Organ chip device capable of simulating physiological microenvironment in joint and application thereof

Abstract

The invention discloses an organ chip capable of simulating a physiological microenvironment in a joint and application of the organ chip. The organ chip comprises a cell culture layer 1, an elastic film 2, an air pressure control layer 3, a cell culture channel 4, a cell culture fluid perfusion channel 5, a cell suspension inlet 6, a cell suspension outlet 7, a culture fluid perfusion inlet 8, a culture fluid perfusion outlet 9, a through hole 10, a through hole 11, a first air pressure groove 12, a second air pressure groove 13, a pneumatic chamber 14, a pneumatic chamber 15, a cell bearing platform 16 and a matrix 17. The organ chip can simulate physical factors such as dynamic compression and relative sliding in joint movement in vitro, and the physical factors are combined with chemical stimulation factors.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an organ chip device capable of simulating the physiological microenvironment in a joint and its application. Background Art

[0002] An organ chip is a miniature cell culture device used to simulate the functional units of human organs in vitro. The device can simulate the physiological activities of organisms and their physiological microenvironment in vitro. Compared with traditional cell culture technology, this technology can more realistically simulate the real state in the body and is easy to observe and characterize.

[0003] Osteoarthritis is the most common intra-articular disease and one of the chronic degenerative diseases. The most typical clinical manifestations are usually joint stiffness, swelling, pain, and limited or lost mobility, which may seriously affect the patient's quality of life. According to statistics, osteoarthritis affects the health of more than 240 million people worldwide and has caused nearly 27 million people to become disabled, becoming a major public health issue. The occurrence of osteoarthritis is often accompanied by damage to the articular cartilage, but its clear pathogenesis is still unclear and there is a lack of effective treatment. Articular cartilage bears the main functions of the joint, providing lubrication and cushioning for joint compression and rotation movements, and can effectively transfer loads. Therefore, the physiological microenvironment of articular cartilage is crucial to studying the pathogenesis and treatment strategies of arthritis and other related diseases. There is an urgent need to develop a model that can simulate the physiological microenvironment of articular cartilage in vitro and can be precisely controlled.

[0004] At present, the models commonly used to construct tissues or organs in vivo include 2D cell culture, 3D cell culture and experimental animals. The experimental animal model has a long experimental cycle and high cost. In addition, rats, mice and rabbits, which are often used as experimental animals, have significant differences from humans in anatomy, biomechanics and genetics, such as the size, weight and walking mode (bipeds and quadrupeds) between animals and humans, which may lead to limited transformation from animal experiments to clinical experiments. 2D cell culture technology has been developed for nearly a century. So far, it still has a wide range of applications and value in the biomedical field. The technology is mature, but 2D cell culture still has certain limitations, such as the lack of tissue-specific multiple cell types and cell-to-cell interactions, and it is difficult to accurately simulate complex biological processes in the body. Based on this, 3D cell culture has gradually developed. 3D cell culture models can provide the geometric environment required for cell growth and have higher application value in studying tissue and organ functions and molecular mechanisms.

[0005] In the field of in vitro modeling of biological diseases, the development of organ chip technology will greatly contribute to the study of the pathogenesis and treatment strategies of the corresponding diseases. Organ chip technology relies on microfluidics technology, involving multiple cross-disciplinary subjects such as materials science, biomedical engineering, and mechanics. It mainly uses related technologies such as photolithography to design and prepare chip structures with different functions, so as to achieve the precise establishment of in vitro models. Therefore, organ chip technology has good prospects in constructing articular cartilage models to study the pathogenesis and drug screening of diseases such as osteoarthritis. The microenvironment of articular chondrocytes in the body mainly includes physical stimulation and chemical stimulation. Physical stimulation is mainly caused by pressure and shear force caused by its own weight and movement, and chemical stimulation is mainly protein and inflammatory cytokines. The imbalance of these factors together leads to pathological changes in articular chondrocytes. Therefore, to study diseases such as osteoarthritis caused by articular cartilage damage, it is necessary to establish a model that can effectively simulate and control the above factors.

[0006] In the prior art, there is an integrated cell-loaded microfluidic chip model that uses a concentration gradient generator and a perfusion-based three-dimensional cell culture module to evaluate the proliferation of rabbit articular chondrocytes in the presence of basic fibroblast growth factor and insulin-like growth factor-1 and their combination. This achievement is the first to use microfluidic chip technology in cartilage tissue engineering research, but it is limited to chemical stimulation such as growth factors and cannot better simulate the complex dynamic microenvironment of cartilage in the body.

