WO2022134294A1

WO2022134294A1 - Detachable and reusable hydrophobic or super-hydrophobic microfluidic organ-on-a-chip

Info

Publication number: WO2022134294A1
Application number: PCT/CN2021/077101
Authority: WO
Inventors: 张秀莉; 丛烨; 罗勇
Original assignee: 苏州大学
Priority date: 2020-12-25
Filing date: 2021-02-20
Publication date: 2022-06-30
Also published as: CN112280678B; CN112280678A

Abstract

Provided is a detachable and reusable hydrophobic or super-hydrophobic microfluidic organ-on-a-chip. A microfluidic organ-on-a-chip is constructed by using a hydrophobic or super-hydrophobic surface having a low critical surface tension. Specifically, it is possible to construct a heart-on-a-chip, a liver-on-a-chip, a brain-on-a-chip, a tumor-on-a-chip, a kidney-on-a-chip, a gut-on-a-chip, a skin-on-a-chip, a fat-on-a-chip, a vessel-on-a-chip, a womb-on-a-chip, an eye-on-a-chip, a nose-on-a-chip, a bone-on-a-chip, a periodontal chip, an islet-on-a-chip, a spleen-on-a-chip, a placenta-on-a-chip, a lung-on-a-chip, a muscle-on-a-chip, a larynx-on-a-chip, a bone marrow-on-a-chip, a diabetic chip and a multi-organ-on-a-chip. The organ-on-a-chip constructed by means of the present invention is detachable and reusable, such that the application cost of a microfluidic organ-on-a-chip is greatly reduced.

Description

A detachable and reusable hydrophobic or superhydrophobic microfluidic organ chip

technical field

The invention relates to the technical field of microfluidic chips, in particular to a detachable and reusable hydrophobic or superhydrophobic microfluidic organ chip.

Background technique

Microfluidic organ chip is a cutting-edge emerging technology, which refers to the co-cultivation of multiple mammalian cells in a microfluidic chip, controlling the three-dimensional spatial arrangement of cells, fluid shear force and signal molecule concentration, simulating real Organ microenvironment, technology to achieve real organ function. In 2016, the World Davos Conference selected it as one of the "Top Ten Emerging Technologies" in the world, which is believed to have an impact on human life in the future.

Microfluidic organ chips have been developed for nearly 10 years. At this stage, they have entered the stage of industrialization. In China, special microfluidic organ chip companies have begun to try to industrialize organ chips. However, compared with the vigorous academic research, the development of the microfluidic organ chip industry is still relatively lagging behind. A major problem is that traditional microfluidic organ chips are mainly based on polydimethylsiloxane (PDMS) elastic materials, and the processing requires the use of light. The engraving technology has complicated procedures and lengthy process, and the PDMS chip can only be used a limited number of times (in most cases, it is used once), resulting in a high cost of the microfluidic organ chip itself.

The main reason why the PDMS microfluidic organ chip is not durable is that the microchannels and chambers in the PDMS microfluidic organ chip are a closed micron-scale space, and the adsorption effect on the surface of PDMS is very serious. The embedded cells, three-dimensional glue and other substances are cleaned, which affects the second use.

Therefore, there are two main problems that need to be solved urgently in the field of microfluidic organ chips: 1. Extend the matrix of the microfluidic chip to other materials with lower cost and easier processing, such as rigid polymethacrylic acid methyl ester (PMMA); 2. Development of reusable organ chips.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a detachable, reusable hydrophobic or superhydrophobic microfluidic organ chip, which is provided with a functionalized surface, and the feature of the functionalized surface is that it has low adhesion. , low surface energy and hydrophobic or superhydrophobicity, whereby the material of organ-on-a-chip can be expanded from traditional more expensive PDMS to a variety of lower cost and easily processable rigid or elastic materials, and greatly increase the microfluidic The number of times the organ chip can be reused.

The first object of the present invention is to provide a microfluidic organ chip, comprising a substrate, the substrate has a functionalized surface, the critical surface tension of the functionalized surface is between 14-25 dynes/cm, and the contact angle with water is between 14 and 25 dynes/cm. at 110-180 degrees.

The functionalized surface of the present invention has the characteristics of low adhesion, low surface energy and hydrophobicity or superhydrophobicity, and the material for constructing the functionalized surface can be polyhexafluoropropylene, polytetrafluoroethylene, polyperfluoroethylene propylene , polytrifluoroethylene, polyvinylidene fluoride, superhydrophobic coatings, silanes, metals, metal oxides, metal inorganic salts, ceramics, waxes, oils or materials with surface micro-nano structures.

Further, the substrates are at least two layers, a porous membrane is arranged between two adjacent substrates, and the porous membrane is in close contact with the functionalized surface.

Further, the porous membrane has a plurality of micropores, and the pore diameter of the micropores is 10 μm or less.

Further, the material of the porous membrane includes polycarbonate, polydimethylsiloxane, polyethylene membrane, PES (polyethersulfone), cellulose and its derivatives, polyvinyl chloride, polyvinylidene fluoride PVDF, polysulfone , polyacrylonitrile, polyamide, polysulfone amide, sulfonated polysulfone, cross-linked polyvinyl alcohol, modified acrylic polymer, polytetrafluoroethylene (PTFE) porous membrane, porous polyurethane membrane, hollow fiber ultrafiltration membrane, Quantifoil copper mesh porous membrane, quantifoil silica support membrane, quantifoil carbon membrane, porous alumina membrane or inorganic ceramic membrane.

Further, one or more of organ-related cells, tissues and organoids are cultured in the microfluidic organ chip.

Further, the microfluidic organ chip also stores other materials for assisting cell culture, such as oxygen generators and oxygen depletion agents.

Further, the material of the substrate is rigid plastic, elastic plastic, glass, quartz, silicon, ceramic or metal.

Further, rigid plastics include but are not limited to polymethyl methacrylate, polycarbonate, polystyrene and other materials. Elastomeric plastics include, but are not limited to, polydimethylsiloxane, polyethylene terephthalate, high density polyethylene, polyvinyl chloride, and other materials.

As an embodiment of the present invention, the microfluidic organ chip includes a first upper substrate, a first porous membrane, a first middle substrate, a second porous membrane, and a first lower substrate, which are arranged in close contact in sequence, and the first upper substrate The lower surface of the substrate, the upper and lower surfaces of the first middle-layer substrate and the upper surface of the first lower-layer substrate are provided with functionalized surfaces, the first upper-layer substrate and the first lower-layer substrate are respectively provided with fluid channels, and the first middle-layer substrate is provided with a fluid channel. There is one through hole or a plurality of through holes, the first porous membrane and the second porous membrane cover at least a part of the fluid channel and cover all the through holes, and the fluid channel and through holes of the first upper substrate pass through the first porous membrane In fluid communication, the fluid channel and the through hole of the first lower substrate are in fluid communication with each other through the second porous membrane, and the through hole, the first porous membrane and the second porous membrane constitute a cell culture chamber.

Further, when the first middle-layer substrate is provided with a plurality of through holes, the plurality of through holes may communicate with each other, or some of the through holes may communicate with each other, and some of the through holes may be isolated from other through holes.

As another embodiment of the present invention, the microfluidic organ chip includes a second upper substrate, a third porous membrane, and a second lower substrate that are in close contact with each other in sequence, a lower surface of the second upper substrate and an upper surface of the second lower substrate. The surface is provided with a functionalized surface, the second upper substrate and the second lower substrate are respectively provided with fluid channels, the third porous membrane completely separates the fluid channels on the second upper substrate and the second lower substrate, and the third porous membrane The upper and lower surfaces are used as cell culture chambers, respectively.

In the present invention, one or more of organ-related cells, cell spheroids, tissues and organoids are cultured in the cell culture chamber, and may also be tumor cells. They can exchange substances through fluids within porous membranes and fluidic channels on the substrate, and cells, spheroids, tissues, or organoids within these connected chambers can also communicate with each other.

