CN117229914A - Microfluidic chip and method for constructing force-chemical-biological coupling microenvironment model of chondrocyte by using microfluidic chip - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a microfluidic chip and a method for constructing a force-chemical-biological coupling microenvironment model of chondrocytes by using the microfluidic chip. According to the invention, the air pressure channel layer in the chip structure is provided with the plurality of parallel air channels, so that the mechanical compression of the cells of the cell three-dimensional culture layer can be accurately regulated and controlled with high flux. According to the invention, a cartilage model is constructed by biological materials such as hydrogel, corresponding cells such as macrophages are inoculated at different positions of the microfluidic chip, the real local mechanical microenvironment and the immune microenvironment where the articular cartilage is positioned are accurately simulated, the force-chemical-biological coupling microenvironment model of the three-dimensional cultured chondrocytes is successfully constructed by utilizing the microfluidic chip, the pathogenesis of the joint diseases is explored from the single cell level, and a convenient, rapid and economic drug screening platform is built, so that support is provided for the treatment of the joint diseases.

Description

Microfluidic chip and use of microfluidic chip to construct force-chemical-biological coupling of chondrocytes Microenvironment model approach

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a microfluidic chip and a method of using the microfluidic chip to construct a force-chemistry-biogenesis coupled microenvironment model of chondrocytes.

Background technique

Articular cartilage is an important part of the movement system, providing support, cushioning, lubrication and other functions during joint movements of the human body. Articular cartilage is a thin layer (about 2 to 4 mm) of avascular connective tissue on the joint surface. It is mainly composed of sparsely distributed chondrocytes and dense extracellular matrix. Chondrocytes are the only cell type in articular cartilage, accounting for only Tissue volume is less than 5% to 10%. However, due to the lack of self-healing ability of articular cartilage, it is difficult to repair itself after damage, and it is very prone to many irreversible disabling diseases, such as osteoarthritis and rheumatoid arthritis, causing the entire joint to lose its ability to move, seriously affecting people's health. Therefore, analyzing the mechanism of cartilage homeostasis maintenance is of great significance for the treatment of joint diseases such as osteoarthritis and rheumatoid arthritis.

Although there has been a large amount of research on articular cartilage, the mechanism of articular cartilage damage is still not completely clear, and there is a lack of effective treatments and preventive measures. One of the main reasons for this situation can be attributed to the location of articular cartilage. Complex force-chemistry-biogenesis coupled microenvironment. From the perspective of mechanical microenvironment, chondrocytes are in a dynamic mechanical stimulation microenvironment, including compression, shear, stretch, osmotic pressure and other different types of mechanical stimulation generated by daily joint activities. Chondrocytes sense and respond to external mechanical stimuli through the process of mechanotransduction, and subsequently initiate biochemical reactions. The biomechanical microenvironment in which chondrocytes exist affects the development, functional maintenance, and even the occurrence and development of diseases by regulating cell adhesion, migration, proliferation, and differentiation (W.Xu, J.Zhu, J.Hu, L.Xiao , Engineering the biomechanical microenvironment of chondrocytes towards articular cartilaginous issue engineering, Life Sciences, 2022 Nov; (15) 309:121043.doi:10.1016/j.lfs.2022.121043.). In addition to mechanical loading, the complex biochemical environment within the joint also has an important impact on cartilage. These biochemical factors include immune cells such as macrophages and lymphocytes, as well as cytokines secreted by these cells. These biochemical factors affect cartilage homeostasis through different pathways. For example, there are many macrophages in the synovial membrane and synovial fluid around articular cartilage. These macrophages are activated by the microenvironment in the joint and can be polarized into different cell phenotypes, that is, pro-inflammatory macrophages. phagocytes and anti-inflammatory macrophages. Among them, inflammatory factors, growth factors, and matrix metalloproteinases (MMPs) secreted by pro-inflammatory macrophages can interact with chondrocytes through paracrine effects, leading to subsequent cartilage degradation and destruction, and affecting the occurrence of diseases such as osteoarthritis. and development plays a vital role. In addition, the accumulation of pro-inflammatory macrophages in the synovium is also closely related to synovial inflammation, and the occurrence of synovial inflammation can also affect the phenotypic polarization of macrophages and the damage of chondrocytes. Similarly, cell debris, aggregated proteins, etc. produced by chondrocyte damage caused by mechanical loading can be used as danger-associated molecular patterns (DAMPs) to stimulate macrophage activation and promote their secretion of inflammatory cytokines and chemokines, etc., and induce glomerulonephritis. inflammation, etc. (H. Zhang, D. Cai, . The mutual influence and interaction between these cells under the combined action of mechanical stimulation and biochemical stimulation will form a vicious cycle in the joint cavity, inducing or even accelerating the occurrence and development of joint diseases.

Although the existing in vitro 2D cell models commonly used to study joint diseases have certain advantages, such as low cost and simple operation, they lack physiological relevance and key features. These key characteristics of the three-dimensional growth environment of articular chondrocytes greatly affect the cell phenotype, behavior and response to test drugs. For example, primary chondrocytes will gradually lose their chondrogenic characteristics in 2D culture, thereby affecting the cell's response to test drugs. response to external stimuli. Animal experiments are still an important part of elucidating disease mechanisms and drug trials in joint disease research, but there are many controversies in the construction of animal models in terms of economy and ethics. These traditional in vitro 2D and in vivo animal experiments cannot meet the needs of joint disease research. Therefore, how to construct a model that accurately simulates the physiological characteristics and microenvironment of articular cartilage is currently one of the most important issues in solving the obstacles to studying the pathogenesis of joint diseases and drug screening.

Modeling human tissue in microphysiologically relevant “chips” will increasingly help reveal the mechanisms behind disease and may ultimately speed up the development of drugs and the assessment of individual patient responses to specific drugs. Due to its unique advantages, microfluidic technology can be used as a new means to simulate the microenvironment of articular cartilage to construct microphysiological models of articular cartilage. Microfluidic technology involves many interdisciplinary subjects such as materials, biomedical engineering, micro-electromechanical, etc. It mainly uses advanced micro-nano processing methods to control tiny fluids (nL ~ μL) in the microchannels of chips prepared to achieve specific purposes. Microfluidic chips can be designed and prepared with different structures due to the flexibility of their processing methods. Therefore, by rationally designing the structure of the chip, it is possible to construct models in vitro that can perform long-term cell culture and mechanical stimulation and biochemical stimulation. Combined with three-dimensional cell culture technology and using biological materials as carriers, it simulates the spatial structure and physical and chemical properties of the extracellular matrix, approximately providing cells with a growth environment similar to that in the body, maximizing the simulation of the three-dimensional growth state of chondrocytes in the body, and establishing and The chip microenvironment is similar to the in vivo microenvironment. The devices typically use small volumes (in the microliter range) and therefore require much fewer analytical reagents and cell numbers compared to two- and three-dimensional cell culture models, effectively reducing the consumption of drugs and reagents for experimental research. Therefore, due to its miniaturization, high integration, low consumables, and flexible structure, microfluidic chips can well simulate and reproduce the microenvironment of articular cartilage cells, and are useful in constructing articular cartilage models to study pathogenesis and drug screening. Bigger prospects.

By rationally designing the structure of the microfluidic chip, the mechanical microenvironment of articular cartilage cells can be reproduced and the behavior of chondrocytes under mechanical stimulation can be studied. In addition, the complex biochemical microenvironment of articular chondrocytes can also be simulated and reproduced by rationally designing the structure of the microfluidic chip. In particular, the immune microenvironment also plays a crucial role in the occurrence and development of joint diseases such as rheumatoid arthritis and osteoarthritis. For example, Occhetta et al. (P.Occhetta, A.Mainardi, E.Votta, Q.Vallmajo-Martin, M.Ehrbar, I.Martin, A.Barbero, M.Rasponi, Hyperphysiological compression of articular cartilage induces anosteoarthriticphenotype in a cartilage -on-a-chip model, Nature BiomedicalEngineering, 2019 Jul; 3(7):545-557.doi:10.1038/s41551-019-0406-3) established a chip model to simulate physiological and supraphysiological compression, revealing the pathology Sexual mechanical stimulation promoted matrix metalloproteinase (MMP) production and cartilage destruction, and drug screening was performed. The structure of the chip in this document is shown in Figure 12 below. The chip is made of polydimethylsiloxane (PDMS) and includes two chambers separated by flexible membranes. The upper layer is a culture chamber that holds three-dimensional culture of cells, and the lower layer is is the drive chamber for mechanical loading. For the cell culture chamber, it consists of a 300 μm wide central channel, flanked by side channels for medium replenishment that are separated by microstructures. The microstructure used to space the channels is two parallel rows of suspended microarrays that provide confining boundaries while the cell-hydrogel mixture is seeded and cultured. There is a gap between these suspended microarrays and the underlying PDMS membrane (see Figure 13). When the drive chamber is pressurized, the PDMS flexible membrane bends upward until it is close to the bottom of the microarray, which affects the three-dimensional structure of the cell-hydrogel. Limited compression of the structure.

However, the microphysiological system in the above-mentioned literature precisely controls the mechanical compression level of the cell-hydrogel structure by adjusting the gap below the suspension column from the PDMS elastic mold. It can be seen that the influencing factor that determines the compression performance of the chip in this document is the gap between the PDMS elastic film under the suspension column set during chip preparation, which is the stroke length that defines the control mechanical drive mechanism. Therefore, this document One chip in can only achieve a single level of compression for cells. In addition, there is a gap between the chip culture chamber and the underlying PDMS flexible membrane in this document. When the cell-hydrogel mixture is inoculated in the middle channel of the microstructure for three-dimensional culture of cells, other types of regionalization cannot be performed. Culture, such as immune cells, synovial cells, etc., which are abundant in the joint cavity. These cells are very important when studying joint diseases. For example, the presence of inflammatory cells will aggravate the response of chondrocytes to mechanical stimulation. According to this article Chips cannot meet this need.

Contents of the invention

The object of the present invention is to provide a microfluidic chip and a method of using the microfluidic chip to construct a microenvironment model of force-chemistry-biogenesis coupling of chondrocytes. The microfluidic chip provided by the present invention has the ability to more closely simulate real tissues in the body. With the advantages of high simulation such as its three-dimensional structure and microenvironment, it can simultaneously simulate and reproduce the microenvironment of multiple factors of mechanical stimulation and biochemical stimulation when chondrocytes grow in the body.

In order to achieve the above objects, the present invention provides the following technical solutions:

The invention provides a microfluidic chip, which includes a basal layer 1 and a three-dimensional cell culture layer 2 and an air pressure control layer 3 which are sequentially stacked on the basal layer 1; the basal layer 1 and the three-dimensional cell culture layer 2 and the air pressure control layer 3 are made of transparent material;

The three-dimensional cell culture layer 2 is provided with a three-dimensional cell culture channel 21 and a first cell culture fluid channel 22 and a second cell culture fluid channel 23 located on both sides of the three-dimensional cell culture channel 21. The three-dimensional cell culture channel 21 and The first cell culture medium channels 22 and the three-dimensional cell culture channel 21 and the second cell culture medium channel 23 are separated by a plurality of microstructures. There are gaps between the microstructures. The three-dimensional cell culture channel 21 A cell fluid inlet channel 24 and a cell fluid outlet channel 25 are provided at both ends of the first cell culture fluid channel 22. A first cell culture fluid inlet channel 26 and a first cell culture fluid outlet channel are provided at both ends of the first cell culture fluid channel 22. 27. A second cell culture medium inlet channel 28 and a second cell culture medium outlet channel 29 are provided at both ends of the second cell culture medium channel 23;

The air pressure control layer 3 is provided with a plurality of gas channels perpendicular to the three-dimensional cell culture channel 21. Both ends of each of the gas channels are provided with gas inlets and outlets, and each of the gas channels is provided with all gas channels at both ends. The extruded parts between the gas inlets and outlets are located above the three-dimensional cell culture channel 21. There are three liquid through holes at both ends of the air pressure control layer, and each of the liquid through holes is connected to the cells respectively. fluid-carrying fluid inlet channel 24, cell fluid-carrying fluid outlet channel 25, first cell culture fluid inlet channel 26, first cell culture fluid outlet channel 27, second cell culture fluid inlet channel 28, and first cell culture fluid outlet channel 29 .