[0007] The prior art discloses a chip model that simulates super-physiological compression of articular cartilage and induces osteoarthritis phenotype. Different from the traditional cartilage organ chip that focuses on chemical stimulation factors, this cartilage organ chip model focuses on mechanical biological effects, induces chondrocytes to present osteoarthritis phenotypes through mechanical compression, and better simulates the physiological conditions in vivo. However, its limitation lies in its limited ability to simulate other chemical stimulation factors that may cause osteoarthritis phenotypes (such as signal exchange with other cells, metabolism, inflammatory factors, etc.).

[0008] Therefore, there is an urgent need to provide an organ chip that can simulate physical factors such as dynamic compression and relative sliding in joint movement in vitro and combine them with chemical stimulation factors. Summary of the invention

[0009] In view of the deficiencies of the prior art and actual needs, the present invention provides an organ chip capable of simulating the physiological microenvironment within a joint and its application. The purpose is to use microfluidic technology to construct an organ chip model that simulates the physiological microenvironment of articular cartilage. The model can simulate the biomechanical environment with dynamic compression and relative sliding of the cartilage within the joint and the biochemical environment composed of chemical factors such as cytokines, thereby revealing the disease mechanism of osteoarthritis, realizing efficient screening of new drugs and exploration of effective treatment strategies.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides an organ chip, the organ chip comprising: a cell culture layer 1, an elastic film 2 and an air pressure control layer 3, wherein the elastic film 2 is bonded between the cell culture layer 1 and the air pressure control layer 3 after being treated with plasma;

[0012] The cell culture layer 1 is provided with a cell culture channel 4 and a cell culture fluid perfusion channel 5 located on both sides of the cell culture channel, the cell culture channel 4 and the cell culture fluid perfusion channel 5 are separated by a plurality of microstructures, and there are gaps between the microstructures;

[0013] The two ends of the cell culture channel 4 are provided with a cell suspension inlet 6 and a cell suspension outlet 7, and the two ends of the cell culture fluid perfusion channel 5 are provided with a culture fluid perfusion inlet 8 and a culture fluid perfusion outlet 9;

[0014] The cell culture layer 1 is provided with a through hole 10 and a through hole 11 corresponding to the air pressure control layer;

[0015] The air pressure control layer 3 is provided with a first air pressure groove 12 and a second air pressure groove 13;

[0016] The first air pressure groove 12 and the second air pressure groove 13 correspond to the cell culture channel 4. The first air pressure groove 12 is provided with a pneumatic chamber 14, and the second air pressure groove 13 is provided with a pneumatic chamber 15.

[0017] The pneumatic chamber 14 corresponds to the through hole 10 of the cell culture layer 1, and the pneumatic chamber 15 corresponds to the through hole 11 of the cell culture layer 1;

[0018] The platform formed between the first air pressure groove 12 and the second air pressure groove 13 is a cell supporting platform 16 , and a lubricant is filled between the cell supporting platform 16 and the elastic film 2 .

[0019] The present invention designs and constructs an articular cartilage organ chip that can simulate dynamic compression and relative sliding within a joint in vitro, and stimulates it with biochemical factors, thereby achieving accurate simulation of the physiological microenvironment of the joint. With the help of the articular cartilage organ chip, the pathogenic mechanism of osteoarthritis and other related diseases can be revealed, new therapeutic drugs can be efficiently and accurately screened, and it is helpful to explore more effective treatment strategies.

[0020] Preferably, the cell culture channel 4 has a length of 10-14 mm (eg, 11 mm, 12 mm, 13 mm), a width of 3-5 mm (eg, 3 mm, 4 mm, 5 mm), and a height of 0.4-0.6 mm (eg, 0.4 mm, 0.5 mm, 0.6 mm).

[0021] Preferably, the cell culture fluid perfusion channel 5 has a length of 15-18 mm (eg, 15 mm, 16 mm, 17 mm), a width of 0.5-1 mm (eg, 0.5 mm, 0.8 mm, 1 mm), and a height of 0.4-0.6 mm (eg, 0.4 mm, 0.5 mm, 0.6 mm).

[0022] Preferably, the microstructure is cylindrical in shape, with a bottom diameter of 0.4-0.6mm (e.g. 0.4mm, 0.5mm, 0.6mm), a height of 0.2-0.3mm (e.g. 0.2mm, 0.3mm), and a gap length of 1-2mm (e.g. 1mm, 1.5mm, 2mm) between the microstructures.

