7 with different heights, and provide a fixed ratio of compression and shear for the three-dimensional cells and / or tissue constructs contained in the chambers formed between the new enclosing microcolumns, and the compression ratio is different from that of the existing compression compartment 5, thereby achieving gradient compression.

[0030] Furthermore, the culture medium placed in the nutrient supply chamber 3 corresponds to the three-dimensional cells and / or tissue constructs introduced into the compression compartment 5 and the non-compression compartment 6, respectively.

[0031] Furthermore, the three-dimensional cell and / or tissue construct comprises stem cells and / or somatic cells, wherein the stem cells are at least one of ex vivo embryonic or ex vivo adult stem cells; and the somatic cells are at least one of ex vivo endothelial cells, bone cells, chondrocytes, nerve cells, fat cells, epithelial cells, fibroblasts, myofibroblasts, mesenchymal cells, hepatocytes, pancreatic cells, blood cells or muscle cells.

[0032] Further, the actuation of the actuation chamber 10 is achieved pneumatically, hydraulically, mechanically, electrically, magnetically or thermally.

[0033] An application of a microfluidic device as described above: for generating in vitro tissue models; or for pharmaceutical screening of active ingredients or drugs; or for evaluating the effects of molecules and / or nanoparticles; or for disease modeling; or for drug discovery.

[0034] Further, the application of the microfluidic device as described above comprises the following steps: S1. Install the microfluidic equipment; S2, inoculating cells in the compressed compartment 5 and / or the non-compressed compartment 6; S3, introducing the culture medium corresponding to the inoculated cells into at least one nutrient supply chamber 3 S4, the actuation chamber 10 is actuated, and the actuation chamber 10 drives the flexible partition 12 to bulge upward and deform, and the flexible partition 12 drives the enclosing micro-column 7 and the central micro-column 8 to push upward and contact the limiting plate 4, and the annular closed cavity between the enclosing micro-column 7 and the central micro-column 8 forms a compression compartment 5, and the annular cavity between the enclosing micro-column 7 and the annular side wall 9 forms a non-compression compartment 6; S5, the actuation chamber 10 stops actuating, the flexible partition 12 naturally returns to a horizontal state under the action of gravity, and the central micro-column 8 and the surrounding micro-column 7 are separated from the limiting plate 4; S6. Repeat the above steps S4-S5, the actuation chamber 10 is actuated periodically, thereby driving the flexible partition 12 to deform periodically at a set deformation rate, and the gap formed between the enclosing micro-column 7, the central micro-column 8, the annular side wall 9 and the limiting plate 4 changes periodically, so that the three-dimensional cells and / or tissue constructs introduced into the compression compartment 5 and the non-compression compartment 6 are periodically squeezed.

[0035] The expression "microfluidic device" refers in the present invention to a system equipped with at least one channel for microscale confinement and movement of fluids, including fluid volumes in the unit between milliliters and hundreds of microliters.

[0036] The expression "tissue" in the present invention refers to a three-dimensional cell and / or tissue construct. The tissue has the property of including an elastic component, and when the culture chamber recovers its original shape after deformation, the tissue also recovers its original shape when pressure is lost.

[0037] The expression "three-dimensional cell construct" refers to a cluster of cells structurally bound to each other directly or through a polymer structure. Such a structure may be of natural origin, synthetic origin or a combination thereof.

[0038] The expression "mechanical compression and / or co-culture" in the present invention refers to providing mechanical compression and simulation of a co-culture microenvironment for the tissue in the device.

[0039] Preferably, the three-dimensional tissue construct is mature. The term "mature" in the present invention means that the tissue consists essentially of cells in an advanced differentiation stage and has a behavior similar to the natural behavior of the tissue to be obtained.

[0040] The term "mechanical compression" refers to the application of force to the tissue in the compressed compartment to produce a unidirectional compressive deformation.