Unless otherwise specified, in the present invention, the fluid in the fluid channel includes gas and/or liquid. The liquid can be selected from cell culture fluid, cell culture fluid containing exogenous compounds (such as drugs, poisons, high sugar, etc.). The gas can be selected from one or more of air, oxygen, carbon dioxide and nitrogen. The flow rate and pressure of the fluid can vary or be constant. The fluid channel can be designed in any shape, such as straight, circular, spindle, etc.

The types of cells, cell spheroids, tissues and organoids cultured in the microfluidic organ chip of the present invention determine which organ chip it belongs to. When the cells are heart-related cells, including cardiac vascular endothelial cells, cardiomyocytes, and cardiac fibroblasts When cells, macrophages, nerve cells, and immune cells are used, when the tissue is myocardial tissue, the cell spheroid is a cardiac cell spheroid, or the organoid is a cardiac organoid, the chip is a reusable heart chip. When the cells are tumor-related cells, including tumor vascular endothelial cells, tumor cells, fibroblasts, and immune cells, when the tissue is tumor tissue, the cell sphere is a tumor cell sphere, or the organoid is a tumor organoid, the chip is A reusable tumor chip. By analogy, the present invention also provides a series of reusable liver chips, brain chips, kidney chips, intestinal chips, skin chips, fat chips, blood vessel chips, uterus chips, eye chips, nose chips, bone chips, tooth chips Zhou Chips, Islet Chips, Spleen Chips, Placenta Chips, Lung Chips, Muscle Chips, Laryngeal Chips, and Bone Marrow Chips, all of which are fundamentally characterized by hydrophobic/superhydrophobic surfaces.

The organ referred to in the present invention can be animal or human heart, liver, tumor, skin, brain, intestine, fat, blood vessel, eye, nose, uterus, kidney, periodontal, spleen, islet, lung, larynx, muscle, bone marrow , placenta, bones, and other organs.

When the microfluidic organ chip of the present invention is used as a heart organ chip, the cultured heart-related cells include cardiac vascular endothelial cells, cardiomyocytes, cardiac fibroblasts, macrophages, nerve cells, immune cells, and cells grown in the heart. other cell types.

When used as a liver chip, the cultured liver-related cells include hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, bile duct endothelial cells, nerve cells, immune cells, hepatocytes, and other cells grown in the liver. cell type.

When used as a brain chip, the cultured brain-related cells include neurons, glial cells, fibroblasts, immune cells, vascular endothelial cells, and other cell types growing in the brain.

When used as an intestinal chip, the cultured intestinal-related cells include intestinal epithelial cells, vascular endothelial cells, immune cells, and other cell types growing in the intestinal tissue.

When used as a fat chip, the cultured fat-related cells include adipocytes, fibroblasts, vascular endothelial cells, and other cell types grown in fat.

When used as a skin chip, the cultured skin-related cells include epidermal cells, vascular endothelial cells, immune cells, dermal cells, and other cell types growing in the skin tissue.

When used as a bone chip, the cultured bone-related cells include osteoblasts, vascular endothelial cells, osteoclasts, mesenchymal stromal cells, hematopoietic stem cells, progenitor cells, and other cell types growing in the bone.

When used as a blood vessel chip, the cultured blood vessel-related cells include vascular endothelial cells, smooth muscle cells, immune cells, nerve cells, etc., as well as other cell types growing in blood vessels.

When used as a kidney chip, the cultured kidney-related cells include glomerular vascular endothelial cells, renal tubular epithelial cells, pericytes, peritubular vascular endothelial cells, renal podocytes, and other cells growing in the kidney.

When used as a uterus chip, the cultured uterus-related cells include nerve cells, vascular endothelial cells, endometrial cells, and other cell types grown in the uterus.

When used as an eye chip, the cultured eye-related cells include nerve cells, vascular endothelial cells, conjunctival epithelial cells, immune cells, and other cell types growing in the eye.

When used as a nose chip, the cultured nose-related cells include nerve cells, vascular endothelial cells, immune cells, cells of the olfactory system, and other cell types growing in the nose.

When used as a periodontal chip, the cultured periodontal-related cells include vascular endothelial cells, macrophages, osteoblasts, osteoclasts, gingival epithelial cells, etc., as well as other cell types growing in the periodontal.

When used as a spleen chip, the cultured spleen-related cells include vascular endothelial cells, splenocytes, various immune cells, lymphocytes, nerve cells, and other cell types grown in the spleen.

When used as an islet chip, the cultured islet-related cells include vascular endothelial cells, islet beta cells, islet alpha cells, islet delta cells, islet PP cells, immune cells, nerve cells, and other cell types growing in islets.

When used as a lung chip, the cultured lung-related cells include vascular endothelial cells, alveolar epithelial cells, airway epithelial cells, smooth muscle cells, nerve cells, immune cells, and other cell types growing in the lung.

When used as a bone marrow chip, the cultured bone marrow-related cells include mesenchymal stem cells, red blood cells, granulocytes, and other cell types growing in the bone marrow.

When used as a laryngeal chip, the cultured laryngeal-related cells include vascular endothelial cells, nerve cells, muscle cells, chondrocytes, and other cell types growing in the larynx.

When used as a placenta chip, the cultured placenta-related cells include nerve cells, vascular endothelial cells, trophoblast cells, epithelial cells, and other cell types grown in the placenta.

When used as a primary chip, the cultured cells include primary cells, animal primary cells, human cell lines, animal cell lines, or human cells transformed from stem cells, but are not limited to the above cell sources.

When used as a muscle chip, the cultured muscle-related cells include fibroblasts, muscle cells, vascular endothelial cells, nerve cells, and other cell types growing in the muscle.

When used as a tumor chip, the cultured tumor-related cells include tumor vascular endothelial cells, tumor cells, fibroblasts, immune cells, and other cell types growing in tumors.

Tissues cultured in microfluidic organ-on-chip include heart, liver, tumor, skin, brain, intestine, fat, blood vessel, eye, nose, uterus, kidney, periodontal, spleen, islet, lung, larynx, muscle, bone marrow, placenta Or the living tissue isolated from organs such as bone.

Organoids cultured in microfluidic organoids include heart organoids, liver organoids, tumor organoids, skin organoids, brain organoids, intestinal organoids, fat organoids, blood vessel organoids, eye organoids, and nasal organoids , uterine organoids, kidney organoids, spleen organoids, pancreatic islet organoids, lung organoids, bone marrow organoids or placental organoids.

The cells in the microfluidic organ chip of the present invention can be cultured in three-dimensional culture in matrigel, suspension culture in culture medium, spherical culture, organoid culture or adherent two-dimensional culture, but not limited to the above-mentioned culture methods.

According to the structure of the nephron, the present invention also provides a kidney chip. The kidney chip includes a third upper substrate and a third lower substrate, and a fourth porous membrane arranged at intervals is arranged between the third upper substrate and the third lower substrate. and the fifth porous membrane, the lower surface of the third upper substrate and the upper surface of the third lower substrate are provided with functionalized surfaces, the critical surface tension of the functionalized surfaces is between 14-25 dynes/cm, and the contact with water The angle is between 110-180 degrees, the third upper substrate and the third lower substrate are respectively provided with fluid channels, and the fourth porous membrane and the fifth porous membrane completely separate the fluid channels on the third upper substrate and the third lower substrate On, the upper surface of the fourth porous membrane is used for culturing glomerular vascular endothelial cells, the lower surface of the fourth porous membrane is used for culturing renal podocytes, and the upper surface of the fifth porous membrane is used for culturing peritubular vascular endothelial cells Cells and/or pericytes, the lower surface of the fifth porous membrane is used to culture tubular epithelial cells.

The present invention also provides a multi-organ combination chip. The multi-organ combination chip includes at least two of the microfluidic organ chips of the present invention, and each microfluidic organ chip shares the same substrate.