[0023] Preferably, the cell culture fluid perfusion inlet 8 and the cell culture fluid perfusion outlet 9 are cylindrical in shape, with a bottom diameter of 2-5 mm (eg, 2 mm, 4 mm, 5 mm) and a height of 0.5-2 mm (eg, 0.5 mm, 1 mm, 2 mm).

[0024] Preferably, the elastic film 2 has a thickness of 40-60 μm (eg, 40 μm, 50 μm, 60 μm).

[0025] Preferably, the lengths of the first air pressure groove 12 and the second air pressure groove 13 are independently 14-16 mm (e.g. 14 mm, 15 mm, 16 mm), the widths are independently 0.8-1.2 mm (e.g. 0.8 mm, 1.0 mm, 1.2 mm), and the depths are independently 0.4-0.6 mm (e.g. 0.4 mm, 0.5 mm, 0.6 mm).

[0026] Preferably, the pneumatic chambers 14 and 15 are in the shape of cylinders, with a bottom diameter of 1.8-2.2 mm (eg, 1.8 mm, 2.0 mm, 2.2 mm) and a height of 0.8-1.2 mm (eg, 0.8 mm, 1.0 mm, 1.2 mm).

[0027] Preferably, the cell supporting platform 16 has a length of 14-16 mm (eg, 14 mm, 15 mm, 16 mm) and a width of 1-3 mm (eg, 1 mm, 2 mm, 3 mm), and is filled with a lubricant between the cell supporting platform 16 and the elastic film 2 .

[0028] Preferably, the cell culture layer 1 , the elastic film 2 and the air pressure control layer 3 are all made of polydimethylsiloxane.

[0029] Preferably, the cell culture channel 4 contains a matrix 17, and the matrix includes a cell suspension or methacryloyl gelatin loaded with cells.

[0030] In a second aspect, the present invention provides a method for constructing a differentiated mechanical microenvironment model of chondrocytes cultured in three dimensions using the organ chip described in the first aspect, the method comprising the following steps:

[0031] The cell culture channel 4 and the cell culture fluid perfusion channel 5 are rinsed with ethanol, distilled water and buffer in sequence, fibronectin is injected into the cell culture channel 4 for incubation, and the channel is sealed after the incubation is completed. After sealing, the primary cells or cell line suspension of rat articular chondrocytes are injected into the cell culture channel 4 from the cell suspension inlet 6 until the cells can completely cover the bottom of the channel, and the channel is placed in an incubator for culture. Different positive or negative pressures are introduced into the pneumatic chamber 14 and the pneumatic chamber 15 to stimulate the chondrocytes with different levels of pressure and tensile force.

[0032] Preferably, the concentration of fibronectin is 10-30 μg / mL (eg, 10 μg / mL, 20 μg / mL, 30 μg / mL).

[0033] Preferably, the incubation temperature is 35-38°C (eg, 35°C, 37°C, 38°C), and the incubation time is 1-3h (eg, 1h, 2h, 3h).

[0034] Preferably, the temperature of the incubator is 35-38°C (e.g., 35°C, 37°C, 38°C), CO 2 The concentration is 3%-8% (eg 4%, 6%, 8%).

[0035] Preferably, the pressure of the pneumatic chamber 14 is (-300)-300 mbar;

[0036] Preferably, the pressure of the pneumatic chamber 15 is (-300)-300 mbar;

[0037] Preferably, the pressure difference between the pneumatic chamber 14 and the pneumatic chamber 15 is 0-300 mbar.

[0038] In a third aspect, the present invention provides an application of the organ chip described in the first aspect in simulating dynamic compression and relative sliding of joint motion.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention designs and constructs an articular cartilage organ chip that can simulate dynamic compression and relative sliding in a joint in vitro, and simultaneously stimulates it with biochemical factors, thereby achieving accurate simulation of the physiological microenvironment of the joint. With the help of this articular cartilage organ chip, the pathogenic mechanism of osteoarthritis and other related diseases can be revealed, new therapeutic drugs can be efficiently and accurately screened, and it is helpful to explore more effective treatment strategies;

[0041] (2) The present invention can be used to simulate the physiological microenvironment of articular cartilage in vitro and study the pathogenic mechanism of osteoarthritis and other related diseases. It can also be used for the efficient and accurate screening of new drugs and help explore more effective osteoarthritis treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the organ chip of the present invention;

[0043] Figure 2 is a cross-sectional schematic diagram of the organ chip of the present invention in a static state;

[0044] Figure 3 is a cross-sectional schematic diagram of the organ chip of the present invention in a stretched state;

[0045] Figure 4 It is a cross-sectional schematic diagram of the organ chip of the present invention in a compressed state. DETAILED DESCRIPTION

[0046] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Preparation Example 1

[0049] This preparation example provides an organ chip.