Preferably, it also includes a liquid storage layer stacked on the air pressure control layer 3. The liquid storage layer includes two liquid storage pools 4 arranged on both sides of the gas channel. Each is provided with three liquid storage through holes, and each liquid storage through hole is connected to each of the liquid through holes respectively.

Preferably, the three-dimensional cell culture channel 21, the first cell culture fluid channel 22 and the second cell culture fluid channel 23 are channel structures with an upper part sealing a lower opening; the microstructure is in the shape of a fence, and one end of the microstructure is connected to The basal layer 1 contacts; the cell fluid inlet channel 24, the cell fluid outlet channel 25, the first cell culture fluid inlet channel 26, the first cell culture fluid outlet channel 27, the second cell culture fluid inlet channel 28 and the first The cell culture solution outlet channel 29 is a through hole that penetrates up and down.

Preferably, the material of the three-dimensional cell culture layer 2 is polydimethylsiloxane;

The thickness of the three-dimensional cell culture layer 2 is 200-400 μm, the width of the three-dimensional cell culture channel 21 is 1-1.5 mm; the width of the first cell culture solution channel 22 and the second cell culture solution channel 23 is 1-2 mm. ; The depth of the three-dimensional cell culture channel 21, the first cell culture fluid channel 22 and the second cell culture fluid channel 23 is 70 to 300 μm.

Preferably, the gas channel is a structure in which the upper part seals the lower opening; the number of the gas channels is 3;

The material of the air pressure control layer 3 is polydimethylsiloxane; the width of the gas channel is 1 to 1.5 mm, and the depth is 50 to 200 μm. The width of the extruded part is greater than the width of the gas channel.

Preferably, the material of the base layer 1 is glass; the thickness of the base layer 1 is 0.13~0.17mm;

The material of the liquid storage layer is polydimethylsiloxane, and the diameter of the liquid storage through hole is 1.5-3.5 mm.

The present invention provides a method for constructing a differentiated mechanical microenvironment model of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution, which includes the following steps:

Inject the chondrocyte-hydrogel mixture into the three-dimensional cell culture channel 21 of the three-dimensional cell culture layer 2, and perform photocuring under ultraviolet irradiation to obtain a solidified chondrocyte-hydrogel; then inject the chondrocyte culture solution into the three-dimensional cell culture channel 21. Cartilage cells are cultured in the first cell culture fluid channel 22 and the second cell culture fluid channel 23 of the culture layer 2. During the chondrocyte culture process, different pressures are introduced into the gas channels of the air pressure control layer 3. The horizontal compressed air exerts different levels of mechanical force stimulation on the solidified chondrocytes in different areas of the cell three-dimensional culture channel 21 - the chondrocytes in the hydrogel.

The present invention provides a method for constructing a microenvironment model of force-chemical-biogenesis coupling of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution, which includes the following steps:

1) Surface-modify the surface of the basal layer 1 that is in contact with the first cell culture medium channel 22 and the second cell culture medium channel 23 of the three-dimensional cell culture layer 2 with a coating material;

2) Inject the chondrocyte-hydrogel mixture into the three-dimensional cell culture channel 21 of the three-dimensional cell culture layer 2, and perform photocuring under ultraviolet irradiation to obtain solidified chondrocyte-hydrogel;

3) Inject the composite cell culture fluid into the first cell culture fluid channel 22 and the second cell culture fluid channel 23 with the modified closed bottom surface. The composite cell culture fluid contains cells related to chondrocytes, cells related to chondrocytes. One or more of biochemical factors, chondrocyte-related chemokines and pharmaceutical reagents are used for cell co-culture; during the process of cell co-culture, the gas channel of the air pressure control layer 3 is passed into Compressed air is used to mechanically stimulate the chondrocytes in the solidified chondrocyte hydrogel in different areas of the three-dimensional cell culture channel 21; or compressed air is introduced into the gas channel of the air pressure control layer 3 to stimulate the three-dimensional cell culture channel. The solidified chondrocytes in different areas in 21 - the chondrocytes in the hydrogel are mechanically stimulated; then the composite cell culture fluid is injected into the first cell culture fluid channel 22 and the second cell culture fluid channel 23 with the modified closed bottom surface. , the composite cell culture medium contains one or more of chondrocyte-related cells, chondrocyte-related biochemical factors, chondrocyte-related chemokines and pharmaceutical reagents for cell co-culture.

Preferably, the pressure of the compressed air is 0-1500 mbar.

Preferably, the chondrocyte-hydrogel mixture includes chondrocytes and a hydrogel solution; the density of the chondrocytes is 2×10 ⁵ to 4×10 ⁶ cells/mL; the hydrogel solution is methyl Acryloyl gelatin solution, the mass content of the methacryloyl gelatin solution is 10 to 15%; during the light curing, the power of ultraviolet light is 3W, and the curing time is ≤10s.

Compared to "Hyperphysiological compression of articular cartilage induces osteoarthritic phenotype in a cartilage-on-a-chip model" (P.Occhetta,A.Mainardi,E.Votta,Q.Vallmajo-Martin,M.Ehrbar,I.Martin, A.Barbero, M.Rasponi, Nature Biomedical Engineering, 2019 Jul;3(7):545-557.doi:10.1038/s41551-019-0406-3), the microfluidic chip provided by the present invention is In addition to meeting the functions of three-dimensional cell culture and mechanical compression of three-dimensional cultured cells, it also has the following advantages:

The chip provided by the present invention can control the mechanical compression level of cells by regulating the air pressure in real time, that is, the same chip can achieve different levels of mechanical compression and can be accurately controlled.

The microfluidic chip provided by the present invention is designed with multiple air pressure channels perpendicular to the three-dimensional cell culture channel 21 of the three-dimensional cell culture layer 2 in the air pressure control layer 3. Each air pressure channel can be controlled by regulating the compression of the gas channel. The level of air causes compression deformation in different areas of the three-dimensional cell culture channel 21, thereby exerting mechanical force on the chondrocytes in the hydrogel in the three-dimensional cell culture channel 21; and each gas channel exerts pressure on the cells when air pressure is applied. A part of the hydrogel in the three-dimensional culture channel 21 is compressed, thereby achieving controllability of the compressed area in the same chip. Moreover, multiple gas channels in the air pressure control layer 3 can be controlled individually or jointly, and different control schemes can be designed, such as applying air pressure to only one channel, applying air pressure to three channels together, or applying dynamic air pressure to the three channels in turn. The design of multiple gas channels can make the mechanical stimulation faced by cells in the microfluidic chip provided by the present invention more diverse, can better simulate the in vivo environment, and can also study more sets of different air pressure output schemes separately. Therefore, the present invention can construct different levels of local mechanical microenvironments in the same microfluidic chip, and can realize flux or localized research on mechanical stimulation of articular cartilage.

The biochemical microenvironment of articular cartilage in the body includes immune cells such as macrophages, lymphocytes, and cytokines secreted by various cells. These biochemical factors can affect the homeostasis of chondrocytes, and cell debris and aggregated proteins produced by chondrocytes caused by damage caused by mechanical stimulation and other factors can stimulate the activation of other cells such as macrophages and prompt them to secrete cytokines. The crosstalk between cells in these force-chemistry-biogenesis coupled microenvironments in the joint cavity is very important to the occurrence and development of joint diseases, and the microfluidic chip designed using the present invention can build the force of chondrocytes. - A model of a chemical-biological coupled microenvironment, in which the cytokines among the bio-chemical factors can be realized by directly adding to the cell culture fluid in the first cell culture fluid channel 22 and the second cell culture fluid channel 23 , the present invention realizes the co-culture of multiple cells in bio-chemical factors by cleverly designing the structure of the microfluidic chip.

In summary, the microfluidic chip provided by the present invention is based on a layer-by-layer classic structure and takes into account the functions of multi-cell co-culture and mechanical stimulation. The establishment of a cartilage bionic microfluidic chip can not only regulate the mechanical stimulation of cartilage tissue, but also Achieve co-culture of a variety of cells, achieve mechanical stimulation of joint tissue and co-construction of immune microenvironment.

The present invention provides a method for constructing a microenvironment model of force-chemical-biogenesis coupling of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution. The present invention is based on the flexibility of the modular design of the above-mentioned microfluidic chip, which provides an opportunity to analyze the mechanical stimulation in the joint microenvironment and the impact of various immune cells on the homeostasis of articular cartilage. By rationally designing the structure of the microfluidic chip, the present invention can realize the biochemical and mechanical microenvironment that articular cartilage receives in the body, and establish a microphysiological system that better simulates the force-chemical-biological coupling microenvironment in which articular cartilage is located. The model accurately reproduces the physiological and pathological activities of human organs and various biological behaviors of the body.

Description of drawings

Figure 1 is a schematic structural diagram of a multifunctional microfluidic chip used for three-dimensional culture of articular cartilage cells and construction of mechanical microenvironment provided by the present invention;

In Figure 1: 1: basal layer, 2: three-dimensional cell culture layer; 21: three-dimensional cell culture channel; 22: first cell culture fluid channel; 23: second cell culture fluid channel; 24: cell fluid inlet channel; 25: Cell fluid outlet channel; 26: First cell culture medium inlet channel; 27: First cell culture medium outlet channel; 28: Second cell culture medium inlet channel; 29: Second cell culture medium outlet channel; 3 : Air pressure control layer; 31: first gas channel; 32: second gas channel; 33: third gas channel; 311: first extrusion part; 321: second extrusion part; 331: third extrusion part; 34～39: Liquid through hole; 4: Liquid storage tank; 41～46: Liquid storage through hole;

Figure 2 shows the changes in fluorescence intensity of chondrocytes labeled with TMRM probe under ultraviolet irradiation (P=3W) as a function of irradiation time;

Figure 3 is a fluorescence diagram of cell activity after 7 days of culture in the present invention;

Figure 4 is a schematic diagram of three-dimensional cultured chondrocytes uniformly distributed in the hydrogel in the present invention and a schematic diagram of in-situ cells deformed before and after mechanical compression is applied, in which green is the living cell tracer labeled fluorescent dye CFSE;

Figure 5 is a schematic diagram of the co-culture of simulated three-dimensional cultured cells and 2D cultured cells in the present invention, in which the green color is the primary chondrocytes labeled with the fluorescent dye CFSE, and the red color is the macrophage RAW264.7 labeled with the fluorescent dye Dil;

Figure 6 is a typical schematic diagram of the local compression effect of the hydrogel in the present invention. A in Figure 6 is a diagram of a single gas channel in the present invention; B in Figure 6 is a longitudinal view of the hydrogel when two adjacent gas channels apply air pressure at the same time. Deformation diagram, C in Figure 6 is the upper surface of the hydrogel when air pressure is applied, D in Figure 6 is the lower surface of the hydrogel when air pressure is applied, and the green fluorescence is polystyrene fluorescent particles with a diameter of 100nm;