The present invention also provides a multi-organ combination chip to simulate the human body. The multi-organ combination chip is formed by coupling at least two single-organ chips through a fluid pipeline, and at least one single-organ chip is the above-mentioned microfluidic device of the present invention. Control organ chip, each single organ chip is provided with at least one fluid inlet and one fluid outlet, along the fluid flow direction of the fluid pipeline, a fluid outlet of the previous single organ chip is connected to a fluid inlet of the latter single organ chip, and finally A single organ chip is a kidney chip. A fluid outlet of the kidney chip is connected to the fluid inlet of the first single organ chip to form a circuit. At least one peristaltic pump is arranged in the circuit to drive the fluid to circulate in the circuit. There is a metabolic outlet, which is used for the excretion of metabolites in the multi-organ chip.

The microfluidic organ chip of the present invention can be a single organ chip, such as a heart chip, a liver chip, a brain chip, a tumor chip, a kidney chip, an intestinal chip, a skin chip, a fat chip, a blood vessel chip, a uterus chip, an eye chip, and a nose chip. Chip, bone chip, periodontal chip, islet chip, spleen chip, placenta chip, lung chip, muscle chip, throat chip or bone marrow chip, and can also be a human chip containing multiple organs.

Further, sampling holes are provided at the positions of the fluid inlet and the fluid outlet of the single-organ chip, and the cell culture fluid can be extracted through the sampling holes for component analysis.

By means of the above scheme, the present invention has at least the following advantages:

The present invention proposes a new idea of constructing a detachable microfluidic organ chip with a hydrophobic or superhydrophobic interface with low surface energy and low adhesion, and expands the material of the organ chip from the traditional more expensive PDMS to a variety of lower cost ones. Hard or elastic materials that are easy to process, the microfluidic organ chip can be disassembled, simply cleaned, and reused, which greatly increases the number of reusable microfluidic organ chips, and based on this, the reusable use is proposed. A variety of organ chips, which greatly improves the processing efficiency of microfluidic organ chips, greatly reduces the processing cost of microfluidic organ chips, and promotes the standardization process of microfluidic organ chips, thereby helping microfluidic organ chips. large-scale industrialization.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the description, the following description is given with the preferred embodiments of the present invention and the detailed drawings.

Description of drawings

1 is a schematic structural diagram of a microfluidic organ chip according to an embodiment of the present invention;

2 is a schematic structural diagram of a microfluidic organ chip according to an embodiment of the present invention;

3 is a schematic structural diagram of a kidney chip according to an embodiment of the present invention;

4 is a schematic structural diagram of a multi-organ combination chip according to an embodiment of the present invention;

5 is a schematic structural diagram of a multi-organ combination chip according to an embodiment of the present invention;

6 is a schematic structural diagram of a heart chip of the present invention and a volcano diagram of differentially expressed proteins in a drug-added group and a control group;

7 is a schematic structural diagram of a liver chip of the present invention;

8 is a component diagram of a brain chip of the present invention;

Fig. 9 is a component diagram of a diabetes chip of the present invention;

Fig. 10 is the part diagram of the periodontal chip, intestinal chip, fat chip, uterine chip, eye chip or bone chip of the present invention;

Figure 11 is a component diagram of a kidney chip of the present invention;

Fig. 12 is the component diagram of the skin chip, blood vessel chip or nose chip of the present invention;

Figure 13 is the relationship between the transmittance of FITC on the skin chip over time;

14 is a schematic structural diagram of a reusable multi-organ chip based on the combination of a single-organ chip in Example 16;

15 is a schematic structural diagram of a reusable multi-organ chip based on a single chip in Example 17;

Description of reference numbers:

100-first upper substrate; 111-first porous membrane; 200-first middle substrate; 222-second porous membrane; 300-first lower substrate; 123-lower surface of the first upper substrate; 124- The upper surface of the first middle-layer substrate; 125-the lower surface of the first middle-layer substrate; 126-the upper surface of the first lower-layer substrate; 400-the second upper-layer substrate; 333-the third porous membrane; 500-the second lower-layer substrate ; 127 - the lower surface of the second upper substrate; 128 - the upper surface of the second lower substrate; 600 - the third upper substrate; 700 - the third lower substrate; 444 - the fourth porous membrane; 555 - the fifth porous membrane ; 129 - lower surface of the third upper substrate; 130 - upper surface of the third lower substrate; 101 - peristaltic pump; 102 - fluid inlet; 103 - fluid outlet; 104 - syringe pump; 105 - metabolic outlet; 106 - vascular endothelium cell; 107-cell, cell spheroid, tissue or organoid; 108-porous membrane; 109-functionalized surface; 110-reservoir and peristaltic pump; 800-multi-organ combination chip upper substrate; 900-multi-organ combination Chip middle-layer substrate; 1000-multi-organ combination chip lower substrate; 1310-multi-organ combination chip upper surface substrate; 1320-multi-organ combination chip middle-layer substrate upper surface; 1330-multi-organ combination chip middle-layer substrate lower surface; 1340 - the upper surface of the lower substrate of the multi-organ combination chip; 131 - the upper substrate; 101 - the first porous membrane of the chip; 132 - the middle substrate; 102 - the second porous membrane of the chip; 133 - the lower substrate; 210 - lower surface of upper substrate; 211 - upper surface of middle substrate; 212 - lower surface of middle substrate; 213 - upper surface of lower substrate; 2000 - liver module; 3000 - heart module; 4000 - tumor module.

Detailed ways

Hereinafter, the critical surface tension of the functionalized surface is between 14-25 dynes/cm and the contact angle with water is between 110-180 degrees. The functionalized surfaces are all located on the substrate where no fluid channels or vias are provided.

As shown in FIG. 1 , wherein (A) is a three-dimensional schematic diagram of a disassembled state, and (B) is a cross-sectional view, as an embodiment of the present invention, the microfluidic organ chip includes a first upper substrate 100 , a first upper substrate 100 , a first upper substrate 100 , a A porous membrane 111, a first middle-layer substrate 200, a second porous membrane 222, and a first lower-layer substrate 300, the lower surface 123 of the first upper-layer substrate, the upper surface 124 of the first middle-layer substrate, and the lower surface of the first middle-layer substrate The surface 125 and the upper surface 126 of the first lower-layer substrate are provided with functionalized surfaces, the first upper-layer substrate 100 and the first lower-layer substrate 300 are respectively provided with fluid channels, and the first middle-layer substrate 200 is provided with three interconnected through holes, The first porous membrane 111 and the second porous membrane 222 cover at least a part of the fluid channel and cover all the through holes, the fluid channel and the through holes of the first upper substrate 100 are in fluid communication through the first porous membrane 111, and the first lower layer The fluid channel and the through hole of the substrate 300 are in fluid communication with each other through the second porous membrane 222, and the through hole, the first porous membrane 111 and the second porous membrane 222 form three interconnected cell culture chambers a, b, c .

As shown in FIG. 2 , wherein (A) is a three-dimensional schematic diagram of a disassembled state, and (B) is a cross-sectional view, as another embodiment of the present invention, the microfluidic organ chip includes a second upper layer substrate 400, The third porous membrane 333 and the second lower substrate 500, the lower surface 127 of the second upper substrate and the upper surface 128 of the second lower substrate are provided with functionalized surfaces, and the second upper substrate 400 and the second lower substrate 500 are respectively provided with functionalized surfaces Fluid channels, the third porous membrane 333 completely separates the fluid channels on the second upper substrate 400 and the second lower substrate 500, and the upper and lower surfaces of the third porous membrane 333 are respectively used as cell culture chambers.