[0050] A polymethyl methacrylate positive mold template is prepared by mechanical processing, and a polydimethylsiloxane part including a cell culture layer 1 and an air pressure control layer 3 is prepared by soft etching technology.

[0051] Among them, the cell culture channel 4 of the cell culture layer 1 is 12.00mm long, 3.50mm wide and 0.50mm high; the cell culture fluid perfusion channel 5 is 16.00mm long, 0.75mm wide and 0.50mm high; the cell culture channel 4 and the cell culture fluid perfusion channel 5 are separated by a plurality of microstructures, the microstructures are cylindrical, with a bottom diameter of 0.50mm and a height of 0.25mm; there is a gap between each microstructure, and the gap is 1.50mm long; a cell suspension inlet 6 and a cell suspension outlet 7 are provided at both ends of the cell culture channel 4, and a culture fluid perfusion inlet 8 and a culture fluid perfusion outlet 9 are provided at both ends of the cell culture fluid perfusion channel 5, the cell suspension inlet 6, outlet 7 and the cell culture fluid perfusion inlet 8, outlet 9 are cylindrical, with a bottom diameter of 4.00mm and a height of 1.00mm; the cell culture layer 1 is provided with through holes 10 and 11 corresponding to the air pressure control layer.

[0052] The elastic film 2 has a thickness of 50 μm.

[0053] The air pressure control layer 3 is provided with a first air pressure groove 12 and a second air pressure groove 13, the two air pressure grooves 12 and 13 are 15.00 mm long, 1.00 mm wide and 0.50 mm deep, and each of the two air pressure grooves is provided with a pneumatic chamber 14, 15, the pneumatic chamber 14 and the pneumatic chamber 15 are cylindrical, with a bottom diameter of 2.00 mm and a height of 1.00 mm; the two pneumatic chambers 14, 15 of the air pressure control layer 3 correspond to the two through holes 10, 11 of the cell culture layer 1 respectively; the platform formed in the middle of the two air pressure grooves is a cell bearing platform 16, the cell bearing platform 16 is 15.00 mm long and 2.00 mm wide; a lubricant is filled between the cell bearing platform 16 and the elastic film 2; the two air pressure grooves 12, 13 and the pneumatic chambers 14, 15 are each connected by a gas channel, the gas channel is 13.00 mm long, 1.00 mm wide and 0.50 mm high.

[0054] The cell culture layer 1 and the air pressure control layer 3 are made by pouring polydimethylsiloxane into a polymethyl methacrylate positive mold template. The polydimethylsiloxane main agent and the curing agent polymethyl methacrylate are mixed in a mass ratio of 10:1, placed in a vacuum drying oven and evacuated for 10 minutes, then poured into the template, and then evacuated for 30 minutes, and demolded after curing at 80°C for 2 hours.

[0055] According to the attached Figure 1 The cell culture layer 1, the elastic film 2 and the air pressure control layer 3 are bonded after being treated by an oxygen plasma cleaning machine in the following order to form an organ chip model. The cell culture channel 4 is aligned with the cell supporting platform 16, with a layer of elastic film 2 sandwiched between them; the through hole 10 of the cell culture layer 1 is connected to the pneumatic chamber 14 of the air pressure control layer 3, the through hole 11 of the cell culture layer 1 is connected to the pneumatic chamber 15 of the air pressure control layer 3, and the elastic PDMS film in the middle is opened.

[0056] The schematic diagram of the organ chip prepared by the present invention is as follows Figure 1 shown.

[0057] The cross-sectional schematic diagram of the organ chip of the present invention in a static state is as follows Figure 2 As shown, an elastic film 2 is bonded between the cell culture layer 1 and the air pressure control layer 3, the air pressure control layer 3 includes an air pressure groove 12 and an air pressure groove 13, a matrix 17 is passed into the cell culture layer 1, and the matrix loads primary chondrocytes 18, and the matrix includes a cell suspension or methacryloyl gelatin loaded with cells.