[0041] The term "co-culture" refers to the communication between cells between different tissues in the multiple compartments by direct contact or culture fluid. The cell matrix comprises natural or synthetic polymers or a combination thereof, more preferably a fluid polymer solution and / or a gel, and even more preferably a hydrogel. The hydrogel is selected from fibrin gel, collagen, collagen gelatin, hyaluronic acid, elastin, agarose, chitosan, alginate, etc. or a combination thereof, more preferably a GelMA hydrogel.

[0042] The term "compressed compartment" in the context of the present invention means that the tissue is compressed to a defined ratio in this compartment.

[0043] The term "non-compressed compartment" in the present invention means that the tissue in this compartment is not compressed or is less compressed than the tissue in the compressed compartment.

[0044] The deformation of the culture chamber refers to the process in which the micro-pillars on the culture chamber are completely fitted to the limiting plate, and this process completes the compression of the tissue in the compressed compartment.

[0045] After the culture chamber is deformed, the tissue is filled into the non-compressed compartment, and after the culture chamber is restored to its original shape, the tissue maintains its original shape, so that the next time the culture chamber is deformed, it is not compressed or is slightly compressed. Filling the tissue means that the tissue construct containing cells is injected into the compartment and then the shape is fixed.

[0046] The restoration to the original state means that the culture chamber loses the power of the actuation chamber after deformation and restores the position before deformation.

[0047] In order to compare the degree of compression of the constrained tissue after compression relative to the original state of the tissue, the ratio of the height of the microcolumn in the culture chamber and the height of the microcolumn plus the height difference between the microcolumn and the restraining plate is used as a method to calculate the compression ratio, which is also called the compression ratio or deformation rate.

[0048] The term "limiting plate" in the present invention refers to a structural element common to the culture chamber and the nutrient supply chamber of the device, which completes the compression of the device when it is in full contact with the micro-columns in the culture chamber.

[0049] Preferably, the limiting plate is a porous single-layer or composite multi-layer film / plate.

[0050] Preferably, the deformation of the limiting plate under the pressure of the actuating chamber is much smaller than the deformation of the culture chamber.

[0051] Preferably, the limiting plate is composed of a layer of a densely porous thin polydimethylsiloxane (PDMS) film and a layer of polymethyl methacrylate (PMMA) containing pores larger than those of the PDMS film, and more preferably, the pores of both layers are circular.

[0052] Preferably, the PDMS film has a thickness of 20-200 um and a pore size of 20-400 um, more preferably a thickness of 100 um and a pore size of 100 um.

[0053] Preferably, the PMMA layer has a thickness of 500-1000 um and a pore size of 300-1000 um, more preferably a thickness of 1000 um and a pore size of 1000 um.

[0054] Preferably, the circular holes of the PDMS film and the PMMA layer are arranged in a circle with a diameter of 13000 um.

[0055] In order to facilitate communication between tissues in different compartments, there are gaps between the circularly arranged enclosure microcolumns. Preferably, the nutrient supply compartments should be interconnected so that they can communicate with each other.

[0056] The shape suitable for the compression compartment preferably has a regular geometric shape, more preferably the enclosing micro-pillars are arranged in a circular and central micro-pillar. Preferably, the culture chamber includes a plurality of enclosing micro-pillars, which are equidistant from each other and have the same size.

[0057] Preferably, the micro-columns surrounding the culture chamber are trapezoidal, the micro-columns in the center are circular, the height of the micro-columns is 100-500um, more preferably 500um, the width and height of the trapezoid are 500um, and the diameter of the central cylinder is 2000um. The trapezoidal shape balances the lateral expansion of the compressed tissue under compression and the direct contact and communication between the non-compressed tissue, so that the two functions are optimally solved.

[0058] The shape suitable for the non-compressed compartment preferably has a regular geometric shape, more preferably a ring consisting of circularly arranged enclosing micro-pillars and annular side walls.

[0059] Preferably, the outer diameter of the circular ring of the compression compartment is 6000um and the inner diameter is 2000 microns.