As shown in FIG. 3 , wherein (A) is a three-dimensional schematic diagram of a disassembled state, and (B) is a cross-sectional view. According to the structure of the nephron, the present invention also provides a kidney chip. The kidney chip includes a third upper substrate 600 and a third The third lower layer substrate 700, the fourth porous membrane 444 and the fifth porous membrane 555 arranged at intervals between the third upper layer substrate 600 and the third lower layer substrate 700, the lower surface 129 of the third upper layer substrate and the third lower layer substrate The upper surface 130 is provided with a functionalized surface, the critical surface tension of the functionalized surface is between 14-25 dynes/cm, and the contact angle with water is between 110-180 degrees, the third upper substrate 600, the third lower substrate 700 are respectively provided with fluid channels, the fourth porous membrane 444 and the fifth porous membrane 555 completely separate the fluid channels on the third upper substrate 600 and the third lower substrate 700, and the upper surface of the fourth porous membrane 444 is For culturing glomerular vascular endothelial cells, the lower surface of the fourth porous membrane 444 is used for culturing renal podocytes, the upper surface of the fifth porous membrane 555 is used for culturing peritubular vascular endothelial cells and pericytes, and the fifth porous membrane 555 is used for culturing perivascular endothelial cells and pericytes. The lower surface of membrane 555 is used for culturing renal tubular epithelial cells.

As shown in FIG. 4 , the present invention provides a multi-organ combination chip to simulate the human body. The multi-organ combination chip is formed by coupling multiple single-organ chips through fluid pipelines. Each single-organ chip is as shown in FIG. 1 . As shown in the microfluidic organ chip, each single organ chip is provided with at least one fluid inlet 102 and one fluid outlet 103. Along the fluid flow direction of the fluid pipeline, one fluid outlet 103 of the previous single organ chip is connected to the latter one. One fluid inlet 102 of the organ chip, the last single organ chip is the kidney chip, and one fluid outlet 103 of the kidney chip is connected to the fluid inlet 102 of the first single organ chip to form a circuit, and at least one peristaltic pump 101 is arranged in the circuit to The driving fluid circulates in the loop, the upper porous membrane 108 in each single organ chip is loaded with vascular endothelial cells 106, and the kidney chip is also provided with a metabolic outlet 105, which is used for the excretion of metabolites in the multi-organ combination chip . The arrows in Figure 4 represent the direction of fluid flow.

As shown in FIG. 5 , the present invention also provides another multi-organ combination chip. The multi-organ combination chip is obtained by integrating multiple microfluidic organ chips shown in FIG. The upper-layer substrate, the middle-layer substrate and the lower-layer substrate in the organ chip are respectively connected into a whole. Among them, the

fluid channels

1, 2, and 3 are connected and are in the same fluid circuit. The

fluid channels

4, 5, and 6 are not communicated with each other. Each fluid channel has its own fluid circuit. Chambers A, B , C, D, E, F, etc. to culture cells, spheroids, tissues or organoids.

The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Example 1: A reusable cardiac chip based on a hydrophobic surface

The heart is the blood supply organ of the human body, mainly including cardiomyocytes (Cardiomyocytes), cardiac fibroblasts (Fibroblast), vascular endothelial cells (Endothelial cells) and macrophages (Macrophage). The heart chip is an in vitro model of the heart, which is used to investigate the toxicity or efficacy of drugs to the heart. In toxicity or efficacy evaluation experiments, it is often necessary to measure changes in different types of cellular proteins and genes. Several kinds of cells are mixed and cultured. After administration, it is difficult to separate these different cells for gene and protein detection. Therefore, the present invention provides a heart chip that is particularly suitable for proteome and genome detection.

As shown in FIGS. 6(A) and 6(B) , the heart chip is formed by laminating the upper layer substrate 131, the chip first porous membrane 101, the middle layer substrate 132, the chip second porous membrane 102 and the lower layer substrate 133 which are closely attached in sequence. As a result, the upper substrate 131 has a fluid channel structure, and the fluid channel is designed as a spindle type, the middle layer substrate 132 is provided with three through holes, and the three through holes are connected with each other, and the lower layer substrate 133 has a fluid channel structure, and the fluid channel is designed as a spindle type. The lower surface 210 of the upper substrate is plated with PTFE, the upper surface 211 of the middle substrate and the lower surface 212 of the middle substrate are plated with PTFE, and the upper surface 213 of the lower substrate is plated with PTFE. The critical surface tension and the contact angle with water of the above-mentioned polytetrafluoroethylene are 18 dynes/cm and 114 degrees, respectively. The positions of the first porous membrane 101 of the chip and the second porous membrane 102 of the chip cover the three through holes on the middle-layer substrate, so the three through holes and the two porous membranes constitute three chambers a, b and c, Cardiac vascular endothelial cells are cultured on the first porous membrane 101 of the chip, cardiomyocytes, cardiac fibroblasts and macrophages are cultured in three dimensions in the three chambers a, b and c, respectively. The cells in c can exchange substances and nutrients through the two porous membranes and the fluid in the fluid channel on the upper substrate and the lower substrate to maintain their activity. These three chambers are also interconnected, and the cells inside can communicate with each other.

The upper, middle and lower substrates of the heart chip are all made of polymethyl methacrylate (PMMA). The inner surface of the fluid channel on the substrate, the lower substrate, and the side surface of the through hole on the middle substrate are still slightly hydrophilic PMMA. Due to the spacing of the porous membranes, it is difficult to closely adhere between the lower surface of the upper substrate and the upper surface of the middle substrate, as well as the lower surface of the middle substrate and the upper surface of the lower substrate. However, because these surfaces are superhydrophobic, when pouring into the microchannel When the cell culture medium is used, the cell culture medium will only be transported in the slightly hydrophilic PMMA channel, and will not leak into the small gap between the lower surface of the upper substrate and the upper surface of the middle substrate, and between the lower surface of the middle substrate and the lower substrate , thus ensuring that the heart-on-a-chip experiment can be carried out smoothly. If there is no superhydrophobic coating on the upper, middle, and lower substrates, and there is only a porous film between the three, leakage is easy to occur. When an experiment is completed, the screws and nuts can be unscrewed, the upper, middle and lower substrates can be disassembled, and wiped with alcohol cotton to remove the splashed cell culture medium, cells, etc. in the channel and on the PTFE surface. These three substrates can be reused, because the oil and water on the PTFE surface is not sticky and easy to clean, and the three substrates can be reused more than 200 times. Considering the extremely low cost of the PMMA material itself, the manufacturing cost of the heart chip is extremely low, and the industrialization prospect is promising.

In the heart chip, although the four kinds of heart cells are cultured separately, the culture medium is connected, and these four kinds of cells can still communicate with each other, so the heart chip still has good bionics, and the drug is added through the fluid channel. In the heart chip, the drug will interact with four kinds of heart cells. After the action is completed, these four kinds of cells can be taken out for subsequent proteome and genome analysis, so as to analyze the toxicity and efficacy of the drug in a deeper level. Figure 6(C) is a volcano plot of the protein level of fibroblasts in the heart chip before and after adding a certain drug. It can be seen that the protein group of fibroblasts changed significantly after the drug was added, indicating that the heart The toxicity to fibroblasts also occupies a certain proportion in the toxicity. This conclusion provides an important clue for the further study of the cardiotoxicity mechanism of this drug.

Example 2: A reusable liver chip based on a hydrophobic surface

The liver is the largest metabolic organ in the body, mainly including hepatocytes, fibroblasts, stellate cells, hepatic endothelial cells, bile duct epithelial cells and Kuppfer Cells, etc. The liver chip is an in vitro model of the liver, which is used to investigate the metabolism of drugs in the liver and the toxicity of drugs to the liver. In the metabolism and toxicity evaluation experiments, it is necessary to measure the changes of different types of cells at the protein and gene levels. For the purpose of biomimetic chips, these kinds of cells are often mixed and cultured. After administration, it is difficult to separate these kinds of cells for gene and protein detection. Liver microarray for genomic testing.