[0058] The cross-sectional schematic diagram of the organ chip of the present invention in a stretched state is as follows: Figure 3 As shown, negative pressure (-300)-0 mbar is introduced into the air pressure groove 12, and negative pressure (-300)-0 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force exerted on the articular cartilage during movement.

[0059] The cross-sectional schematic diagram of the organ chip of the present invention in a compressed state is as follows Figure 4 As shown, a positive pressure of 0-300 mbar is introduced into the air pressure groove 12, and a positive pressure of 0-300 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force on the articular cartilage during movement.

[0060] Preparation Example 2

[0061] This preparation example provides an organ chip.

[0062] A polymethyl methacrylate positive mold template is prepared by mechanical processing, and a polydimethylsiloxane part including a cell culture layer 1 and an air pressure control layer 3 is prepared by soft etching technology.

[0063] Among them, the cell culture channel 4 of the cell culture layer 1 is 10.00mm long, 3.0mm wide and 0.40mm high; the cell culture fluid perfusion channel 5 is 15.00mm long, 0.5mm wide and 0.40mm high; the cell culture channel 4 and the cell culture fluid perfusion channel 5 are separated by a plurality of microstructures, the microstructures are cylindrical, with a bottom diameter of 0.40mm and a height of 0.2mm; there is a gap between each microstructure, and the gap is 1mm long; a cell suspension inlet 6 and a cell suspension outlet 7 are provided at both ends of the cell culture channel 4, and a culture fluid perfusion inlet 8 and a culture fluid perfusion outlet 9 are provided at both ends of the cell culture fluid perfusion channel 5, the cell suspension inlet 6, outlet 7 and the cell culture fluid perfusion inlet 8, outlet 9 are cylindrical, with a bottom diameter of 2.00mm and a height of 0.5mm; the cell culture layer 1 is provided with through holes 10 and 11 corresponding to the air pressure control layer.

[0064] The elastic film 2 has a thickness of 40 μm.

[0065] The air pressure control layer 3 is provided with a first air pressure groove 12 and a second air pressure groove 13, the two air pressure grooves 12 and 13 are 14.00 mm long, 0.8 mm wide and 0.40 mm deep, and each of the two air pressure grooves is provided with a pneumatic chamber 14, 15, the pneumatic chamber 14 and the pneumatic chamber 15 are cylindrical, with a bottom diameter of 1.8 mm and a height of 0.8 mm; the two pneumatic chambers 14, 15 of the air pressure control layer 3 correspond to the two through holes 10, 11 of the cell culture layer 1 respectively; the platform formed in the middle of the two air pressure grooves is a cell bearing platform 16, the cell bearing platform 16 is 14.00 mm long and 1.00 mm wide; a lubricant is filled between the cell bearing platform 16 and the elastic film 2; the two air pressure grooves 12, 13 and the pneumatic chambers 14, 15 are each connected by a gas channel, the gas channel is 13.00 mm long, 1.00 mm wide and 0.50 mm high.

[0066] The cell culture layer 1 and the air pressure control layer 3 are made by pouring polydimethylsiloxane into a polymethyl methacrylate positive mold template. The polydimethylsiloxane main agent and the curing agent polymethyl methacrylate are mixed in a mass ratio of 10:1, placed in a vacuum drying oven and evacuated for 10 minutes, then poured into the template, and then evacuated for 30 minutes, and demolded after curing at 80°C for 2 hours.

[0067] According to the attached Figure 1The cell culture layer 1, the elastic film 2 and the air pressure control layer 3 are bonded after being treated by an oxygen plasma cleaning machine in the order of forming an organ chip model. The cell culture channel 4 is aligned with the cell supporting platform 16, with a layer of elastic film 2 sandwiched in between; the through hole 10 of the cell culture layer 1 is connected to the pneumatic chamber 14 of the air pressure control layer 3, the through hole 11 of the cell culture layer 1 is connected to the pneumatic chamber 15 of the air pressure control layer 3, and the elastic polydimethylsiloxane film in the middle is opened.

[0068] The schematic diagram of the organ chip prepared by the present invention is as follows Figure 1 shown.

[0069] The cross-sectional schematic diagram of the organ chip of the present invention in a static state is as follows Figure 2 As shown, an elastic film 2 is bonded between the cell culture layer 1 and the air pressure control layer 3, the air pressure control layer 3 includes an air pressure groove 12 and an air pressure groove 13, a matrix 17 is passed into the cell culture layer 1, and the matrix loads primary chondrocytes 18, and the matrix includes a cell suspension or methacryloyl gelatin loaded with cells.