Figure 7 is a quantitative result diagram of the local compression effect of the hydrogel in the present invention. A in Figure 7 is a quantitative result of the corresponding local compression deformation of the hydrogel when a single air pressure channel is introduced into different levels of compressed air in the present invention; Figure B in 7 is a quantitative result diagram of the impact on the local compression deformation of the corresponding hydrogel below the adjacent compression channel when a single air pressure channel is introduced into different levels of compressed air in the present invention;

Figure 8 shows the immunofluorescence experimental results of cartilage biological function evaluation using matrix synthesis of chondrocytes under the differentiated mechanical environment constructed by the present invention, where Static means no mechanical compression; PC means cells undergo physiological dose deformation; HPC means cells undergo deformation. Deformation at supraphysiological doses;

Figure 9 shows the results of immunofluorescence experiments for evaluating the cartilage biological function of matrix-destroying enzymes of chondrocytes under the differentiated mechanical environment constructed by the present invention, where Static means not subject to mechanical compression; PC means cells deformed at physiological doses; HPC means cells Supraphysiological dose of deformation occurs;

Figure 10 is the result of an immunofluorescence experiment using the present invention to explore the content of the pro-inflammatory marker iNOS in macrophages in chondrocytes subjected to mechanical compression at a supraphysiological dose;

Figure 11 is a flow cytometry detection result using the present invention to explore the content of the pro-inflammatory marker iNOS in macrophages in chondrocytes subjected to mechanical compression at a supraphysiological dose;

Figure 12 shows the prior art "Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model" (P.Occhetta, A.Mainardi, E.Votta, Q.Vallmajo-Martin, M.Ehrbar, I .Martin, A.Barbero, M.Rasponi, Nature Biomedical Engineering, 2019 Jul; 3(7):545-557.doi:10.1038/s41551-019-0406-3) chip model disclosed in;

Figure 13 shows the prior art "Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model" (P.Occhetta, A.Mainardi, E.Votta, Q.Vallmajo-Martin, M.Ehrbar, I .Martin, A.Barbero, M.Rasponi, Nature Biomedical Engineering, 2019 Jul; 3(7):545-557.doi:10.1038/s41551-019-0406-3) The suspended microarray in the chip model and Schematic diagram of the gap between the lower PDMS films.

Detailed ways

The invention provides a microfluidic chip, which includes a basal layer 1 and a three-dimensional cell culture layer 2 and an air pressure control layer 3 which are sequentially stacked on the basal layer 1; the basal layer 1 and the three-dimensional cell culture layer 2 and the air pressure control layer 3 are made of transparent material;

The three-dimensional cell culture layer 2 is provided with a three-dimensional cell culture channel 21 and a first cell culture fluid channel 22 and a second cell culture fluid channel 23 located on both sides of the three-dimensional cell culture channel 21. The three-dimensional cell culture channel 21 and The first cell culture medium channels 22 and the three-dimensional cell culture channel 21 and the second cell culture medium channel 23 are separated by a plurality of microstructures. There are gaps between the microstructures. The three-dimensional cell culture channel 21 A cell fluid inlet channel 24 and a cell fluid outlet channel 25 are provided at both ends of the first cell culture fluid channel 22. A first cell culture fluid inlet channel 26 and a first cell culture fluid outlet channel are provided at both ends of the first cell culture fluid channel 22. 27. A second cell culture medium inlet channel 28 and a second cell culture medium outlet channel 29 are provided at both ends of the second cell culture medium channel 23;

The air pressure control layer 3 is provided with a plurality of gas channels perpendicular to the three-dimensional cell culture channel 21. Both ends of each of the gas channels are provided with gas inlets and outlets, and each of the gas channels is provided with all gas channels at both ends. The extruded parts between the gas inlets and outlets are located above the three-dimensional cell culture channel 21. There are three liquid through holes at both ends of the air pressure control layer, and each of the liquid through holes is connected to the cells respectively. fluid-carrying fluid inlet channel 24, cell fluid-carrying fluid outlet channel 25, first cell culture fluid inlet channel 26, first cell culture fluid outlet channel 27, second cell culture fluid inlet channel 28, and first cell culture fluid outlet channel 29 .

In the present invention, unless otherwise specified, all preparation raw materials/components are commercially available products well known to those skilled in the art.

The schematic structural diagram of the microfluidic chip provided by the present invention is shown in Figure 1. The microfluidic chip provided by the present invention will be described in detail below in conjunction with Figure 1.

The microfluidic chip provided by the invention includes a base layer 1 . In the present invention, the basal layer 1 serves as the base of the entire microfluidic chip and encapsulates the three-dimensional cell culture layer 2.

As one or more embodiments of the present invention, the base layer 1 is made of glass.

As one or more embodiments of the present invention, the thickness of the base layer 1 is 0.13-0.17 mm.

In the present invention, the basal layer 1 uses ultra-thin glass with a thickness of 0.13 to 0.17 mm. Due to its thin thickness, transparency and other advantages, ultra-thin glass can allow three-dimensional cell culture in the three-dimensional culture layer 2 to be realized under a microscope. In situ observation of cells at the single-cell level and analysis of their mechanical behavior.

The microfluidic chip provided by the present invention includes a three-dimensional cell culture layer 2 disposed on the basal layer 1 . The three-dimensional cell culture layer 2 is provided with a three-dimensional cell culture channel 21 and a first cell culture fluid channel 22 and a second cell culture fluid channel 23 located on both sides of the three-dimensional cell culture channel 21. The three-dimensional cell culture channel 21 and The first cell culture medium channels 22 and the three-dimensional cell culture channel 21 and the second cell culture medium channel 23 are separated by microstructures. There are gaps between the microstructures. Both sides of the three-dimensional cell culture channels 21 A cell fluid inlet channel 24 and a cell fluid outlet channel 25 are provided at both ends of the first cell culture fluid channel 22. A first cell culture fluid inlet channel 26 and a first cell culture fluid outlet channel 27 are provided at both ends. A second cell culture medium inlet channel 28 and a second cell culture medium outlet channel 29 are provided at both ends of the second cell culture medium channel 23 . In the present invention, the function of the three-dimensional cell culture layer 2 is to perform cell culture.

As one or more embodiments of the present invention, the three-dimensional cell culture channel 21, the first cell culture fluid channel 22, and the second cell culture fluid channel 23 are all structures in which the upper part (top) seals the lower opening.

As one or more embodiments of the present invention, the microstructure is a linearly arranged micro-cylinder with gaps between adjacent micro-cylinders. The microstructure is used to connect the three-dimensional cell culture channel 21 with the first cell culture channel. The liquid channel 22 is separated from the second cell culture liquid channel 23, allowing the passage and exchange of cells and nutrients between the channels.

As one or more embodiments of the present invention, the microstructure is in the shape of a fence, and one end of the microstructure is in contact with the base layer 1 .

As one or more embodiments of the present invention, the cell fluid inlet channel 24, the cell fluid outlet channel 25, the first cell culture fluid inlet channel 26, the first cell culture fluid outlet channel 27, the second cell culture fluid The inlet channel 28 and the first cell culture medium outlet channel 29 have a through-hole structure that penetrates up and down.

As one or more embodiments of the present invention, the width of the three-dimensional cell culture channel 21 is 1 to 1.5 mm. The widths of the first cell culture fluid channel 22 and the second cell culture fluid channel 23 are 1 to 2 mm. The depths of the three-dimensional cell culture channel 21, the first cell culture fluid channel 22 and the second cell culture fluid channel 23 are 70-300 μm.

As one or more embodiments of the present invention, the cell fluid inlet channel 24, the cell fluid outlet channel 25, the first cell culture fluid inlet channel 26, the first cell culture fluid outlet channel 27, the second cell fluid The diameters of the culture medium inlet channel 28 and the first cell culture medium outlet channel 29 are 0.5 to 1.5 mm.

As one or more embodiments of the present invention, the three-dimensional cell culture layer 2 is made of polydimethylsiloxane (PDMS) and has a thickness of 200-400 μm. In the present invention, the difference between the thickness of the three-dimensional cell culture layer 2 and the depth of the three-dimensional cell culture channel 21 is the thickness of the PDMS film that deforms when the three-dimensional cell culture layer 2 is subjected to compressed air. The thickness of the PDMS film affects subsequent A key factor in the compressive effect of compressed air on the cell-hydrogel mixture during use.

In the present invention, when the three-dimensional cell culture layer 2 is used, a mixture of chondrocytes and hydrogel is injected from the cell carrier fluid inlet channel 24. Since the three-dimensional cell culture channel 21 and the first cell culture fluid channel 22 and the third There is a microstructure between the two cell culture fluid channels 23, so the mixture of chondrocytes and hydrogel will be confined in the three-dimensional cell culture channel 21. After the hydrogel is solidified, the purpose of three-dimensional culture of chondrocytes in the hydrogel is achieved in the three-dimensional cell culture channel 21 . The cell culture medium is injected through the first cell culture medium inlet channel 26 and the second cell culture medium inlet channel 28 .

The microfluidic chip provided by the present invention includes an air pressure control layer 3 disposed on the three-dimensional cell culture layer 2 . The air pressure control layer 3 is provided with a plurality of gas channels perpendicular to the three-dimensional cell culture channel 21. Both ends of each of the gas channels are provided with gas inlets and outlets, and each of the gas channels is provided with all gas channels at both ends. The extruded parts between the gas inlets and outlets are located above the three-dimensional cell culture channel 21. There are three liquid through holes at both ends of the air pressure control layer, and each of the liquid through holes is connected to the cells respectively. fluid-carrying fluid inlet channel 24, cell fluid-carrying fluid outlet channel 25, first cell culture fluid inlet channel 26, first cell culture fluid outlet channel 27, second cell culture fluid inlet channel 28, and first cell culture fluid outlet channel 29 . In the present invention, the function of the air pressure control layer 3 is to realize the mechanical compression function of the mechanical loading area of the three-dimensional cell culture layer 2 in the microfluidic chip, including the force-chemical-biogenesis coupling microenvironment of articular cartilage cells. The construction simulates the mechanical compression stimulus that cells are subjected to.

As one or more embodiments of the present invention, the number of the gas channels is three. As shown in FIG. 1 , they are a first gas channel 31 , a second gas channel 32 and a third gas channel 33 respectively. The first gas channel 31 is provided with a first squeeze portion 311 located between the gas inlets and outlets at both ends. The second gas channel 31 is provided with a second squeeze portion 321 located between the gas inlets and outlets at both ends. The third gas channel 31 is provided with a third squeeze portion 331 located between the gas inlets and outlets at both ends. The gas channel is a structure in which the upper part (top) seals the lower opening. The width of the gas channel is 1 to 1.5 mm and the depth is 50 to 200 μm. The width of the squeeze portion is greater than the width of the gas channel. The shape of the pressing part is rectangular or circular. The extrusion part is used to exert mechanical pressure on the three-dimensional cell culture layer 2 .

In the present invention, gas inlets and outlets are provided at both ends of each gas channel. The shape of the gas inlet and outlet is circular. The diameter of the gas inlet and outlet is preferably 0.7mm. Each of the gas channels passes the gas inlet and outlet through an external air pressure output device to pass compressed air into the gas channels of the air pressure control layer 3, and the squeezed portion of the gas channels causes the three-dimensional cell culture channel 21 in the three-dimensional cell culture layer 2 to deform, and The pipe connecting the air pressure output device and the gas inlet is equipped with an Elveflow microfluidic OB1 pressure flow controller. The Elveflow microfluidic OB1 pressure flow controller can achieve ultra-accurate and fast-response air pressure flow control on the value of compressed air. Each air pressure channel can longitudinally compress and deform the hydrogel in the three-dimensional cell culture channel 21 by regulating the amount of compressed air flowing into the gas channel, thereby affecting the three-dimensional growth of cartilage evenly mixed in the hydrogel. Cells exert mechanical compression and deform. At the same time, each gas channel of the present invention will compress a part of the hydrogel in the three-dimensional cell culture channel 21 when air pressure is applied, thereby achieving controllability of the compression area in the same chip. Several gas channels in the air pressure control layer can be controlled individually or jointly, and different control schemes can be designed. Specifically: only apply air pressure to one of the gas channels, apply air pressure to three channels together, or apply dynamic air pressure to the three channels in turn. The design of multiple gas channels in the present invention can make the mechanical stimulation faced by cells in the chip more diverse, can better simulate the in vivo environment, and can also study more sets of different air pressure output schemes separately.