As shown in FIGS. 7(A) and 7(B) , the liver chip is formed by laminating the upper substrate 131, the first porous membrane 101 of the chip, the middle substrate 132, the second porous membrane 102 of the chip and the lower substrate 133, which are closely attached in sequence. The upper substrate 131 has a fluid channel structure, the fluid channel is designed as a spindle type, and the middle substrate 132 is provided with two through holes, which are connected to each other. Along the height direction of the through holes, one of the through holes has a larger diameter at both ends. , the middle diameter is small, the lower substrate 133 has a fluid channel structure, and the fluid channel is designed as a spindle type. The lower surface 210 of the upper substrate is plated with PFEP, the upper surface 211 of the middle substrate and the lower surface 212 of the middle substrate are plated with PFEP, and the upper surface 213 of the lower substrate is plated with PFEP. The critical surface tension and contact angle with water of the above-mentioned polyperfluoroethylene propylene are 20 dynes/cm and 168.1 degrees, respectively. The positions of the first porous membrane 101 of the chip and the second porous membrane 102 of the chip cover the two through holes on the middle-layer substrate, so the two through holes and the two porous membranes form two connected chambers a and b, Hepatic vascular endothelial cells and Kupffer cells are cultured on the first porous membrane 101 of the chip, the upper part of chamber a is cultured three-dimensionally cultured hepatic stellate cells, the lower part is cultured three-dimensionally cultured hepatocytes, and the chamber b is cultured Fibroblasts are cultured in three dimensions, bile duct epithelial cells are cultured on the second porous membrane 102 of the chip, and the three types of cells in the two chambers a and b can pass through the two porous membranes and the fluid in the fluid channels on the upper and lower substrates Substance and nutrient exchange are carried out to maintain its activity.

The upper, middle and lower substrates of the liver chip are made of polycarbonate (PC). The inner surface of the upper fluid channel and the sides of the through holes on the middle substrate are still slightly hydrophilic PC. When the three layers of rigid substrates are pressed together by screws and nuts with two porous membranes spaced apart, due to the The spacers, the lower surface of the upper substrate and the upper surface of the middle substrate, and the lower surface of the middle substrate and the upper surface of the lower substrate are difficult to adhere closely, but because these surfaces are superhydrophobic, when the cell culture medium is perfused into the microchannel, the cells The culture medium will only be transported in the slightly hydrophilic PC channel, and will not leak into the small gap between the lower surface of the upper substrate and the upper surface of the middle substrate, and between the lower surface of the middle substrate and the lower substrate, thus ensuring the liver chip. The experiment can proceed smoothly. If there is no superhydrophobic coating on the upper, middle, and lower substrates, and there is only a porous film between the three, leakage is easy to occur. When an experiment is completed, the screws and nuts can be unscrewed, the upper, middle and lower substrates can be disassembled, wiped gently with alcohol cotton to remove the cell culture fluid, cells, etc. splashed in the channel and on the surface of PFEP , these three substrates can be reused, because the oil and water on the surface of PFEP are not sticky and easy to clean, and the three substrates can be reused more than 200 times. Considering the extremely low cost of the PC material itself, the production cost of the liver chip is extremely low, and the industrialization prospect is promising.

In the liver chip, although the six kinds of liver cells are cultured separately, the culture medium is connected, and these six kinds of cells can still communicate with each other, so the liver chip still has good bionics, and the drug is added through the fluid channel. In the liver chip, the drug will interact with six kinds of liver cells. After the action is completed, these six kinds of cells can be taken out for subsequent proteomic and genomic analysis, so as to analyze the metabolism and toxicity of the drug in a deeper level. Using the liver chip to study the results of protein level clustering analysis of stellate cells before and after adding a certain drug, it was found that the protein expression of stellate cells changed significantly before and after adding the drug, indicating that the drug's hepatotoxicity is moderately important to the liver. The toxicity of fibroblasts also occupies a certain proportion. This conclusion provides an important clue for the further study of the mechanism of this drug's liver toxicity.

Example 3: A reusable tumor chip based on a hydrophobic surface

Tumor is one of the major diseases of human beings. Tumor tissue mainly includes tumor cells (Cancer cells), fibroblasts (Fibroblasts), vascular endothelial cells (Endothelial cells) and immune cells (Immune cells). The tumor chip is an in vitro model of tumors, which is used to investigate the anti-tumor activity of drugs. In drug efficacy evaluation experiments, it is necessary to measure the changes of different types of cells at the protein and gene levels. Traditional tumor chips are often used for biomimetic purposes. After being mixed and cultured, it is difficult to separate these different cells for gene and protein detection. Therefore, the present invention provides a tumor chip that is particularly suitable for proteome and genome detection.

The structure of the tumor chip is the same as that of the heart chip in Example 1, but the cells cultured in the tumor chip are different. The three chambers a, b and c are three-dimensionally cultured tumor cells, cancer fibroblasts and immune cells. , the tumor vascular endothelial cells are cultured on the first porous membrane 101 of the chip. And the critical surface tension and the contact angle with water of the functionalized surface of each substrate surface were 25 dynes/cm and 178 degrees, respectively. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the tumor chip can be used repeatedly for many times.

In the tumor chip, although the four tumor cells are cultured separately, the culture medium is connected, and the four kinds of cells can still communicate with each other, so the tumor chip still has good biomimetic properties. The drug is added through the fluid channel. On the tumor chip, the drug will interact with four tumor cells. After the effect is completed, these four kinds of cells can be taken out for subsequent proteome and genome analysis, so as to analyze the anti-cancer activity of the drug in a deeper level.

Example 4: A reusable brain chip based on a hydrophobic surface

The brain is the commander of human body functions. The brain tissue mainly contains neurons (Neurons), astrocytes (Astrocytes), cerebral vascular endothelial cells (Endothelial cells), ependymal cells (Ependymal cells), microglia ( Microglia), Oligodendrocytes. The brain chip is an in vitro model of the brain, which is used to investigate the neurotoxicity or efficacy of drugs. In toxicity and efficacy evaluation experiments, it is necessary to measure the changes of different types of cells at the protein and gene levels. Traditional brain chips are often used for bionics. Several kinds of cells are mixed and cultured. After administration, it is difficult to separate these different cells for gene and protein detection. Therefore, the present invention provides a brain chip that is particularly suitable for proteome and genome detection.

The basic structure of the brain chip is the same as that of the heart chip in Embodiment 1. The difference is that the middle-layer substrate 132 is provided with 4 through-holes that communicate with each other, and the cavities a, b, c and d formed by the 4 through-holes Neurons, astrocytes, microglia and oligodendritic glial cells are respectively cultured in three dimensions, cerebral vascular endothelial cells are cultured on the upper surface of the first porous membrane 101 of the chip, and ependymal cells are cultured on the lower surface. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the brain chip can be used repeatedly for many times. The part diagram of the chip is shown in Figure 8.

In the brain chip, although the six kinds of cells are cultured separately, the culture medium is connected, and the six kinds of cells can still communicate with each other, so the brain chip still has good bionics, adding drugs to the brain through fluid channels On the chip, the drug will interact with five kinds of brain cells. After the action is completed, these five kinds of cells can be taken out for subsequent proteome and genome analysis, so as to analyze the toxicity and efficacy of the drug in a deeper level.

Example 5: A reusable diabetes chip based on a hydrophobic surface

Diabetes is a serious disease that plagues modern people. Early studies believed that diabetes was only related to the impairment of pancreatic islet function. Later studies found that diabetes is actually closely related to liver, fat, muscle, pancreatic islets, heart and intestines. The in vitro model of diabetes should include the above-mentioned multiple organs, not just pancreatic islets. Therefore, the present invention provides an advanced diabetes in vitro model including 7 organs—diabetes chip.