[0070] The cross-sectional schematic diagram of the organ chip of the present invention in a stretched state is as follows: Figure 3 As shown, negative pressure (-300)-0 mbar is introduced into the air pressure groove 12, and negative pressure (-300)-0 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force exerted on the articular cartilage during movement.

[0071] The cross-sectional schematic diagram of the organ chip of the present invention in a compressed state is as follows Figure 4 As shown, a positive pressure of 0-300 mbar is introduced into the air pressure groove 12, and a positive pressure of 0-300 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force on the articular cartilage during movement.

[0072] Preparation Example 3

[0073] This preparation example provides an organ chip.

[0074] A polymethyl methacrylate positive mold template is prepared by mechanical processing, and a polydimethylsiloxane part including a cell culture layer 1 and an air pressure control layer 3 is prepared by soft etching technology.

[0075] Among them, the cell culture channel 4 of the cell culture layer 1 is 14.00 mm long, 5 mm wide and 0.60 mm high; the cell culture fluid perfusion channel 5 is 18.00 mm long, 1 mm wide and 0.60 mm high; the cell culture channel 4 and the cell culture fluid perfusion channel 5 are separated by multiple microstructures, and the microstructure is a cylinder with a bottom diameter of 0.60 mm and a height of 0.3 mm; there is a gap between each microstructure, and the gap is 2 mm long; a cell suspension inlet 6 and a cell suspension outlet 7 are provided at both ends of the cell culture channel 4, and a culture fluid perfusion inlet 8 and a culture fluid perfusion outlet 9 are provided at both ends of the cell culture fluid perfusion channel 5, and the cell suspension inlet 6, outlet 7 and the cell culture fluid perfusion inlet 8, outlet 9 are cylindrical with a bottom diameter of 5.00 mm and a height of 2.00 mm; the cell culture layer 1 is provided with through holes 10 and 11 corresponding to the air pressure control layer.

[0076] The elastic film 2 has a thickness of 60 μm.

[0077] The air pressure control layer 3 is provided with a first air pressure groove 12 and a second air pressure groove 13, the two air pressure grooves 12 and 13 are 16.00 mm long, 1.2 mm wide and 0.60 mm deep, and each of the two air pressure grooves is provided with a pneumatic chamber 14, 15, the pneumatic chamber 14 and the pneumatic chamber 15 are cylindrical, with a bottom diameter of 2.2 mm and a height of 1.2 mm; the two pneumatic chambers 14, 15 of the air pressure control layer 3 correspond to the two through holes 10, 11 of the cell culture layer 1 respectively; the platform formed in the middle of the two air pressure grooves is a cell bearing platform 16, the cell bearing platform 16 is 16.00 mm long and 3.00 mm wide; a lubricant is filled between the cell bearing platform 16 and the elastic film 2; the two air pressure grooves 12, 13 and the pneumatic chambers 14, 15 are each connected by a gas channel, the gas channel is 13.00 mm long, 1.00 mm wide and 0.50 mm high.

[0078] The cell culture layer 1 and the air pressure control layer 3 are made by pouring polydimethylsiloxane into a polymethyl methacrylate positive mold template. The polydimethylsiloxane main agent and the curing agent polymethyl methacrylate are mixed in a mass ratio of 10:1, placed in a vacuum drying oven and evacuated for 10 minutes, then poured into the template, and then evacuated for 30 minutes, and demolded after curing at 80°C for 2 hours.

[0079] According to the attached Figure 1The cell culture layer 1, the elastic film 2 and the air pressure control layer 3 are bonded after being treated by an oxygen plasma cleaning machine in the order of forming an organ chip model. The cell culture channel 4 is aligned with the cell supporting platform 16, with a layer of elastic film 2 sandwiched in between; the through hole 10 of the cell culture layer 1 is connected to the pneumatic chamber 14 of the air pressure control layer 3, the through hole 11 of the cell culture layer 1 is connected to the pneumatic chamber 15 of the air pressure control layer 3, and the elastic polydimethylsiloxane film in the middle is opened.

[0080] The schematic diagram of the organ chip prepared by the present invention is as follows Figure 1 As shown,.