[0060] Preferably, the distance between all micro-pillars and the limiting plates in the culture chamber before deformation is between 10-100 um, more preferably between 10-50 um, and most preferably 50 um.

[0061] Preferably, the culture chamber has a height of 100-600 um, more preferably 500 um.

[0062] Preferably, the device is suitable for cyclic compression of tissue, more preferably irregular compression with a deformation rate (compression ratio) of less than 10%.

[0063] Preferably, the actuation chamber is separated from the culture chamber by a flexible partition.

[0064] Preferably, the actuation chamber supports the culture chamber via support columns, so that the flexible partition is initially in a horizontal state.

[0065] Advantageously, the microfluidic device comprises one or more openings for injection of tissue, and openings for entry and exit of culture medium.

[0066] Preferably, the microfluidic device comprises at least one inlet for applying pressure and / or for introducing fluid and / or gas into the actuation chamber.

[0067] Preferably, the device is obtained by micro-nano processing technology, more preferably using CNC engraving, laser engraving and soft lithography technology, wherein it is preferably obtained in layers and then combined into a whole by a bonding method.

[0068] In a preferred embodiment, the device is assembled after the production of the various components obtained by micro-nanofabrication techniques.

[0069] Preferably, the step of compressing the tissue is repeated at least until a three-dimensional cell construct, more preferably a three-dimensional tissue construct, and even more preferably until a mature three-dimensional tissue construct is obtained.

[0070] Preferably, the controlled compression is achieved by deformation of the culture chamber and contact with the limiting plate.

[0071] Preferably, co-cultivation is achieved by direct contact between the gaps between the micro-pillars of the culture chamber and fluid exchange between the culture media.

[0072] Preferably, the tissue comprises specialized cells or unspecialized cells or a combination thereof.

[0073] Preferably, the tissue comprises prokaryotic or eukaryotic cells, more preferably eukaryotic cells, even more preferably mammalian cells, most preferably human cells.

[0074] Preferably, the cells are stem cells or somatic cells, or a combination thereof.

[0075] Preferably, the stem cells are selected from the group comprising embryonic and adult stem cells, preferably pluripotent cells and induced pluripotent cells, more preferably obtained from placenta, umbilical cord, adipose tissue, neural tissue, muscle tissue, cardiac tissue, connective tissue, epidermal tissue and bone marrow tissue, and combinations thereof.

[0076] Preferably, the somatic cells are selected from the group comprising cells derived from mesoderm, endoderm and ectoderm, preferably endothelial cells, osteocytes, chondrocytes, neural cells, adipocytes, epithelial cells, fibroblasts, myofibroblasts, mesenchymal cells, hepatocytes, pancreatic cells, blood cells, muscle cells and combinations thereof.

[0077] Preferably, the three-dimensional cell and / or tissue construct is selected from the group consisting of a cartilage construct, a muscle construct, a bone construct, a neural construct, a connective construct, a fibrotic construct and combinations thereof, more preferably a cartilage construct.

[0078] Preferably, the tissue construct obtained is a mature three-dimensional tissue construct.

[0079] The invention will now be further illustrated by way of examples of embodiments provided below.

[0080] Each part of the microfluidic device is made in layers, wherein the actuation chamber is made of a purchased 100um thick PDMS film by laser engraving and punching to make a template, which is then placed on a silicon wafer and injected with PDMS prepolymer; the culture chamber is made by CNC engraving an aluminum alloy mold into a mold and injecting PDMS prepolymer; the first limiting plate is made by laser engraving and punching a 100um thick PDMS membrane; the second limiting plate is formed by laser engraving and punching a PMMA sheet purchased; the nutrient supply chamber is made by laser engraving a PMMA sheet to make a mold and then injecting PDMS; all of the above molds need to be sprayed with a release agent before injecting PDMS, and the bonding of each of the above layers is achieved by plasma treatment and silanization treatment.