The basic structure of the diabetes chip is the same as that of the heart chip in Example 1. The difference lies in that the middle-layer substrate 132 is provided with 5 through holes that communicate with each other, and the cavities a, b, c, d formed by the 5 through holes Pancreatic beta cells, liver parenchymal cells, muscle cells, adipocytes and cardiomyocytes are cultured in three dimensions in and e respectively, vascular endothelial cells are cultured on the upper surface of the first porous membrane 101 of the chip to simulate the vascular barrier, and the second porous membrane 102 of the chip is cultured on the upper surface Intestinal epithelial cells. And the critical surface tension and the contact angle with water of the functionalized surface of each substrate surface are 14 dynes/cm and 135 degrees, respectively. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the diabetes chip can be used repeatedly for many times. The part diagram of the chip is shown in FIG. 9 .

When in use, first add high sugar to the culture medium in the fluid channel of the upper substrate to destroy islet beta cells, and interact with other cells in the chip to form a pathological environment for diabetes, and then add drugs that can treat diabetes to detect The recovery status of pancreatic islet β cells, and also detect the changes in the proteome and genome of other organ cells before and after the drug is added, so as to judge the efficacy of the drug and analyze its mechanism in depth.

In the diabetes chip, seven organ cells are connected through simulated blood flow and can communicate with each other, so the diabetes chip has good bionics, and the chip can not only observe the efficacy of drugs, but also explore the pharmacology in depth. , has great potential in diabetes research.

Example 6: A reusable periodontal chip based on a hydrophobic surface

Periodontitis is a common disease, and patients will feel very painful. At present, the only in vitro models of periodontitis are animal models such as beagle dogs, which greatly limits the discovery of periodontitis drugs. The emergence of organ-on-a-chip may change this status quo. The invention provides a periodontal chip capable of simulating periodontitis.

The basic structure of the periodontal chip is the same as the structure of the heart chip in Example 1, the difference is that there is only one through hole on the middle-layer substrate 132, and the bottom of the chamber a formed by this through hole, that is, the second most A piece of bone is placed on the upper surface of the porous membrane 102, osteoblasts and osteoclasts are cultured on the bone piece, vascular endothelial cells and macrophages are cultured on the upper surface of the first porous membrane 101 of the chip, and gingival epithelial cells are cultured on the lower surface. Chamber a Filled with LPS solution or gingival fluid in periodontal patients. Since the periodontal chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the periodontal chip can be used repeatedly for many times. The part diagram of the chip is shown in Figure 10.

The chip simulates the periodontal tissue structure and the pathological state of periodontitis. In this state, osteoclasts are dominant over osteoblasts and will gradually devour the bone fragments used to simulate the alveolar bone. After the drug, the bone chips stopped erosion or recovered, indicating that the drug is effective for the treatment of periodontitis.

Example 7: A reusable kidney chip based on hydrophobic surface

Kidney is the main elimination organ of human beings. Kidney tissue mainly contains glomerular vascular endothelial cells (Renal endothelial cells), renal podocytes (Podocytes), peritubular vascular endothelial cells (Renal peritubular endothelial cells), and renal tubular epithelial cells (Renal tubular epithelial cells). epithelial cells) and pericytes (Renal pericytes). The kidney chip is an in vitro model of the kidney, which is used to investigate the nephrotoxicity or efficacy of drugs. In toxicity and efficacy evaluation experiments, it is necessary to measure the changes in proteins and genes of different types of cells. Traditional kidney chips are often used for bionics. All kinds of cells are mixed and cultured. After administration, it is difficult to separate these different cells for gene and protein detection. Therefore, the present invention provides a kidney chip which is particularly suitable for proteome and genome detection.

The design of the kidney chip is shown in FIG. 3 , the kidney chip includes a third upper substrate 600 and a third lower substrate 700 , and the third upper substrate 600 and the third lower substrate 700 are both made of PMMA. A fourth porous membrane 444 and a fifth porous membrane 555 are arranged at intervals between the third upper substrate 600 and the third lower substrate 700. The fourth porous membrane 444 and the fifth porous membrane 555 have a pore diameter of 1 micron of polycarbonate film. The lower surface 129 of the third upper layer substrate and the upper surface 130 of the third lower layer substrate are provided with PTFE coating, the third upper layer substrate 600 and the third lower layer substrate 700 are respectively provided with fluid channels, and the cell culture fluid flows in the fluid channels . The fourth porous membrane 444 and the fifth porous membrane 555 completely separate the fluid channels on the third upper substrate 600 and the third lower substrate 700, and the upper surface of the fourth porous membrane 444 culture glomerular vascular endothelial cells, Renal podocytes are cultured on the lower surface of the fourth porous membrane 444 , peritubular vascular endothelial cells and pericytes are cultured on the upper surface of the fifth porous membrane 555 , and renal tubular epithelial cells are cultured on the lower surface of the fifth porous membrane 555 . Since the kidney chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the kidney chip can be used repeatedly for many times. The parts diagram of the chip is shown in Figure 11.

In this kidney chip, although the five kinds of cells are cultured separately, but the culture medium is connected, these five kinds of cells can still communicate with each other. When the drug is added to the kidney chip through the fluid channel, the drug will interact with the five kinds of brain cells. After the action is completed, these five kinds of cells can be taken out for subsequent proteome and genome detection, so as to analyze the nephrotoxicity and efficacy of the drug in a deeper level.

Example 8: A reusable intestinal chip based on a hydrophobic surface

Intestine is the main digestive and absorptive organ of human beings. Intestinal tissue mainly contains Intestine epithelial cells, Endothelial cells, and Macrophages. The intestinal chip is an in vitro model of the intestine, which is used to investigate the absorption of drugs or nutrients and the role of intestinal flora. Traditional intestinal chips are mostly based on PDMS materials and are disposable. In this example, the intestinal chip Using PC rigid plastic and superhydrophobic organ-on-a-chip technology, the intestinal chip can be reused.

The basic structure of the intestinal chip is the same as that of the heart chip in Example 1, the difference is that the middle-layer substrate 132 has only one through hole, and vascular endothelial cells are cultured on the upper surface of the first porous membrane 101 of the chip. Intestinal epithelial cells are cultured on the lower surface of the porous membrane 101, intestinal epithelial cells are cultured on the upper surface of the second porous membrane 102 of the chip, vascular endothelial cells are cultured on the lower surface of the second porous membrane 102 of the chip, and the chamber a is filled with simulated intestinal fluid. When the chip is running, a periodically changing pressure is applied in the fluid channel. Under the action of this pressure, the porous membrane will vibrate periodically, simulating the peristalsis of the intestine. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the intestinal chip can be used repeatedly for many times. The part diagram of the chip is shown in Figure 10.

In the intestinal chip, although the two kinds of cells are cultured separately, but the culture medium is connected, the two kinds of cells can still communicate with each other, and the chip simulates the peristalsis of the intestine, which is conducive to the subsequent inoculation of intestinal flora .

Example 9: A reusable skin chip based on a hydrophobic surface

The skin is the tissue on the surface of the body wrapped around the muscles, and is one of the important components of the human external image. The skin tissue mainly includes epidermal cells, dermal cells, vascular endothelial cells and macrophages. Cells (Macrophages) etc. The skin chip is an in vitro model of the skin, which is used to investigate the absorption, health care effect and toxicity of cosmetics. Traditional skin chips are mostly based on PDMS materials, which are disposable and expensive. In this embodiment, the chip is made of PMMA rigid plastic. And superhydrophobic organ-on-a-chip technology, which can realize the reuse of skin chips.

The basic structure of the skin chip is shown in FIG. 2 , epidermal cells and dermal cells are cultured on the upper surface of the third porous membrane 333 , vascular endothelial cells and macrophages are cultured on the lower surface of the third porous membrane 333 , and the second upper substrate 400 Air circulates in the fluid channel of the second lower layer substrate 500 to differentiate epidermal cells, and cell culture fluid circulates in the fluid channel of the second lower substrate 500 to provide nutrients for the cells on the third porous membrane 333 . Since the skin chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the skin chip can be used repeatedly for many times. The parts diagram of the chip is shown in Figure 12, the permeability of the skin chip is shown in Figure 13, and the transwell is the control group in the figure. .