[0081] The cross-sectional schematic diagram of the organ chip of the present invention in a static state is as follows Figure 2 As shown, an elastic film 2 is bonded between the cell culture layer 1 and the air pressure control layer 3, the air pressure control layer 3 includes an air pressure groove 12 and an air pressure groove 13, a matrix 17 is passed into the cell culture layer 1, and the matrix loads primary chondrocytes 18, and the matrix includes a cell suspension or methacryloyl gelatin loaded with cells.

[0082] The cross-sectional schematic diagram of the organ chip of the present invention in a stretched state is as follows: Figure 3 As shown, negative pressure (-300)-0 mbar is introduced into the air pressure groove 12, and negative pressure (-300)-0 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force exerted on the articular cartilage during movement.

[0083] The cross-sectional schematic diagram of the organ chip of the present invention in a compressed state is as follows Figure 4 As shown, a positive pressure of 0-300 mbar is introduced into the air pressure groove 12, and a positive pressure of 0-300 mbar is introduced into the air pressure groove 13, so that the polydimethylsiloxane elastic film 2 is deformed, causing mechanical force stimulation to the chondrocytes 18, thereby simulating the pressure and tensile force on the articular cartilage during movement.

[0084] Example 1

[0085] In this embodiment, the cell culture channel 4 and the cell culture fluid perfusion channel 5 were rinsed with 75% medical alcohol, distilled water, and PBS in sequence, and then 20 μg / mL fibronectin was injected into the cell culture fluid channel and incubated at 37° C. for 2 h. After the incubation, the cell culture fluid channel was blocked with a 3% bovine serum albumin PBS solution.

[0086] Primary cells of rat articular chondrocytes were obtained and added with DMEM / F12 medium to an appropriate cell density (about 2×10 6The cell suspension was injected into the cell culture channel 4 from the cell suspension inlet 6 until the cells could completely cover the bottom of the channel. The cell suspension was placed in a 37°C, 5% CO 2 Culture in an incubator to allow cells to adhere.

[0087] By using the organ chip of the present invention, a positive pressure of 300 mbar is introduced into the pneumatic chamber 14 and a positive pressure of 300 mbar is introduced into the pneumatic chamber 15, so that the polydimethylsiloxane elastic film is deformed, causing mechanical force stimulation to the chondrocytes, thereby simulating the pressure and tensile force on the articular cartilage during movement.

[0088] Example 2

[0089] This embodiment simulates the pressure and tensile force test on the articular cartilage during movement.

[0090] In this embodiment, the cell culture channel 4 and the cell culture fluid perfusion channel 5 were rinsed with 75% medical alcohol, distilled water, and PBS in sequence, and then 20 μg / mL fibronectin was injected into the cell culture fluid channel and incubated at 37° C. for 2 h. After the incubation, the cell culture fluid channel was blocked with a 3% bovine serum albumin PBS solution.

[0091] Take rat articular chondrocyte cell line and add DMEM / F12 medium to the appropriate cell density (about 2×10 6 The precursor solution was slowly injected into the cell culture channel 4 from the cell suspension inlet 6, taking care not to break through the microstructure and flow into the cell culture fluid perfusion channel 5, and then UV-cured for 30 seconds. DMEM / F12 culture medium was injected from the culture fluid perfusion inlet 8 to provide sufficient nutrition for cell growth and prevent the hydrogel from losing water and drying up.

[0092] By using the organ chip of the present invention, negative pressure (-300)-0 mbar is introduced into the pneumatic chamber 14, and negative pressure (-300)-0 mbar is introduced into the pneumatic chamber 15, so that the polydimethylsiloxane elastic film is deformed, causing mechanical force stimulation to the chondrocytes, thereby simulating the pressure and tensile force exerted on the articular cartilage during movement.

[0093] In summary, the organ chip of the present invention can simulate physical factors such as dynamic compression and relative sliding in joint movement in vitro, and combine them with chemical stimulation factors.