By providing multiple gas channels in the air pressure control layer 3, the present invention can construct local mechanical microenvironments of different levels in the same chip, and can realize flux or localized research on mechanical stimulation of articular cartilage.

As one or more embodiments of the present invention, the air pressure control layer 3 is made of PDMS.

As one or more embodiments of the present invention, the diameter of the liquid through hole on the air pressure control layer 3 is 1.2 mm.

The microfluidic chip provided by the present invention preferably further includes a liquid storage layer stacked on the gas pressure control layer 3. The liquid storage layer includes two liquid storage pools 4 arranged on both sides of the gas channel. The liquid storage tank 4 is provided with three liquid storage through holes, and each of the liquid storage through holes is connected to each of the liquid through holes respectively. In the present invention, the function of the liquid storage layer is mainly to provide more storage space for the culture medium of cell culture. In addition, the liquid storage layer can provide more liquid storage space when processing cells such as immunofluorescence experiments for biochemical analysis.

As one or more embodiments of the present invention, the diameter of the liquid storage through hole is 1.5 to 3.5 mm.

As one or more embodiments of the present invention, the liquid storage layer is made of PDMS.

The microfluidic chip provided by the present invention preferably includes from bottom to top: a basal layer 1, a three-dimensional cell culture layer 2, an air pressure control layer 3 and a liquid storage layer. The air pressure control layer 3 of the microfluidic chip provided by the present invention includes a number of gas channels, which can realize mechanical compression of different areas of the three-dimensional cell culture channel, thereby forming multiple mechanically compressed cell culture areas; in order to realize the coexistence of multiple cells, The culture uses microstructurally spaced channels. Different layers of the present invention are combined through irreversible bonding. The microfluidic chip provided by the present invention is prepared using PDMS and has optical transparency, gas permeability, deformability and ease of manufacturing, which allows real-time observation of the response of cells and tissues to mechanical stimulation and the like.

The present invention provides a method for preparing the microfluidic chip described in the above technical solution, which includes the following steps:

(1) Design and prepare masks for the three-dimensional cell culture layer 2 and the air pressure control layer 3;

(2) Use a mask to prepare a silicon wafer containing the structure of the three-dimensional cell culture layer 2 and the air pressure control layer 3 through photolithography technology;

(3) Use the above silicon wafer to prepare the PDMS-based three-dimensional culture layer 2 and air pressure control layer 3;

(4) After bonding the three-dimensional cell culture layer 2 and the air pressure control layer 3, bond them to the basal layer 1.

The present invention prepares templates for the three-dimensional cell culture layer 2 and the air pressure control layer 3. In the present invention, the specific preparation method of the template preferably includes: using CAD to draw the structural diagram of the three-dimensional cell culture layer 2, the air pressure control layer 3 and the liquid storage layer; according to the structural diagram drawn by CAD, using software on the silicon wafer. The photolithography method is used to prepare the templates of the three-dimensional culture layer 2, the control layer 3 and the liquid storage layer of the air-pressure cells, and then the templates are subjected to hydrophobic treatment before use.

The present invention uses a template to prepare the air pressure cell three-dimensional culture layer 2, the air pressure control layer 3 and the liquid storage layer. The preparation method of the air pressure control layer 3 and the liquid storage layer preferably includes: pouring the mixed and defoamed PDMS liquid into the formwork, and turning the mold over after solidification to obtain the air pressure control layer 3 and the liquid storage layer. The film casting liquid is preferably Dow Corning Sylgard 184 PDMS, including a polymer and a cross-linking agent, and the mass ratio of the polymer and the cross-linking agent is 10:1. The preparation method of the three-dimensional cell culture layer 2 preferably includes: applying a uniform and controllable film of uniform thickness on the template by spin coating the mixed and defoamed PDMS, and turning over the mold after solidification to obtain the three-dimensional cell culture layer 2. . The spin coating preferably includes low-speed spin coating and high-speed spin coating in sequence. The rotation speed of the low-speed spin coating is preferably 100 rpm, and the time is preferably 60 s; the rotation speed of the high-speed spin coating is preferably >100 rpm, and more preferably 200 to 300 rpm. , the time is preferably 60s. The curing temperature is preferably 65°C, and the curing time is preferably 3 to 12 hours. In the present invention, the three-dimensional cell culture layer 2 is preferably prepared by spin coating. The thickness can be controlled by the rotation speed parameters of the spin coating and the ratio of PDMS, because the total thickness of the spin-coated three-dimensional cell culture layer 2 is minus the channel depth. The last is the thickness of the PDMS membrane that is deformed by the compressed air. The thickness of the PDMS membrane is a key factor affecting the compression effect of the compressed air on the cell-hydrogel mixture during subsequent use.

After obtaining the three-dimensional cell culture layer 2, the air pressure control layer 3 and the liquid storage layer, the present invention bonds the air pressure cell three-dimensional culture layer 2 and the control layer 3, then bonds it to the basal layer 1, and finally bonds it to the liquid storage layer. The microfluidic chip was obtained. In the present invention, the specific implementation of the bonding preferably includes: performing surface modification treatment on the three-dimensional cell culture layer 2, the air pressure control layer 3 and the liquid storage layer in sequence using a plasma cleaning machine, and then performing permanent bonding on them. On the transparent base layer 1. The preferred steps for bonding each layer of the chip are: first use a hole punch to punch holes at both ends of the air pressure channel of the air pressure control layer 3, and then use tape to remove the dust on the surface of the air pressure control layer and the three-dimensional cell culture layer, and then bond the With the sides facing up together, place them in a plasma cleaning machine for surface modification treatment, bond the three-dimensional cell culture layer 2 and the air pressure control layer 3 together, and place them on an 80°C heating station for heating for ≥1 hour; use The hole puncher punches the two ends of the channel into which the three-dimensional cell culture layer 2 and the air pressure control layer 3 are bonded together. Then use tape to remove the dust on the surface and bond them together. With the side facing up, place it and the base layer 1 in a plasma cleaning machine for surface modification treatment, bond them together, and place them on an 80°C heating station for heating for ≥1 hour; after finally bonding the liquid storage layer, Place on 80℃ heating platform for heating, heating time ≥2h.

The present invention provides a method for constructing a differentiated mechanical microenvironment model of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution, which includes the following steps:

Inject the chondrocyte-hydrogel mixture into the three-dimensional cell culture channel 21 of the three-dimensional cell culture layer 2, and perform photocuring under ultraviolet irradiation to obtain a solidified chondrocyte-hydrogel; then inject the chondrocyte culture solution into the three-dimensional cell culture channel 21. Chondrocytes are cultured in the first cell culture fluid channel 22 and the second cell culture fluid channel 23 of the culture layer 2. During the process of chondrocyte culture, different levels of air are introduced into the gas channels of the air pressure control layer 3. Compressed air is used to perform different levels of mechanical stimulation on the solidified chondrocytes in different areas of the cell three-dimensional culture channel 21 - the chondrocytes in the hydrogel.

In the present invention, the microfluidic chip is preferably sterilized before use. In the present invention, the specific operation of the sterilization process is preferably: using a pipette gun to inject 75% sterilizing alcohol into the three-dimensional cell culture channel 21, the first cell culture solution channel 22 and the second cell culture channel of the three-dimensional cell culture layer 3 In the liquid channel 23, let it stand at room temperature for 1 to 3 hours; then, use a pipette to inject the PBS solution into the three-dimensional cell culture channel 21, the first cell culture liquid channel 22 and the second cell culture liquid channel 23 of the three-dimensional cell culture layer 3 , let it stand at room temperature; then dry the washed chip. The drying temperature is preferably 37-65°C, the drying is performed on a hot stage or in an oven, and the drying is to remove the liquid in the microfluidic chip. Finally, place it on the cell operating table for UV irradiation for ≥1h. The chondrocyte-hydrogel mixture preferably includes chondrocytes and a hydrogel solution; the density of chondrocytes is preferably 2×10 ⁵ to 4×10 ⁶ cells/mL. The chondrocytes are selected from primary chondrocytes or cell lines isolated from articular cartilage of humans, mice or rats. The hydrogel solution is a methacrylated gelatin (GelMA) solution, and the mass content of the methacrylated gelatin solution is preferably 10 to 15%, more preferably 10%. In specific embodiments of the present invention, the GelMA solution is preferably GelMA60. During the photocuring, the power of ultraviolet light is preferably 3W, and the curing time is preferably ≤10s, and more preferably 10s. The pressure of the compressed air is preferably 0 to 1500 mbar. In a specific embodiment of the present invention, the present invention achieves ultra-accurate and fast-response air pressure flow control by using an external Elveflow microfluidic OB1 pressure flow controller to regulate the value of compressed air flowing into the air pressure control layer 3 to cause the PDMS membrane to deform. The Elveflow microfluidic OB1 pressure flow controller is a multi-channel programmable constant pressure pump that achieves precise microfluidic pressure control. It allows pulse-free flow within millisecond response time and can output diverse waveforms of air pressure through ESI software. The pressure waveform of the compressed air preferably includes sine, square wave, triangle wave, ramp and customized waveform. The number of pressure output channels of the Elveflow microfluidic OB1 pressure flow controller can be customized from 1 channel to 4 channels, enabling throughput of chip operation. In a specific embodiment of the present invention, the pressure waveform of the compressed air is preferably a 0.5Hz sine wave.

The present invention provides a method for constructing a microenvironment model of force-chemical-biogenesis coupling of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution, which includes the following steps:

The surface of the basal layer 1 that is in contact with the first cell culture fluid channel 22 and the second cell culture fluid channel 23 of the three-dimensional cell culture layer 2 is surface-modified with a coating material; then the chondrocyte-hydrogel mixture is injected into the three-dimensional cell culture layer 2 In the three-dimensional cell culture channel 21 of the culture layer 2, light curing is performed under ultraviolet irradiation to obtain a solidified chondrocyte-hydrogel; then the composite cell culture medium is injected into the first cell culture medium channel 22 and the first cell culture medium channel 22 with the modified closed bottom surface. In the second cell culture medium channel 23, the composite cell culture medium contains one or more of chondrocyte-related cells, chondrocyte-related biochemical factors, chondrocyte-related chemokines and pharmaceutical reagents, Carry out cell co-culture; during the process of cell co-culture, compressed air is introduced into the gas channel of the air pressure control layer 3, and the solidified chondrocytes in different areas of the cell three-dimensional culture channel 21-hydrogel are Chondrocytes are mechanically stimulated.

Alternatively, the present invention provides a method for constructing a force-chemistry-biogenesis coupled microenvironment model of three-dimensional cultured chondrocytes using the microfluidic chip described in the above technical solution, including the following steps:

The surface of the basal layer 1 that is in contact with the first cell culture fluid channel 22 and the second cell culture fluid channel 23 of the three-dimensional cell culture layer 2 is surface-modified with a coating material; then the chondrocyte-hydrogel mixture is injected into the three-dimensional cell culture layer 2 In the three-dimensional cell culture channel 21 of the culture layer 2, light curing is performed under ultraviolet irradiation to obtain a solidified chondrocyte-hydrogel; compressed air is then introduced into the gas channel of the air pressure control layer 3 to culture the cells three-dimensionally. The solidified chondrocytes in different areas of the channel 21 - the chondrocytes in the hydrogel are mechanically stimulated; finally, the composite cell culture fluid is injected into the first cell culture fluid channel 22 and the second cell culture fluid channel 23 after the closed bottom surface has been modified , the composite cell culture medium contains one or more of chondrocyte-related cells, chondrocyte-related biochemical factors, chondrocyte-related chemokines and pharmaceutical reagents for cell co-culture.