[0081] A microfluidic device for mechanical compression and / or co-culture is manufactured using polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA), comprising an actuation chamber made of PDMS, a culture chamber, a first limiting plate made of PDMS (100um thick, 100um pore size), a nutrient supply chamber and a second limiting plate made of PMMA (1mm thick, 1mm pore size), the first limiting plate and the second limiting plate are stacked together to form a composite, above the limiting plate is a nutrient supply chamber with a square chamber, the bottom is a gas actuation chamber (circular with a diameter of 13mm and a height of 100um), which contains a support column flush with the chamber height, above the actuation chamber is a culture chamber, the bottom of the culture chamber is a flexible partition (1mm thick), above the flexible partition is a circular central microcolumn (2mm in diameter, 450um high), the central microcolumn is surrounded by a circle of trapezoidal enclosure microcolumns (450um high) arranged in a circle, and the outer periphery of the enclosure microcolumns is separated by a certain gap and an annular side wall (500um high).

[0082] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A microfluidic device for mechanical compression and / or co-culture, comprising a housing, a limiting plate (4) and a flexible partition (12), characterized in that: A plurality of support columns (11) are arranged on the bottom surface of the shell; A limit plate (4) is arranged in the horizontal direction at an upper position in the middle of the inner cavity of the shell, a plurality of through holes are arranged on the limit plate (4), and at least one nutrient supply chamber (3) is arranged in the inner cavity of the shell above the limit plate (4); A flexible partition (12) is arranged horizontally at a lower middle position of the shell cavity, at least two culture chambers (2) are arranged in the shell cavity between the flexible partition (12) and the limiting plate (4), the culture chamber (2) comprising a compression chamber (5) and a non-compression chamber (6), the culture chamber (2) and the nutrient supply chamber (3) are connected via a through hole, and the shell cavity below the flexible partition (12) is arranged as an actuation chamber (10); the lower surface of the flexible partition (12) is placed above the support column (11), an annular side wall (9) is arranged at an edge position of the upper surface of the flexible partition (12), a cylindrical central micro-column (8) is arranged at the center of the upper surface of the flexible partition (12), a plurality of enclosure micro-columns (7) are evenly distributed outside the central micro-column (8) along the circumferential direction, and the heights of the central micro-column (8) and the enclosure micro-columns (7) are not The height of the enclosing microcolumns (7) is higher than the annular side wall (9), and the cross-sections of the enclosing microcolumns (7) are all set to be trapezoidal, and gaps are set between adjacent enclosing microcolumns (7); when the actuation chamber (10) is actuated, the actuation chamber (10) drives the flexible partition (12) to bulge upward and deform, and the flexible partition (12) drives the enclosing microcolumns (7) and the central microcolumn (8) to push up and contact with the limit plate (4), and the annular closed cavity between the enclosing microcolumns (7) and the central microcolumn (8) forms a compression compartment (5), and the compression compartment (5) is used to accommodate three-dimensional cells and / or tissue constructs that are subjected to mechanical compression, and the annular cavity between the enclosing microcolumns (7) and the annular side wall (9) forms a non-compression compartment (6), and the non-compression compartment (6) is used to accommodate three-dimensional cells and / or tissue constructs that are not subjected to or are subjected to a compression strength far lower than that in the compression compartment (5); When the actuation chamber (10) stops actuating, the flexible baffle (12) naturally returns to a horizontal state under the action of gravity, the central micro-column (8) and the surrounding micro-column (7) are separated from the limiting plate (4), and the actuation chamber (10) is actuated periodically to drive the flexible baffle (12) to deform periodically at a set deformation rate.

2. A mechanical compression and / or co-culture microfluidic device according to claim 1, characterized in that: The limiting plate (4) is made of a single chemically inert material, or is made of a composite of chemically inert materials, wherein the chemically inert material is one or more of silicone rubber, fluorinated rubber, polystyrene, polymethyl methacrylate, polycarbonate, ester, glass, silicon or polyethylene glycol.