The skin chip is simple to manufacture and low cost, and is expected to be used on a large scale.

Example 10: A reusable fat chip based on a hydrophobic surface

The adipose tissue in humans and animals is composed of fat cells, which are related to obesity, diabetes and some cardiovascular and cerebrovascular diseases. The present invention provides a fat chip, which can realize this kind of fat chip due to the use of PMMA hard plastic and super-hydrophobic organ chip technology. Reuse of fat chips.

The basic structure of the fat chip is the same as that of the heart chip in Example 1, the difference is that the middle substrate only has one through hole, the vascular endothelial cells are cultured on the upper surface of the first porous membrane of the chip, and the vascular endothelial cells are cultured in suspension in the chamber a. Figure 10 shows the parts diagram of the adipocytes, the chip.

When the drug is added to the fat chip through the fluid channel, the drug will pass through the vascular endothelial cell layer and enter the chamber a to interact with the fat cells. Hierarchical analysis of pharmacology and toxicology.

Example 11: A reusable vascular chip based on a hydrophobic surface

Blood vessels refer to a series of pipes through which blood flows. According to different structures and functions, they are divided into three types: arteries, veins and capillaries. The cardiovascular circulatory system plays a vital role in maintaining homeostasis in the human body. It is a closed network of arteries, veins, and capillaries that allow blood to circulate throughout the body for gas exchange and large-scale nutrient delivery. Luck is the core element to maintain the vitality of organs. Vascular chips can simulate the characteristics and functions of blood vessels in vitro by patterning, and enable a variety of blood vessels to be physiologically interconnected and connected to multiple organ units, which can be used as a supplement to more complete disease model drug screening and other platforms. In this embodiment, the chip adopts PMMA rigid plastic and super-hydrophobic organ chip technology, which can realize the repeated use of the blood vessel chip.

The basic structure of the vascular chip is shown in Figure 2. Smooth muscle cells are cultured on the upper surface of the third porous membrane, vascular endothelial cells and sugar jaws are cultured on the lower surface of the third porous membrane, and the fluid of the second upper substrate and the second lower substrate is The cell culture fluid circulates in the channel to provide nutrients for the cells on the third porous membrane. The part diagram of the chip is shown in Figure 12.

The blood vessel chip can be used to study some cardiovascular diseases and to screen drugs for cardiovascular diseases.

Example 12: A reusable uterine chip based on a hydrophobic surface

The uterus is the main organ that secretes estrogen and reproduces sexually in humans and most other mammals. The uterine wall consists of three layers: the endometrium, the myometrium, and the perimetrium. The uterus, as an important organ specialized in reproductive function in the human body, needs to construct a reasonable and effective in vitro research model. The uterus chip can construct an in vitro uterine culture system in vitro, and simultaneously analyze functions such as matrix decidualization and vascular barrier formation under controlled physiological conditions, which also verifies its ability to examine physiological reproductive processes. Agents or environmental poisons that improve health or reproductive dysfunction.

The basic structure of the uterus chip is the same as that of the heart chip in Example 1. The difference is that the middle-layer substrate only has one through hole, the vascular endothelial cells are cultured on the upper surface of the first porous membrane of the chip, and the bottom of the first porous membrane of the chip is cultured. Endometrial cells are cultured on the surface, embryos can be cultured in the chamber a, and cell culture fluid is perfused in the channels of the upper substrate and the lower substrate. The chip part diagram is shown in Figure 10.

In this embodiment, the chip adopts PMMA rigid plastic and super-hydrophobic organ chip technology, which can realize the repeated use of the uterus chip, and analyze the proteome and genome of cells or embryos, so as to analyze the toxicity and efficacy of drugs in a deeper level. .

Example 13: A reusable eye chip based on a hydrophobic surface

The eye is an organ of the visual system and a complex part of the human body, which can provide vision and the ability to receive and process visual details, and to achieve a variety of response functions that are independent and felt. The human eye is approximately spherical, and the eyeball includes the eyeball wall, contents, nerves, blood vessels and other tissues. The ocular chip can reproduce the ocular surface and tear system in vitro, simulate ocular surface infection and watery dry eye disease caused by inflammation, and provide a new platform for ocular surface pathophysiology research and topical drug screening. Traditional eye chips are mostly based on glass or PDMS materials, which are disposable and expensive. In this embodiment, the chip adopts PMMA rigid plastic and super-hydrophobic organ chip technology, which can be reused.

The basic structure of the eye chip is the same as that of the heart chip in Example 1. The difference is that the intermediate layer substrate only has one through hole. The lacrimal gland cell spheroid, the part diagram of the chip is shown in Figure 10.

In this eye chip, although the two kinds of cells are cultured separately, the culture medium is connected, and the two kinds of cells can still communicate with each other. When the topical drug is added to the conjunctival cell layer, the drug will interact with the conjunctival cells or lacrimal gland cell spheroids. After the action is completed, it can be taken out for subsequent proteomic and genomic testing, so as to analyze the toxicity and efficacy of the drug in a deeper level.

Example 14: A reusable nose chip based on a hydrophobic surface

The olfactory system is the sensory system used for smell, and most mammals and reptiles have a primary olfactory system and a secondary olfactory system. The peripheral olfactory system is mainly composed of the nostrils, ethmoid, nasal cavity and olfactory epithelium. The main components of the epithelial tissue layer are the mucosa, olfactory glands, olfactory neurons, and olfactory nerve fibers. The nose chip can imitate the olfactory system in vitro, the interaction between odor molecules and the cells expressing the olfactory system, and the generated odor molecules can be monitored in real time through fluorescent signals.

The basic structure of the nose chip is shown in Figure 2. Human skin epithelial cells are cultured on the upper surface of the third porous membrane, and hOR cells expressing the olfactory system are cultured on the lower surface of the third porous membrane. Or the air of odor molecules, the cell culture fluid circulates in the fluid channel of the second lower substrate to provide nutrients for the cells on the third porous membrane. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the nose chip can be used repeatedly for many times. The part diagram of the chip is shown in Figure 12.

In the nose chip, although the two kinds of cells are cultured separately, the culture medium is connected, and the two kinds of cells can still communicate with each other. When gaseous drugs are added to the upper channel, the drugs will interact with the cells of the olfactory system. After the action is completed, they can be taken out for subsequent proteome and genome analysis, so as to analyze the toxicity and efficacy of the drugs in a deeper level.

Example 15: A reusable bone chip based on a hydrophobic surface

Bone is a rigid organ that supports and protects various organs in the body, produces red and white blood cells, stores minerals, and provides structural support to the body. Bone tissue is composed of different types of osteocytes, including inactive osteoblasts and osteoclasts involved in bone tissue resorption. There are also hematopoietic stem cells in the bone marrow. The construction of bone-related organ models in vitro is crucial for the study of skeletal muscle dynamics, osteocyte growth and differentiation, the physiological mechanism of intercellular communication, the study of bone-related pathological mechanisms, and the evaluation of drug activity.

The basic structure of the bone chip is the same as that of the heart chip in Example 1. The difference is that there is only one through hole in the middle-layer substrate. Bone slices, osteoblasts and osteoclasts are cultured on the bone slices, vascular endothelial cells and macrophages are cultured on the upper surface of the first porous membrane of the chip, and mesenchymal stem cells are cultured in the chamber a. Since the chip adopts the hydrophobic/superhydrophobic surface technology of the present invention, the bone chip can be used repeatedly for many times. The parts diagram of the bone chip is shown in Figure 10.

The bone chip simulates the physiological balance of osteogenesis and osteoclast. If an exogenous drug is added, the effect of the drug on the bone can be judged by the erosion or recovery of the bone chip.