In the present invention, the sterilization treatment performed before using the microfluidic chip is preferably the same as above, and will not be described again. In the present invention, the biochemical microenvironment of articular cartilage in the body includes immune cells such as macrophages, lymphocytes, synovial cells, and cytokines secreted by various cells. These biochemical factors can affect the homeostasis of chondrocytes, and cell debris and aggregated proteins produced by chondrocytes caused by damage caused by mechanical stimulation and other factors can stimulate the activation of other cells such as macrophages and prompt them to secrete cytokines. The crosstalk between cells in these force-chemistry-biogenesis coupled microenvironments in the joint cavity is very important to the occurrence and development of joint diseases. Therefore, the microfluidic chip designed in the present invention is used to construct the force of chondrocytes. - Models of coupled chemical-biological microenvironments are extremely valuable. In the present invention, the cytokines in the bio-chemical factors can be directly added to the cell culture medium, and the cells in the bio-chemical factors can be co-cultured with multiple cells by cleverly designing the structure of the microfluidic chip. . This invention distributes other cells in the joint in the form of 2D growth in the channels on both sides of the three-dimensional cell culture layer of the chip. In order to inoculate other related cells in the joint such as macrophages in the chip, the present invention coats the surface of the basal layer 1 that is in contact with the first cell culture fluid channel 22 and the second cell culture fluid channel 23 of the cell three-dimensional culture layer 2 with Materials are surface modified. In the present invention, the coating material preferably includes fibronectin or RGD, more preferably fibronectin. In the present invention, fibronectin is used to modify the surface of the glass substrate in the channel to make it suitable for cell adhesion and growth. In the present invention, the specific implementation of the surface treatment preferably includes: taking the sterilized and dried chip and placing it in a sterile petri dish, first flushing the channel with sterile water, and then using a pipette to absorb 10 μL of coating The liquid is injected into the microchannel from one end of the channel to coat the glass surface at the bottom of the channel. The chip containing the coating liquid is incubated in a clean bench for 1 hour and the remaining coating liquid is sucked out, and then dried naturally for 1 hour before use. In the present invention, the preparation method of the solidified chondrocyte hydrogel is the same as described above and will not be described again. The composite cell culture fluid of the present invention is injected into the first cell culture fluid channel 22 and the second cell culture fluid channel 23 with modified closed bottom surfaces to perform cell co-culture. In the present invention, the composite cell culture medium preferably contains one or more of chondrocyte-related cells, chondrocyte-related biochemical factors, chondrocyte-related chemokines and pharmaceutical reagents. The chondrocyte-related cells preferably include immune cells, and the immune cells are particularly preferably macrophages and/or lymphocytes. The chondrocyte-related biochemical factors preferably include cytokines secreted by cells related to chondrocytes. The chondrocyte-related cells are selected from primary cells or cell lines isolated from humans, mice or rats. In a specific embodiment of the present invention, the composite cell culture medium is specifically a culture medium containing cells related to chondrocytes, and the density of cells related to chondrocytes in the culture medium containing cells related to chondrocytes is preferably It is 2×10 ⁵ to 4×10 ⁶ cells/mL. The pressure of the compressed air is preferably 0 to 1500 mbar. In a specific embodiment of the present invention, the present invention achieves ultra-accurate and fast-response air pressure flow control by using an external Elveflow microfluidic OB1 pressure flow controller to regulate the value of compressed air flowing into the air pressure control layer 3 to cause the PDMS membrane to deform. The Elveflow microfluidic OB1 pressure flow controller is a multi-channel programmable constant pressure pump that achieves precise microfluidic pressure control. It allows pulse-free flow within millisecond response time and can output diverse waveforms of air pressure through ESI software. The pressure waveform of the compressed air preferably includes sine, square wave, triangle wave, ramp and customized waveform. The number of pressure output channels of the Elveflow microfluidic OB1 pressure flow controller can be customized from 1 channel to 4 channels, enabling throughput of chip operation. In a specific embodiment of the present invention, the pressure waveform of the compressed air is preferably a 0.5Hz sine wave.

The microfluidic chip of this embodiment can be reused. In the present invention, the hydrogel GelMA lysis solution is preferably used to lyse the hydrogel in the microstructure channel of the three-dimensional cell culture layer, and then trypsin is used to digest the cells in the chip channel, and PBS is used to clean the channel, followed by cell inoculation. sterilization treatment.

The invention provides a multifunctional microfluidic chip capable of realizing multi-cell co-culture and mechanical compression research. The present invention uses micro-nano processing technology such as photolithography to construct a cartilage-like chip based on microfluidic technology. By setting multiple parallel gas channels in the air pressure channel layer of the chip structure, it can realize high-throughput processing of cells in the three-dimensional cell culture layer. of precisely adjustable mechanical compression. Construct an articular cartilage model using biomaterials such as hydrogel, and inoculate corresponding cells such as macrophages at different locations on the chip to accurately simulate the real local mechanical microenvironment and immune microenvironment of articular cartilage, and explore joints from the single cell level. The pathogenesis of the disease, building a convenient, fast and economical drug screening platform to provide support for the treatment of joint diseases.

In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the protection scope of the present invention.

The following embodiments of the present invention use the microfluidic chip with the structure shown in Figure 1 to conduct experiments. The microfluidic chip is divided into four parts: basal layer 1, three-dimensional cell culture layer 2, air pressure control layer 3 and the uppermost liquid reservoir layer. The three-dimensional cell culture layer 2 is three channels divided by microstructure. The width of the three-dimensional cell culture channel 21 is 1.5mm. The width of the first cell culture medium channel 22 and the second cell culture medium channel 23 is 2mm. The depth is 100 μm, the number of gas channels in the upper air pressure control layer 3 is 3, the width of the gas channel is 1.5 mm, the depth is 100 μm, and the liquid reservoir is a rectangular parallelepiped with a circular through hole structure of 3.5 mm in diameter.

The preparation method of the microfluidic chip with the structure shown in Figure 1 includes: (1) using CAD to draw the chip structure diagram and preparing a mask, using soft photolithography to prepare templates for each structural layer on the silicon wafer, and After the template is hydrophobically treated, it is ready for use. (2) Use polydimethylsiloxane (PDMS) to pour, dry, and flip the silicon wafer template of the air pressure control layer 3 and the liquid storage layer to prepare the air pressure control layer and liquid storage layer of PDMS used to prepare the chip. layer. (3) The three-dimensional cell culture layer 2 needs to be prepared by the spin coating method, in which the ratio of PDMS polymer and cross-linking agent is 10:1, the curing temperature of PDMS is 65°C, and the curing time is 3 hours. According to the present invention The spin coating used in is divided into two steps: the thickness of the three-dimensional cell culture layer 2 is 200 μm, the spin coating parameters are 100rpm 60s and 300rpm 60s, and the mold is turned over after drying. (4) The three-dimensional cell culture layer 2, the air pressure control layer 1 and the liquid storage layer 4 are sequentially surface modified using a plasma cleaning machine, and then permanently bonded to the transparent substrate 1. The substrate used in the following examples It is a light-transmitting ultra-thin glass slide of 0.15±0.02mm. The steps for bonding each layer of the chip are: first use a hole punch with a diameter of 0.7mm to punch holes at both ends of the air pressure channel of the air pressure control layer, and then use tape to remove the dust on the surfaces of the air pressure control layer 3 and the three-dimensional cell culture layer 2. Place the surfaces that need to be bonded face up, place them in a plasma cleaning machine for surface modification treatment for 1 minute, bond the air pressure control layer 3 and the three-dimensional cell culture layer 2 together, and bake them on a heating table at 80°C for 1 hour; Use a hole punch with a diameter of 1.2mm to punch holes at both ends of the channels for injecting cells into the three-dimensional culture layer 2 of the air pressure control layer 3 and the three-dimensional cell culture layer 2 bonded together, and then use tape to remove the dust on the surface. With the bonded sides facing up, place it and the ultra-thin glass slide (thickness 0.15±0.02mm) in a plasma cleaning machine for surface modification treatment for 1 minute, bond them together, and bake on a heating table at 80°C for 1 hour. ;After finally bonding the liquid storage layer, place it on a heating table at 80°C for ≥2 hours.

The microfluidic chip with the structure shown in Figure 1 used in the following examples needs to be sterilized before being used in all cell experiments. The specific operation is: use a pipette to inject 75% sterile alcohol into the channel of the three-dimensional cell culture layer 2, and let it stand at room temperature for 3 hours. Then, use a pipette to inject the PBS solution into the channel of the three-dimensional cell culture layer 2 and let it stand at room temperature. 1h. Then dry the liquid in the chip on a 65°C hot stage or oven, place it on the cell operating table for UV irradiation for 1 hour, and then it can be used for cell culture. In the following examples, the power and irradiation time of the ultraviolet light source were determined as follows: Tetramethylrhodamine methyl ester TMRM probe (Invitrogen, Cat. No. I34361) was selected as the mitochondrial membrane potential indicator. The TMRM probe is a permeable Selective staining of cell membranes Fluorescent dyes for mitochondria in living cells can quickly pass through the cell membrane and be captured by active mitochondria in just a few minutes. They can be selectively located in mitochondria and are not toxic to cells; TMRM probes Strong fluorescence allows the use of low probe concentrations, thereby avoiding aggregation, making the TMRM probe one of the best fluorescent dyes for dynamic and quantitative in situ measurements. Quickly and sensitively detect changes in mitochondrial membrane potential through the intensity of the fluorescence signal: a weakening of the fluorescence signal indicates a decrease in mitochondrial membrane potential. The specific experimental operations are as follows: wash the chondrocytes digested with trypsin twice with PBS to remove serum; add TMRM working dye diluted with serum-free medium to the cell suspension, and incubate at 37°C in a 5% CO ₂ incubator Incubate for 30 minutes; wash the cells twice with PBS; then collect the cells by centrifugation, count them, and resuspend the cells in 10% GelMA60 solution preheated at 37°C; inject the TMRM probe-labeled cells and hydrogel mixture into the chip channel. After irradiating with ultraviolet light source (P=3W) for different times, the fluorescence intensity of the cells was detected using a laser confocal microscope. Figure 2 shows the changes in fluorescence intensity of primary chondrocytes labeled with the fluorescent probe TMRM as a function of UV light source irradiation time. As can be seen from Figure 2, under the refraction of an ultraviolet light source with power P = 3W, the fluorescence intensity of the cells is not affected when the exposure time is less than 10s; however, when the exposure time is greater than 10s, the fluorescence intensity of the cells decreases, indicating that the cell state is affected, so the selection The duration of UV irradiation is set to 10s.