[0094] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An organ chip, characterized in that: The organ chip comprises: a cell culture layer (1), an elastic film (2) and an air pressure control layer (3); the elastic film (2) is bonded between the cell culture layer (1) and the air pressure control layer (3) after being subjected to plasma treatment; The cell culture layer (1) is provided with a cell culture channel (4) and cell culture fluid perfusion channels (5) located on both sides of the cell culture channel, the cell culture channel (4) and the cell culture fluid perfusion channel (5) are separated by a plurality of microstructures, and there are gaps between the microstructures; The cell culture channel (4) is provided with a cell suspension inlet (6) and a cell suspension outlet (7) at both ends, and the cell culture fluid perfusion channel (5) is provided with a culture fluid perfusion inlet (8) and a culture fluid perfusion outlet (9) at both ends; The cell culture layer (1) is provided with a through hole (10) and a through hole (11) corresponding to the air pressure control layer; The air pressure control layer (3) is provided with a first air pressure groove (12) and a second air pressure groove (13); The first air pressure groove (12) and the second air pressure groove (13) correspond to the cell culture channel (4); the first air pressure groove (12) is provided with a pneumatic chamber (14), and the second air pressure groove (13) is provided with a pneumatic chamber (15); The pneumatic chamber (14) corresponds to the through hole (10) of the cell culture layer (1), and the pneumatic chamber (15) corresponds to the through hole (11) of the cell culture layer (1); The platform formed between the first air pressure groove (12) and the second air pressure groove (13) is a cell supporting platform (16), and a lubricant is filled between the cell supporting platform (16) and the elastic film (2).

2. The organ chip according to claim 1, characterized in that: The cell culture channel (4) has a length of 10-14 mm, a width of 3-5 mm, and a height of 0.4-0.6 mm; Preferably, the cell culture fluid perfusion channel (5) has a length of 15-18 mm, a width of 0.5-1 mm, and a height of 0.4-0.6 mm; Preferably, the microstructure is in the shape of a cylinder, with a bottom diameter of 0.4-0.6 mm and a height of 0.2-0.3 mm, and the length of the gaps between the microstructures is 1-2 mm.

3. The organ chip according to claim 1 or 2, characterized in that: The cell culture fluid perfusion inlet (8) and the cell culture fluid perfusion outlet (9) are cylindrical in shape, with a bottom diameter of 2-5 mm and a height of 0.5-2 mm.

4. The organ chip according to any one of claims 1 to 3, characterized in that: The thickness of the elastic film (2) is 40-60 μm.

5. The organ chip according to any one of claims 1 to 4, characterized in that: The length of the first air pressure groove (12) and the second air pressure groove (13) are independently 14-16 mm, the width is independently 0.8-1.2 mm, and the depth is independently 0.4-0.6 mm.

6. The organ chip according to any one of claims 1 to 5, characterized in that: The pneumatic chamber (14) and the pneumatic chamber (15) are in the shape of a cylinder, with a bottom diameter of 1.8-2.2 mm and a height of 0.8-1.2 mm; Preferably, the cell-carrying platform (16) has a length of 14-16 mm and a width of 1-3 mm, and is filled with a lubricant between the cell-carrying platform (16) and the elastic film (2).

7. The organ chip according to any one of claims 1 to 6, characterized in that: The cell culture layer (1), the elastic film (2) and the air pressure control layer (3) are all made of polydimethylsiloxane; Preferably, the cell culture channel (4) contains a matrix (17), and the matrix includes a cell suspension or methacryloyl gelatin loaded with cells.

8. A method for constructing a differentiated mechanical microenvironment model of chondrocytes cultured in three dimensions using the organ chip according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: The cell culture channel (4) and the cell culture fluid perfusion channel (5) are rinsed with ethanol, distilled water, and buffer in sequence, and fibronectin is injected into the cell culture channel (4) for incubation. After the incubation is completed, the channel is sealed. After sealing, a cell suspension of primary cells or cell lines of rat articular chondrocytes is injected into the cell culture channel (4) from a cell suspension inlet (6) until the cells can completely cover the bottom of the channel. The channel is placed in an incubator for culture, and different positive or negative pressures are introduced into the pneumatic chamber (14) and the pneumatic chamber (15) to stimulate the chondrocytes with different levels of pressure and tensile force.

9. The microfluidic chip according to claim 8, characterized in that: The concentration of the fibronectin is 10-30 μg / mL; Preferably, the incubation temperature is 35-38°C, and the incubation time is 1-3h; Preferably, the temperature of the incubator is 35-38°C and the CO2 concentration is 3%-8%; Preferably, the pressure of the pneumatic chamber (14) is (-300)-300 mbar; Preferably, the pressure of the pneumatic chamber (15) is (-300)-300 mbar; Preferably, the pressure difference between the pneumatic chamber (14) and the pneumatic chamber (14) is 0-300 mbar.

10. Use of the organ chip according to any one of claims 1 to 7 in simulating dynamic compression and relative sliding of joint motion.

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