3. A mechanical compression and / or co-culture microfluidic device according to claim 1, characterized in that: The flexible partition (12) is made of a flexible, deformable, chemically inert material, wherein the flexible, deformable, chemically inert material is one or more of silicone rubber, fluorinated rubber, or polyethylene glycol.

4. A mechanical compression and / or co-culture microfluidic device according to claim 1, characterized in that: A block or a membrane containing nutrients is placed in the nutrient supply chamber (3) as a culture medium, and the culture medium enters the culture chamber (2) through a through hole provided on the limiting plate (4).

5. A mechanical compression and / or co-culture microfluidic device according to claim 1, characterized in that: The three-dimensional cells and / or tissue constructs introduced into the compression compartment (5) and the non-compression compartment (6) are different. The three-dimensional cells and / or tissue constructs introduced into the compression compartment (5) and the non-compression compartment (6) are periodically squeezed by the gap changes formed between the enclosing microcolumn (7), the central microcolumn (8), the annular side wall (9) and the limiting plate (4).

6. A mechanical compression and / or co-culture microfluidic device according to claim 4 or 5, characterized in that: The culture medium placed in the nutrient supply chamber (3) corresponds to the three-dimensional cells and / or tissue constructs introduced into the compression compartment (5) and the non-compression compartment (6), respectively.

7. A mechanical compression and / or co-culture microfluidic device according to claim 1 or 5, characterized in that: The three-dimensional cell and / or tissue construct comprises stem cells and / or somatic cells, wherein the stem cells are at least one of ex vivo embryonic or ex vivo adult stem cells; and the somatic cells are at least one of ex vivo endothelial cells, bone cells, chondrocytes, nerve cells, fat cells, epithelial cells, fibroblasts, myofibroblasts, mesenchymal cells, hepatocytes, pancreatic cells, blood cells or muscle cells.

8. A mechanical compression and / or co-culture microfluidic device according to claim 1, characterized in that: Actuation of the actuation chamber (10) is achieved pneumatically, hydraulically, mechanically, electrically, magnetically or thermally.

9. A use of the microfluidic device according to claim 1, characterized in that: For the generation of in vitro tissue models; or for pharmaceutical screening of active ingredients or drugs; or for evaluating the effects of molecules and / or nanoparticles; or for disease modeling; or for drug discovery.

10. The use according to claim 9, characterized in that The following steps are involved: S1. Install the microfluidic equipment; S2, seeding the three-dimensional cells and / or tissue constructs in the compression compartment (5) and / or the non-compression compartment (6); S3, introducing a culture medium corresponding to the seeded three-dimensional cells and / or tissue constructs into at least one nutrient supply chamber (3); S4, the actuation chamber (10) is actuated, the actuation chamber (10) drives the flexible baffle (12) to bulge upward and deform, the flexible baffle (12) drives the enclosing micro-column (7) and the central micro-column (8) to be lifted upward and contact the limiting plate (4), the annular closed cavity between the enclosing micro-column (7) and the central micro-column (8) forms a compression compartment (5), and the annular cavity between the enclosing micro-column (7) and the annular side wall (9) forms a non-compression compartment (6); S5, the actuation chamber (10) stops actuating, the flexible partition (12) naturally returns to a horizontal state under the action of gravity, and the central micro-column (8) and the surrounding micro-column (7) are separated from the limiting plate (4); S6. Repeat the above steps S4 to S5, the actuation chamber (10) is actuated periodically, thereby driving the flexible partition (12) to deform periodically at a set deformation rate, and the gap formed between the enclosing micro-column (7), the central micro-column (8), the annular side wall (9) and the limiting plate (4) changes periodically, so that the three-dimensional cells and / or tissue constructs introduced into the compression compartment (5) and the non-compression compartment (6) are squeezed periodically.

Images