Example 16: A reusable multi-organ chip based on single-organ chip combination

The tissues and organs in the human body do not exist in isolation, they are actually in a highly integrated dynamic interactive environment. In this environment, the tissues or organs are connected by circulation such as blood, nerves and lymph. Behavior affects other tissues or organs, and they restrict and complement each other to form an organic whole, a system. Microfluidic chips have the characteristics of flexible combination of various unit operations under fluid-driven conditions, overall controllability and large-scale integration, making multi-organ chips a higher-level target at the organ-on-chip system level. Through the series and parallel connection of different organ chips, the realization of organ-organ interaction is crucial for in vitro physiopathological research and drug activity and toxicity evaluation.

As shown in Figure 14, the chip is composed of a separate intestine chip, liver chip, heart chip, tumor chip, brain chip and kidney chip, wherein the intestine chip and liver chip are connected in sequence, and the fluid outlet 103 of the previous single organ chip is connected to The fluid inlet 102 of the latter single organ chip and the fluid outlet 103 of the liver chip are connected to the inlets of the reservoir and the peristaltic pump 110, and the outlets of the reservoir and the peristaltic pump 110 are respectively connected to the fluid inlets of the heart chip, the tumor chip, and the brain chip. 102, the fluid outlet 103 of the heart chip, the tumor chip, and the brain chip are all connected to another reservoir and the inlet of the peristaltic pump 110, the outlet of the reservoir and the peristaltic pump 110 is connected to the kidney chip, and the fluid outlet 103 of the kidney chip is connected to The fluid inlet 102 of the intestinal chip forms a circuit. The intestinal chip is designed according to the structure of FIG. 2 , the liver chip is designed according to the structure of FIG. 1 , the difference is that the number of through holes is 1, the heart chip is the heart chip in Example 1, and the tumor chip is Example 3 The tumor chip in , the brain chip is designed according to the structure of FIG. 2 , and the kidney chip is designed according to the structure of the kidney chip in Example 7, the difference is that there is a metabolic outlet 105 on the kidney chip. Each single organ chip has a porous membrane loaded with vascular endothelial cells 106 . The chips are connected to each other by pipelines, and each chip contains simulated blood vessels, which are connected with each other to form a circuit. The circuit is provided with a peristaltic pump 101 to simulate real blood circulation. In addition, some organ chips include a separate nutrient supply system, and a peristaltic pump 101 is also set separately in these single organ chips. These single-organ chips all use the hydrophobic surface technology of the present invention and can be reused. Drugs are added from the intestinal chip, absorbed by intestinal cells, then enter the liver chip, metabolized, metabolites and the original drug enter the heart chip, and then distributed in the brain chip and tumor chip, resulting in drug efficacy and toxicity, and finally enter the kidney chip and be excreted , thus completing the entire simulated ADME process.

The multi-organ chip includes the main organs involved in the ADME process of the drug, and can simulate the ADME process of the drug in vitro, thereby realizing the prediction of the pharmacokinetic properties of the drug.

Example 17: A reusable multi-organ chip based on a single chip

As shown in Figure 15, all the simulated organs are integrated in one chip, including the liver module 2000, the heart module 3000 and the tumor module 4000 arranged in sequence. The three modules are designed according to the structure shown in Figure 1. The modules have a common blood circulation system, and each module has its own small circulation system. The chip can study the interaction of liver, heart and tumor during drug action.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the technical principles of the present invention. , these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

A microfluidic organ chip, comprising a substrate, characterized in that: the substrate has a functionalized surface, the critical surface tension of the functionalized surface is between 14-25 dynes/cm, and the contact angle with water is between 110-180 degrees; the material of the substrate is rigid plastic, elastic plastic, glass, quartz, silicon, ceramic or metal; the material for constructing the functionalized surface is polyhexafluoropropylene, polytetrafluoroethylene, polytetrafluoroethylene Fluoroethylene propylene, polytrifluoroethylene, polyvinylidene fluoride, superhydrophobic coatings, silanes, metals, metal oxides, metal inorganic salts, ceramics, waxes, oils or materials with surface micro-nano structures.
The microfluidic organ chip according to claim 1, wherein the substrate is at least two layers, a porous membrane is provided between two adjacent substrates, and the porous membrane is in close contact with the functionalized surface.
The microfluidic organ chip according to claim 2, wherein the porous membrane has a plurality of micropores, and the pore diameter of the micropores is less than 10 μm.
The microfluidic organ chip according to claim 1, wherein the microfluidic organ chip is cultured in one or more of cells, cell spheroids, tissues and organoids.
The microfluidic organ chip according to any one of claims 1 to 4, characterized in that it comprises a first upper substrate, a first porous membrane, a first middle substrate, and a second porous membrane which are arranged in close contact in sequence. and a first lower-layer substrate, the lower surface of the first upper-layer substrate, the upper and lower surfaces of the first middle-layer substrate, and the upper surface of the first lower-layer substrate are provided with the functionalized surface, the The first upper-layer substrate and the first lower-layer substrate are respectively provided with fluid channels, the first middle-layer substrate is provided with a through hole or a plurality of through holes, and the first porous membrane and the second porous membrane cover at least a part of the The fluid channel covers all the through holes, the fluid channels and through holes of the first upper substrate are in fluid communication through the first porous membrane, and the fluid channels and through holes of the first lower substrate pass through all the through holes. The second porous membranes are in fluid communication with each other, and the through holes, the first porous membrane and the second porous membrane constitute a cell culture chamber.
The microfluidic organ chip according to any one of claims 1 to 4, characterized in that it comprises a second upper layer substrate, a third porous membrane and a second lower layer substrate which are arranged in close contact in sequence, and the second upper layer substrate The lower surface of the substrate and the upper surface of the second lower substrate are provided with the functionalized surface, the second upper substrate and the second lower substrate are respectively provided with fluid channels, and the third porous membrane connects the second upper substrate. The fluid channels on the substrate and the second lower substrate are completely separated, and the upper and lower surfaces of the third porous membrane are respectively used as cell culture chambers.
A kidney chip, characterized in that the kidney chip includes a third upper substrate and a third lower substrate, and a fourth porous membrane and a fifth Porous membrane, the lower surface of the third upper substrate and the upper surface of the third lower substrate are provided with functionalized surfaces, the critical surface tension of the functionalized surfaces is between 14-25 dynes/cm, and The contact angle of water is between 110-180 degrees, the third upper substrate and the third lower substrate are respectively provided with fluid channels, and the fourth porous membrane and the fifth porous membrane connect the third upper substrate and the third lower substrate The fluid channels on the substrate are completely separated, the upper surface of the fourth porous membrane is used for culturing glomerular vascular endothelial cells, the lower surface of the fourth porous membrane is used for culturing renal podocytes, and the fifth The upper surface of the porous membrane is used for culturing peritubular vascular endothelial cells and/or pericytes, and the lower surface of the fifth porous membrane is used for culturing renal tubular epithelial cells.
A multi-organ combination chip, characterized in that: the multi-organ combination chip includes at least two microfluidic organ chips according to any one of claims 1-5, and each of the microfluidic organ chips is shared by the microfluidic organ chips the same substrate.
A multi-organ combination chip, characterized in that: the multi-organ combination chip is formed by coupling at least two single-organ chips through a fluid pipeline, and at least one single-organ chip is any one of claims 1-5 In the microfluidic organ chip, each single organ chip is provided with at least one fluid inlet and one fluid outlet, and along the fluid flow direction of the fluid pipeline, one fluid outlet of the former single organ chip is connected to the latter single organ. A fluid inlet of the chip, the last single organ chip is a kidney chip, and a fluid outlet of the kidney chip is connected to the fluid inlet of the first single organ chip to form a circuit, and at least one peristaltic pump is arranged in the circuit to drive The fluid circulates in the circuit, and the kidney chip is further provided with a metabolic outlet, and the metabolic outlet is used for the excretion of metabolites in the multi-organ combination chip.