Example 1

This embodiment uses a microfluidic chip to construct a force-chemistry-biogenesis coupled microenvironment model of three-dimensional cultured chondrocytes, but is not limited to this. In addition to chondrocytes, other force-chemistry-biogenesis coupled microenvironments in somatic cells can be used. The present invention can also be used to realize the construction of complex microenvironments. The specific process is as follows:

(1) Inoculation and culture of chondrocytes on microfluidic chip

Primary mouse chondrocytes isolated from the knee joints of mice aged 3 to 5 days were mixed into hydrogel (10% GelMA60). The density of the cell suspension ranged from 4×10 ⁶ cells/mL. Use a pipette to draw 10 μL of the primary chondrocyte-hydrogel mixture and slowly inject it from the liquid storage through hole 45 of the chip to prevent the speed from breaking through the limitations of the microstructure and leaking into the channels on both sides due to too fast speed, and then UV curing ( Power P=3W, T=10s). After injecting the chondrocyte culture medium from the liquid storage through holes 41 and 43 on both sides, fill the 200 μL pipette tip with the culture medium and insert it vertically into the liquid storage through holes 41, 42, 43, and 44 on both sides of the chip. . Then the chip seeded with cells was placed in an incubator (37°C, 5% CO ₂ ). Insert the pipette tip into the chip channel. On the one hand, it is for liquid sealing to prevent the hydrogel from losing water and drying out. On the other hand, it is to provide sufficient culture medium for cell growth. The chip needs to change the medium during long-term cell culture. The frequency is ≤3 days. This cell culture method does not affect the activity of the cells, avoids the need for external lateral flow pumps on the chip, reduces the risk of cell contamination, and is relatively simple to operate.

Figure 3 is the cell activity analysis of primary mouse chondrocytes after three-dimensional culture for 7 days in the microfluidic chip of the present invention. The cell viability detection reagent is Soleba Calcein-AM/PI Live Cell/Dead Cell Double Staining Kit (Cat. No.: CA1630). The specific measurement method is as follows: First, use a pipette to remove the channels 22 and 23 of the three-dimensional cell culture layer. culture medium and inject PBS for washing twice, and incubate at 37°C for 15 minutes each time; remove residual esterase, and inject the prepared dye solution Calcein-AM working solution into channels 22 and 23, and let it stand in the dark at 37°C. 2h; use a pipette to remove the dye Calcein-AM, add diluted PI working solution, and keep in the dark at room temperature for 10 minutes; after incubation, remove the dye, wash twice with PBS, and incubate at 37°C for 15 minutes during each wash. ; followed by detection using confocal laser microscopy. It can be seen from the experimental results in Figure 3 that primary chondrocytes maintained good cell activity after being cultured in the microfluidic chip of the present invention for 7 days.

(2) Construction of mechanical microenvironment

The microfluidic chip of the present invention can simulate and reproduce the mechanical microenvironment of chondrocytes in the body, especially mechanical compression. In this embodiment, compressed air is introduced into the channel of the air pressure control layer to deform the PDMS film below. The deformed PDMS film will longitudinally compress the hydrogel in the channel of the three-dimensional cell culture layer below and deform it, thereby uniformly The three-dimensional growth of chondrocytes mixed in the hydrogel is subjected to mechanical compression and deforms. Therefore, precise regulation of mechanical stimulation and compression of cells can be achieved by controlling the deformation of the hydrogel in the three-dimensional cell culture layer.

In this embodiment, an external Elveflow microfluidic OB1 pressure flow controller is used to regulate the value of the compressed air flowing into the air pressure control layer to cause the PDMS membrane to deform, achieving ultra-accurate and fast-response air pressure flow control. The Elveflow microfluidic OB1 pressure flow controller is a multi-channel programmable constant pressure pump that realizes precision microfluidic pressure control. It allows pulse-free flow within millisecond response time and can output diverse waveforms of air pressure through ESI software, including sinusoidal , square wave, triangle wave, ramp and custom waveform pressure output control and microfluidic flow control, without any programming operation, can achieve accurate measurement of hydrogel deformation, allowing the simulation of different types of dynamic mechanical compression to which cells are subjected. Mechanical stimulation; The number of pressure output channels of the Elveflow microfluidic OB1 pressure flow controller can be customized from 1 channel to 4 channels, enabling throughput of chip operation. The value of the compressed air used in the present invention is between 0 and 1500 mbar.

To detect in situ whether the cells are deformed due to mechanical compression after deformation of the hydrogel, the required reagent is the live cell tracer labeled fluorescent dye CFSE (Cat. No. HY-D0938). CFSE is a live cell tracer that can penetrate the cell membrane. After entering the cell, the dye is mainly located in the cell membrane, cytoplasm and nucleus. Specifically, discard the culture medium of primary chondrocytes cultured in a culture dish, add trypsin to digest the cells, centrifuge and discard the supernatant, add PBS to wash twice, 5 minutes each time; then add 1 mL of preheated serum-free solution. Cell culture medium or PBS diluted CFSE working solution (5 ~ 10 μM), incubate at room temperature for 15 minutes; then centrifuge and discard the supernatant, add PBS to wash the cells twice, 5 minutes each time; resuspend in 10% GelMA60 preheated at 37°C Stained cells; use a pipette to absorb 10 μL of cell-hydrogel mixture and slowly inject it into the chip channel, and cure with UV (P=3W) for 10 seconds; inject culture medium into the channels on both sides of the cell culture layer; use a laser confocal microscope to Observe below. A in Figure 4 is a diagram of three-dimensional cultured chondrocytes evenly distributed in the hydrogel in this embodiment. B in Figure 4 is a picture of the in situ cells deformed before and after mechanical compression is applied. From Figure 4, it can be seen that after the hydrogel deforms, the cells also deform. This shows that by controlling the deformation of the hydrogel in the three-dimensional cell culture layer of the present invention, precise control of mechanical stimulation and compression of cells can be achieved.

(3) Construction of biochemical microenvironment

The biochemical microenvironment of articular cartilage in the body includes immune cells such as macrophages, lymphocytes, and cytokines secreted by various cells. These biochemical factors can affect the homeostasis of chondrocytes, and cell debris and aggregated proteins produced by chondrocytes caused by damage caused by mechanical stimulation and other factors can stimulate the activation of other cells such as macrophages and prompt them to secrete cytokines. The crosstalk between cells in these force-chemistry-biogenesis coupled microenvironments in the joint cavity is very important to the occurrence and development of joint diseases. Therefore, the microfluidic chip designed in the present invention is used to construct the force of chondrocytes. - Models of coupled chemical-biological microenvironments are extremely valuable. Among them, the cytokines in the bio-chemical factors can be realized by adding directly to the cell culture medium, and the cells in the bio-chemical factors can achieve multi-cell co-culture by cleverly designing the structure of the microfluidic chip.

In this embodiment, other cells in the joint are distributed in the channels on both sides of the three-dimensional cell culture layer of the chip in a 2D growth form. In order to inoculate other related cells in the joint (macrophages in this example) into the chip, this example first requires surface modification of the glass substrates in the channels on both sides of the three-dimensional cell culture area. The material used for surface modification is Fibronectin material. In this embodiment, fibronectin is used to modify the surface of the glass substrate in the channel to make it suitable for cell adhesion and growth. The specific operation is as follows: place the sterilized and dried chip in a sterile petri dish, first rinse the channel with sterile water, and then use a pipette to draw 10 μL of fibronectin coating solution from one end of the channel into the microchannel. The glass surface at the bottom of the channel is coated, and the chip containing the coating solution is incubated in a clean bench for 1 hour and the remaining coating solution is sucked out, and then dried naturally for 1 hour before use.

When seeding cells, first seed the primary chondrocytes that need to be cultured in three dimensions into the middle channel of the chip. The specific operation is as described in step (1); then macrophages are seeded into the channels on both sides of the chip. Specifically, Use a pipette to take 20 μL of macrophage suspension prepared in culture medium (cell density range: 4×10 ⁶ cells/mL) and slowly inject it into the channels on both sides. Under the microscope, make sure that the cells cover the side cell culture channels. Evenly dispersed without accumulation, and allowed to adhere to the wall after being left to incubate in an incubator (37°C, 5% CO ₂ ) for more than 1 hour. Figure 5 is a distribution diagram of the co-culture of simulated three-dimensional cultured primary chondrocytes and 2D cultured macrophages in the present invention, in which the middle channel is the primary cartilage labeled with the green fluorescent probe CFSE (MCE, Cat. No. HY-D0938) Cells, in the channels on both sides are macrophages RAW264.7 labeled with the cell membrane red fluorescent dye Dil (Beyotime, Cat. No. C1991S).

The microfluidic chip of this embodiment can be reused. The hydrogel GelMA lysis solution is used to lyse the hydrogel in the microstructure channel of the three-dimensional cell culture layer, and then the cells in the chip channel are digested with trypsin, the channel is washed with PBS, and then sterilized before cell inoculation. deal with.

Example 2

In this example, a microfluidic chip is used to construct a differentiated mechanical microenvironment for three-dimensional cultured chondrocytes. The specific process is as follows:

(1)Chip cell inoculation and culture

The operation flow is as in step (1) in the embodiment.

(2) Use the function of microfluidic chip to perform local mechanical compression to construct a differentiated mechanical environment for three-dimensional cultured chondrocytes.

In order to enable the microfluidic chip provided by the present invention to achieve flux or localized research on mechanical stimulation of articular cartilage, the present invention designs a hydrogel channel in the air pressure control layer that is perpendicular to the three-dimensional cell culture layer below. Each of the three gas channels can deform the PDMS film and squeeze the hydrogel at the bottom of the chip by adjusting the level of compressed air flowing into the upper gas channel, causing the hydrogel to compress and deform. It is possible to apply mechanical force to the chondrocytes in the hydrogel; each channel will compress a part of the hydrogel in the hydrogel channel when air pressure is applied, thereby achieving controllability of the compression area in the same chip. These gas channels can be controlled individually or jointly, and different control schemes can be designed, such as applying air pressure to only one channel, applying air pressure to three channels together, or applying dynamic air pressure to the three channels in turn. The multi-channel design can make the mechanical stimulation faced by the cells in the chip more diverse, better simulate the in vivo environment, and can also study more sets of different air pressure output schemes separately. Circular areas for drilling are also reserved at both ends of the gas channel to facilitate access to air pressure output devices for control. Therefore, the present invention can construct different levels of local mechanical microenvironments in the same chip.

In this example, to quantify the local deformation of the hydrogel caused by each gas channel in the present invention, traced polystyrene fluorescent particles (Invitrogen, product number F8803) were doped into the hydrogel solution before being injected into the chip channel. The size is 100nm. A confocal microscope was used to conduct a three-dimensional scanning reconstruction of the thickness of the hydrogel in the chip channel before and after compression. The confocal microscope software NI-Elements was used to measure the longitudinal deformation of the hydrogel before and after compression. Figure 6 is a confocal imaging image of a hydrogel labeled with fluorescent particles that is locally compressed in the present invention. The relative positional relationship between a single gas channel and the hydrogel in the chip is shown in A in Figure 6 . B in Figure 6 is the longitudinal deformation diagram of the hydrogel when air pressure is applied to two adjacent gas channels at the same time. When air pressure is applied, the upper surface of the hydrogel is C in Figure 6 and the lower surface is D in Figure 6 . It can be seen from Figure 6 that when compressed air is introduced into each gas channel, the hydrogel below can deform; and the deformation of the hydrogel below a single gas channel is consistent with the longitudinal shape of the hydrogel in the uncompressed area. There are significant differences.

Figure 7 is a quantitative result of the local compression effect of the hydrogel in the present invention. A in Figure 7 is a quantitative result of the local compression deformation of the hydrogel when a single air pressure channel is introduced into different levels of compressed air in the present invention; Figure B in 7 is a quantitative result diagram of the influence on the local compression deformation of the corresponding hydrogel below the adjacent compression channel when a single air pressure channel passes different levels of compressed air in the present invention. It can be seen from A in Figure 7 that by changing the air pressure value, the degree of deformation of the hydrogel in the chip can be accurately controlled. When the chip structural parameters are constant, the compression deformation of the hydrogel gradually increases as the applied air pressure increases. When air pressure is applied to a single channel, the effect on adjacent channels is negligible. Therefore, it is proved that the present invention is used to perform local mechanical compression in the same chip to construct a differentiated mechanical environment.

(3) Detection of cartilage biological functions under differentiated mechanical microenvironment

The main components of articular cartilage extracellular matrix are type II collagen (Collagen II) and aggrecan (Aggrecan), which are characteristic markers of cartilage tissue and are also important indicators for detecting the maintenance of chondrocyte phenotype. In the pathogenesis of the most common joint degenerative diseases such as osteoarthritis (OA), the loss of aggregated proteins caused by reduced chondrocyte synthesis and activation of enzymes that degrade cartilage matrix is considered to be one of the earliest events in the OA process. Therefore, in this embodiment, detecting the synthesis levels of Collagen II and Aggrecan in chondrocytes can evaluate the status of chondrocytes. In addition, a variety of matrix-destroying enzymes produced by chondrocytes after injury, especially matrix metallopeptidase (MMP13) and aggregase 2 (ADAMTS5), are thought to play a key role in early cartilage degradation in osteoarthritis (OA). Responsible for the degradation of cartilage extracellular matrix, its content is overexpressed and the cartilage matrix is gradually degraded, which is also the main feature of OA. Therefore, in this example, chondrocyte damage can be evaluated by detecting the expression levels of MMP13 and ADAMTS5.

In order to explore the impact of different levels of mechanical compression on the phenotype and function of chondrocytes, the present invention was used to construct a differentiated mechanical environment and mechanically stimulate the three-dimensional cultured chondrocytes, and then in situ tested Collagen II, Aggrecan, MMP13 and ADAMTS5. Immunofluorescence staining. Specifically, primary mouse chondrocytes are first inoculated on the chip, and the inoculation method and culture method are as described in step (1) in Example 1. After the cells were seeded, the chip was placed in a 37°C 5% CO ₂ incubator for static culture for 7 days, during which the cells were replaced every other day. The three-dimensionally cultured cells were then subjected to local mechanical compression. The loading period was 14 days, 4 hours a day. The method of mechanical loading was to introduce different levels of compressed air into different gas channels of the air pressure control layer of the chip. The output air pressure was uniform. It is a sinusoidal waveform with a frequency of 0.5HZ, and the mechanical loading in the three gas channels simulates static, physiological dose stimulation and supraphysiological dose (ie, mechanical overload) mechanical stimulation conditions respectively. In this example, the parameters selected in the three channels are 0, 100mbar and 600mbar.

After the mechanical stimulation is completed, the local mechanically stimulated cells are characterized in situ. The specific operation is to use a pipette to suck out the culture medium in the chip channel, and then use a pipette to suck PBS at room temperature, slowly inject it into the chip channel, and wash it three times. 30 minutes each time; pipet up the cell fixative and slowly inject 4% paraformaldehyde into the channel to fix the cells in the chip. Fix at room temperature for 30 minutes; remove the fixative in the channel and wash carefully three times with PBS buffer, 30 minutes each time; use 0.2 Permeate with % TritonX-100 for 30 minutes, wash three times with PBS, 10 minutes each time; block with 5% goat serum blocking buffer at room temperature for 60 minutes; add primary antibody after blocking and incubate at 4°C overnight; wash slowly three times with PBS containing 0.1% Tween. Dilute the secondary antibody with antibody diluent for 30 minutes each time, and keep in the dark at room temperature for 60 minutes; wash three times with PBS buffer, 30 minutes each time; keep the nuclear dye DAPI for 5 minutes at room temperature, in the dark, and wash with PBS buffer three times after staining. 10min; finally take pictures using a laser confocal microscope.

Figure 8 shows the immunofluorescence experimental results of cartilage biological function evaluation using the matrix synthesis of chondrocytes under the differentiated mechanical environment constructed in the present invention. Among them, Static means no mechanical compression; PC (physiological compression) means the deformation of the hydrogel at a physiological dose (here is the deformation of the hydrogel when the compressed air value of 100mbar is introduced into the gas channel); HPC (hyper-physiological compression) It is the deformation of the hydrogel at a supraphysiological dose (here is the deformation of the hydrogel when the compressed air value of 600 mbar is introduced into the gas channel). As can be seen from Figure 8, the expression of Collagen II and Aggrecan in chondrocytes in the same chip has changed after different local mechanical stimulations. There are obvious differences in the expression levels between different areas. Specifically, for Collagen II , all were expressed in cartilage under three different mechanical stimulations. The expression level in the PC group under physiological dose mechanical stimulation was significantly higher than that in the Static group without mechanical stimulation; the expression level in the HPC group under superphysiological dose mechanical stimulation was compared to There was a significant decrease in the PC group, which was slightly lower than the Static group but not significantly. Aggrecan is expressed in both the Static group without mechanical stimulation and the PC group with physiological dose mechanical stimulation, and the expression level in the PC group is significantly higher than that in the Static group; it is basically not expressed in the superphysiological dose mechanical stimulation group, that is, the HPC group. From the above expression results of Collagen II and Aggrecan, it can be seen that mechanical stimulation at physiological doses promotes the formation of cell matrix, while mechanical loading at superphysiological doses inhibits the formation of cell matrix. At the same time, it also shows that the differential mechanical microenvironment constructed in the present invention can be used as a biological function evaluation model to study the impact of different levels of mechanical loading on chondrocyte matrix synthesis.

Figure 9 shows the immunofluorescence experimental results of the cartilage biological function evaluation of matrix-destroying enzymes of chondrocytes under the differentiated mechanical environment constructed by the present invention. As can be seen from Figure 9, the expression of MMP13 and ADAMTS5 in chondrocytes after different local mechanical stimulations on the same chip showed different levels. The expression levels of MMP13 and ADAMTS5 in the area of mechanical stimulation at a supraphysiological dose were both higher than those without mechanical stimulation. The stimulation area is the Static group and the physiological dose mechanical stimulation area is the PC group; while the expression of MMP13 and ADAMTS5 in chondrocytes in the PC area and Static group is low, and there is no significant difference, but the expression levels in both areas are significantly low. in the area of mechanical stimulation at supraphysiological doses. These experimental results indicate that supraphysiological doses of mechanical stimulation promote the formation of matrix-destroying enzymes in chondrocytes. It also shows that the differential mechanical microenvironment constructed in the present invention can be used as a biological function evaluation model to study the secretion of matrix-destroying enzymes after different levels of mechanical loading on chondrocytes after injury.

In summary, the present invention can use the function of local mechanical compression to construct a differentiated mechanical environment for three-dimensional cultured chondrocytes, and can use this differentiated mechanical microenvironment to establish a biological function evaluation model that simulates articular cartilage, and study the effects of mechanical stimulation on The effects of anabolism and catabolism on chondrocytes provide an effective model method, which is expected to help in mechanism research and drug screening of joint degenerative diseases such as osteoarthritis.

Example 3

In this embodiment, the microfluidic chip prepared in Example 1 is used to construct a microenvironment model of force-chemical-biogenesis coupling of three-dimensional cultured chondrocytes. The microenvironment model of force-chemistry-biogenesis coupling of chondrocyte cells constructed in this embodiment can be used to conduct many studies, such as the synthesis and metabolism of chondrocytes under the action of a single factor or a combination of multiple factors in the microenvironment, and mechanically stimulated chondrocytes. Cell behavior and its impact on other cells in the microenvironment, the impact of mechanical stimulation in the joint microenvironment on the immune microenvironment, etc. This example takes the phenotypic polarization of macrophages by chondrocytes under mechanical overload as an example. The specific implementation steps are as follows:

(1) Surface modification and cell seeding of microfluidic chips

The surface in the chip channel is treated with fibronectin, and the specific operation is the same as step (3) in Example 1. The surface-treated chip was dried at 37°C, and then chondrocytes were inoculated. The cell density range was: 4×10 ⁶ cells/mL. The specific operation was the same as step (1) in Example 1. Macrophages were then inoculated, and mouse macrophages RAW264.7 were selected. The cell density range was: 4×10 ⁶ cells/mL. The specific operation was the same as step (3) in Example 1. Then slowly put the chip into the incubator and let it stand for 1 hour to wait for macrophages to adhere to the wall. After observing macrophage adhesion using a microscope, absorb 200 μL of culture medium with a 200 μL pipette tip and vertically insert it into the entrance of the chip channel, and place the chip in an incubator (37°C, 5% CO ₂ ).

(2) Mechanical compression stimulates chondrocytes

Using the microfluidic chip of the present invention, the hydrogel is deformed by 30% by regulating the air pressure of the compressed air flowing into the air pressure control layer channel to simulate the deformation that occurs when articular cartilage in the body is subjected to mechanical overload. Based on what was mentioned in Example 1, in order to deform the hydrogel by more than 30%, the amount of compressed air flowing into the chip is 600 mbar. In order to simulate the walking frequency of adults, a dynamic air pressure frequency of 0.5HZ was used to stimulate chondrocytes for 4 hours. After continuing to culture for 24 hours, the cell phenotype of macrophages was analyzed.

(3) Phenotypic detection of macrophages

Immunofluorescence experiments and flow cytometry were used to test macrophage inflammatory markers (iNOS) for characterization. For the immunofluorescence experiment, the specific operation is: after the cell culture is completed, use a pipette to carefully and slowly suck out the culture medium in the chip channel, then use a pipette to suck PBS at room temperature, slowly inject it into the chip channel, and wash twice, each time 10s; pipette the cell fixative and slowly inject 4% paraformaldehyde into the channel to fix the cells in the chip. Fix at room temperature for 15 minutes; remove the fixative in the channel and wash carefully three times with PBS buffer, 5 minutes each time; use 0.2% TritonX Permeate with -100 for 5 minutes, wash three times with PBS, 5 minutes each time; block with 5% goat serum blocking buffer at room temperature for 60 minutes; after blocking, add the primary antibody iNOS (Proteintech, Cat. No. 22226-1-AP), and incubate at 4°C overnight; PBS Wash slowly three times with 0.1% Tween, 5 minutes each time, dilute the secondary antibody DyLight 488 (Boster, Cat. No. BA1145) with antibody diluent, and keep in the dark at room temperature for 60 minutes; wash three times with PBS buffer, 5 minutes each time; nuclear dye DAPI, at room temperature Protect from light for 3 minutes. After staining, add PBS buffer and wash three times for 3 minutes each time. Observe the iNOS expression of cells under a laser confocal microscope. Figure 10 shows the effects of chondrocytes that have not suffered mechanical overload and chondrocytes that have suffered mechanical overload on the iNOS expression content of the macrophage cell line RAW264.7. From Figure 10, it can be seen that chondrocytes that have suffered mechanical overload induce the expression of macrophages. Elevation of the pro-inflammatory marker iNOS. Flow cytometry can also be used to detect the expression level of macrophage inflammatory marker (iNOS). The specific operation is: use a pipette to suck out the culture medium in the microfluidic chip channel, wash it three times with PBS buffer, and Place the aspirated culture medium and washed PBS buffer in a centrifuge tube to reduce cell loss; inject the transferase into the channel, place the chip at 37°C for 1 minute, use a pipette to suck out the transferase, inject the culture medium containing serum, and repeat Rinse the channels of the chip with serum-containing culture fluid until all macrophages are washed out under a microscope. Place all the liquid in the above-mentioned centrifuge tube and centrifuge the cells at 1000 rpm for 5 minutes; wash twice with PBS buffer; add pre-cooled fixed/ Break membrane reagent (BD, Cat. No. 554714) for 20 minutes at 4°C; wash twice with pre-cooled membrane breaking/washing buffer; block with 5% goat serum, room temperature for 15 minutes; add fluorescent dye direct-labeled antibody (Invitrogen, Cat. No. 17-5920 -80), 4°C for 60 minutes; wash twice with membrane rupture/wash buffer, adjust cell suspension concentration, and analyze cells using flow cytometry. Figure 11 shows the flow cytometric test results of the effects of chondrocytes not subjected to mechanical overload and chondrocytes subjected to mechanical overload on the iNOS expression content of the macrophage cell line RAW264.7. As can be seen from Figure 11, the conclusion is consistent with the results of the immunofluorescence experiment. , chondrocytes that suffered mechanical overload caused an increase in the inflammatory marker iNOS in the mouse macrophage cell line RAW264.7, which further demonstrated that the microfluidic chip of the present invention was used to successfully study the effects of mechanical overload on articular cartilage. The impact on other cells within the articular cartilage further illustrates the mutual influence between the mechanical microenvironment and the biochemical microenvironment in the microenvironment of articular cartilage.

Comparative example 1

"Hyperphysiological compression of articular cartilage induces anosteoarthritic phenotype in a cartilage-on-a-chip model" (P. Occhetta, A. Mainardi, E. Votta, Q. Vallmajo-Martin, M. Ehrbar, I. Martin, A. Barbero , M.Rasponi, Nature Biomedical Engineering, 2019 Jul; 3(7):545-557.doi:10.1038/s41551-019-0406-3) discloses a mechanically compressible microphysiological system for the joint disease osteoarthritis Model building and drug screening. The structure of the chip in this document is shown in Figure 12 below. The three-dimensional cell microstructure of the chip in this document is a microscale system that is mechanically constrained and compressed. Shown in Figure 12, a: The microscale system consists of two separated PDMS microchambers with configurable geometry and separated by a PDMS membrane. As shown in b in Figure 12: the top compartment is subdivided into a central channel (blue part) that accommodates the three-dimensional microstructure and two medium supplement channels (red part) through two rows of T-shaped hanging columns (white part). The bottom chamber (grey part) represents the drive chamber. As shown in c in Figure 12: In the static position, the bottom compartment maintains normal pressure and the membrane remains flat. As shown in d in Figure 12: By pressurizing the bottom compartment, the PDMS membrane deforms, compresses the hydrogel (blue part) in the central channel, and finally clings to both ends of the column, causing restricted compression. e in Figure 12 is a photo of the assembled equipment. F in Figure 12 is a stereomicroscope image showing a T-shaped pillar viewed from above. Figure 13 is a microscope image of the microstructure of the chip in the literature, as well as the gap and size between the PDMS film and the microstructure. Hposts and hgap represent the height of the hanging posts and the height of the gap respectively. The height is set based on the degree of compression. It can be seen from Figures 12 and 13 that the microphysiological system in this document precisely controls the mechanical compression level of the cell-hydrogel structure by adjusting the gap under the suspension column from the PDMS elastic membrane. Therefore, the factor that determines the compression performance of the chip in this document is the gap between the lower surface of the suspension column set during chip preparation and the PDMS elastic mode below. Therefore, one chip in this document can only achieve a single compression level. .

In addition, there is a gap between the chip culture chamber and the underlying PDMS flexible membrane in this document. When the cell-hydrogel mixture is inoculated in the middle channel of the microstructure for three-dimensional culture of cells, other types of cells cannot be cultured. Cultured, such as immune cells, synovial cells, etc., which are abundant in the joint cavity, are very important when studying joint diseases. For example, the presence of inflammatory cells will aggravate the response of chondrocytes to mechanical stimulation. This document The chip in cannot meet this requirement.

The microfluidic chip provided by the present invention can construct a force-chemical-biogenesis microenvironment of chondrocytes. Specifically, by designing the structure of the microfluidic chip, it can be achieved: simulating the three-dimensional growth state of articular cartilage in the body, simulating the mechanical compression stimulation of articular cartilage cells, simulating the microenvironment of multi-cell coexistence of articular cartilage in the body, and simulating mechanical stimulation of articular cartilage. and biochemical synergistic microenvironment. The multifunctional microfluidic chip can be used to study the response of chondrocytes on various scales after being mechanically stimulated. The multifunctional microfluidic chip can be used to study the impact of chondrocytes subjected to different degrees of mechanical compression on other cells in the joint to simulate whether chondrocytes in diseased articular cartilage in the real body will affect the response of other cells in the joint. Aggravate joint disease. In addition, the multifunctional microfluidic chip can be used to study the response of chondrocytes under the synergistic effect of mechanical compression and immune cells such as macrophages. The multifunctional microfluidic chip can be used to establish joint injury models, drug research and development, etc.

Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. Other embodiments can also be obtained according to this embodiment without any inventive step, and these embodiments are all It belongs to the protection scope of the present invention.

Claims (10)

1. The microfluidic chip is characterized by comprising a basal layer (1), and a cell three-dimensional culture layer (2) and an air pressure control layer (3) which are sequentially laminated on the basal layer (1); the basal layer (1), the cell three-dimensional culture layer (2) and the air pressure control layer (3) are made of transparent materials;

the three-dimensional cell culture layer (2) is provided with a three-dimensional cell culture channel (21), a first cell culture fluid channel (22) and a second cell culture fluid channel (23) which are positioned at two sides of the three-dimensional cell culture channel (21), a plurality of microstructures are arranged between the three-dimensional cell culture channel (21) and the first cell culture fluid channel (22) and between the three-dimensional cell culture channel (21) and the second cell culture fluid channel (23) to separate, gaps exist between the microstructures, two ends of the three-dimensional cell culture channel (21) are provided with a cell carrier fluid inlet channel (24) and a cell carrier fluid outlet channel (25), two ends of the first cell culture fluid channel (22) are provided with a first cell culture fluid inlet channel (26) and a first cell culture fluid outlet channel (27), and two ends of the second cell culture fluid channel (23) are provided with a second cell culture fluid inlet channel (28) and a second cell culture fluid outlet channel (29);

The cell three-dimensional culture device comprises a cell three-dimensional culture channel (21), a gas pressure control layer (3), a plurality of gas channels perpendicular to the cell three-dimensional culture channel (21), gas inlets and outlets arranged at two ends of each gas channel, extrusion parts arranged between the gas inlets and outlets at two ends of each gas channel, three liquid through holes arranged at two ends of the gas pressure control layer and respectively communicated with a cell carrier fluid inlet channel (24), a cell carrier fluid outlet channel (25), a first cell culture fluid inlet channel (26), a first cell culture fluid outlet channel (27), a second cell culture fluid inlet channel (28) and a first cell culture fluid outlet channel (29).

2. The microfluidic chip according to claim 1, further comprising a liquid storage layer stacked on the air pressure control layer (3), wherein the liquid storage layer comprises two liquid storage tanks (4) arranged on two sides of the air channel, three liquid storage through holes are respectively arranged in each Chu Yechi (4), and each liquid storage through hole is respectively communicated with each liquid through hole.

3. The microfluidic chip according to claim 1, wherein the cell three-dimensional culture channel (21), the first cell culture fluid channel (22) and the second cell culture fluid channel (23) are of a channel structure with upper portions sealed and lower portions open; the microstructure is in a fence shape, and one end of the microstructure is in contact with the basal layer (1); the cell carrier fluid inlet channel (24), the cell carrier fluid outlet channel (25), the first cell culture fluid inlet channel (26), the first cell culture fluid outlet channel (27), the second cell culture fluid inlet channel (28) and the first cell culture fluid outlet channel (29) are through holes which are communicated up and down.

4. A microfluidic chip according to claim 3, wherein the material of the cell three-dimensional culture layer (2) is polydimethylsiloxane;

the thickness of the cell three-dimensional culture layer (2) is 200-400 mu m, and the width of the cell three-dimensional culture channel (21) is 1-1.5 mm; the width of the first cell culture fluid channel (22) and the second cell culture fluid channel (23) is 1-2 mm; the depth of the cell three-dimensional culture channel (21), the first cell culture fluid channel (22) and the second cell culture fluid channel (23) is 70-300 mu m.

5. The microfluidic chip according to claim 1, wherein the gas channel has a structure in which an upper portion seals a lower portion opening; the number of the gas channels is 3;

the material of the air pressure control layer (3) is polydimethylsiloxane; the width of the gas channel is 1-1.5 mm, the depth is 50-200 mu m, and the width of the extrusion part is larger than the width of the gas channel.

6. The microfluidic chip according to claim 1, wherein the substrate layer (1) is made of glass; the thickness of the substrate layer (1) is 0.13-0.17 mm;

the material of the liquid storage layer is polydimethylsiloxane, and the diameter of the liquid storage through hole is 1.5-3.5 mm.

7. A method for constructing a differential mechanical microenvironment model of a three-dimensionally cultured chondrocyte by using the microfluidic chip according to any one of claims 1 to 6, comprising the following steps:

injecting the mixture of the chondrocyte and the hydrogel into a cell three-dimensional culture channel (21) of a cell three-dimensional culture layer (2), and performing photocuring under ultraviolet irradiation to obtain solidified chondrocyte and hydrogel; and then, injecting a chondrocyte culture solution into a first cell culture solution channel (22) and a second cell culture solution channel (23) of the cell three-dimensional culture layer (2) to perform chondrocyte culture, and introducing compressed air with different pressure levels into the gas channel of the air pressure control layer (3) in the chondrocyte culture process to perform mechanical force stimulation with different levels on chondrocytes in solidified chondrocyte-hydrogel in different areas in the cell three-dimensional culture channel (21).

8. A method for constructing a force-chemo-bio coupled micro-environment model of three-dimensionally cultured chondrocytes using the microfluidic chip according to any one of claims 1 to 6, comprising the steps of:

1) surface-modifying the surface of the basal layer (1) in contact with the first cell culture fluid channel (22) and the second cell culture fluid channel (23) of the cell three-dimensional culture layer (2) with a coating material;

2) Injecting the mixture of the chondrocyte and the hydrogel into a cell three-dimensional culture channel (21) of a cell three-dimensional culture layer (2), and performing photocuring under ultraviolet irradiation to obtain solidified chondrocyte and hydrogel;

3) Injecting a composite cell culture solution into the first cell culture solution channel (22) and the second cell culture solution channel (23) with the modified closed bottom surface, wherein the composite cell culture solution contains one or more of cells related to chondrocytes, biochemical factors related to chondrocytes, chemokines related to chondrocytes and pharmaceutical agents, and performing cell co-culture; in the process of cell co-culture, compressed air is introduced into the air channel of the air pressure control layer (3) to perform mechanical force stimulation on the chondrocytes in the solidified chondrocyte-hydrogel in different areas in the cell three-dimensional culture channel (21); or compressed air is introduced into the air channel of the air pressure control layer (3) to perform mechanical force stimulation on the chondrocytes in the solidified chondrocyte-hydrogel in different areas in the cell three-dimensional culture channel (21); and then injecting a composite cell culture solution into the first cell culture solution channel (22) and the second cell culture solution channel (23) with the modified closed bottom surface, wherein the composite cell culture solution contains one or more of cells related to chondrocytes, biochemical factors related to chondrocytes, chemokines related to chondrocytes and pharmaceutical reagents, and cell co-culture is performed.

9. A method according to claim 7 or 8, wherein the pressure of the compressed air is between 0 and 1500mbar.

10. The method of claim 7 or 8, wherein the chondrocyte-hydrogel mixture comprises chondrocyte and a hydrogel solution; chondrocyte density of 2X 10 ⁵ ～4×10 ⁶ Individual cells/mL; the hydrogel solution is a methacryloylated gelatin solution, and the mass content of the methacryloylated gelatin solution is 10-15%; when the ultraviolet light is cured, the power of ultraviolet light is 3W, and the curing time is less than or equal to 10s.