Micro-fluidic chip model structure for improving yield of cell exosomes and use method of micro-fluidic chip model structure

Abstract

The invention discloses a micro-fluidic chip model structure for improving the yield of cell exosomes, which is characterized in that the micro-fluidic chip model structure is formed by bonding a micro-fluidic chip main body and a bottom substrate, and comprises a cell culture channel with a culture solution inlet and a culture solution outlet; the cell culture channel comprises an upper culture solution flowing layer, a middle cell culture layer and a lower collagen gel layer; the culture solution inlet and the culture solution outlet are connected to a circulating pump system for circulating flow of a culture solution and are used for applying mechanical force stimulation to cells to promote exosome production. According to the invention, the mechanical stimulation effect of a liquid environment in a living body on cells can be simulated, the yield of exosomes can be conveniently increased, the operation process is simplified, the experiment efficiency is improved, and meanwhile, the influence of shear force on the cells can be better controlled by designing a specific structural layout between the cell culture layer and the collagen gel layer, so that the experiment efficiency is improved. Therefore, the cell growth environment is optimized, and the production efficiency and quality of the exosome are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a microfluidic chip model structure for improving the yield of extracellular vesicles and a method for using the same. Background Art

[0002] Almost all cells transmit information and exchange substances between cells by secreting exosomes, thereby achieving a variety of biological functions. Therefore, exosomes are an important cellular product for diagnosing and monitoring diseases. The traditional method of extracting exosomes is to extract exosomes after the culture flask / dish is full of cells. The extraction methods include ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, magnetic bead immunoassay, etc. These methods are very time-consuming and labor-intensive, and the steps are cumbersome and inefficient.

[0003] To this end, the present application specifically proposes a microfluidic chip model structure for increasing the production of cell exosomes, which can simulate the blood flow in blood vessels and simulate the mechanical stimulation of blood on cells, thereby increasing the production of exosomes. At the same time, it is easy to operate and highly efficient to solve the above technical problems. Summary of the invention

[0004] The main purpose of the present invention is to provide a microfluidic chip model structure for improving the yield of cell exosomes, so as to solve the technical problem of low exosome yield in the existing methods proposed in the background technology.

[0005] The present invention adopts the following technical solutions to solve the above technical problems: A microfluidic chip model structure for improving the production of cell exosomes, which is composed of a microfluidic chip body and a bottom substrate bonded together, and includes a cell culture channel with a culture fluid inlet and a culture fluid outlet; The cell culture channel includes an upper culture fluid flow layer, a middle cell culture layer and a lower collagen gel layer; The culture fluid inlet and the culture fluid outlet are both connected to a circulating pump system for circulating the culture fluid, and are used to cooperate with the cell culture channel to apply mechanical force stimulation to the cells in the cell culture layer to promote the production of exosomes.

[0006] Therefore, compared with the static culture adopted in the prior art (a patent with application number 202211270154.3: a tumor microfluidic chip for mesenchymal stem cell migration screening and its preparation and application method, which utilizes the microstructure channel on the chip body to construct a static culture channel, and realizes the synchronous co-culture of various types of mesenchymal stem cells and tumor cells. The operation is simple, and the amount of actual samples is reduced. It has the advantages of simple preparation, portability, economy, high efficiency and accuracy), the microfluidic chip model structure enables cells to grow in a three-dimensional environment that is closer to the body, can target exosome production, enhance cell activity and exosome secretion, and greatly improve the efficiency of exosome production during cell culture.

[0007] Preferably, the cell culture channel is divided into two upper and lower channels at the culture fluid inlet, and is reunited into one channel at the culture fluid outlet. Preferably, the cell culture channel is spiral-shaped, and the culture fluid outlet is located at the center of the spiral.

[0008] Preferably, the cell culture channel is in a broken line shape, and the number of the cell culture channels is set to 2.

[0009] Preferably, the main body material of the microfluidic chip includes but is not limited to PDMS, and can also be replaced by other existing materials such as 3D printing materials, glass, acrylic plates, etc.

[0010] Preferably, the material of the bottom substrate includes but is not limited to glass, and other existing materials may also be used instead.

[0011] A method for using a microfluidic chip model, which is used to use the above-mentioned microfluidic chip model structure for increasing the yield of cell exosomes, comprising: S1. Prepare a matrix solution suitable for cell growth and with appropriate pH and nutrient conditions: take out a centrifuge tube and place it on ice, add collagen type I to the centrifuge tube placed in an ice bath, add NaOH to adjust the pH to 7.4, and then mix immediately; S2. Add 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel of the microfluidic chip model; S3. After the gel is solidified, a circulation pump system is connected to the culture medium inlet, and the circulation pump is started until the culture medium just reaches the inlet; S4. Use a medical needle to take 200 μL of endothelial cell suspension and add it into the microfluidic chip model from the culture medium outlet. Then connect the culture medium outlet to the circulation pump system, start the circulation pump until the culture medium completely covers the entire cell culture channel, and put the microfluidic chip body into a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall. S5. Start the circulation pump system to culture the cells in a fixed shear force system.

[0012] A method for using a microfluidic chip model, which is used to use the above-mentioned microfluidic chip model structure for increasing the yield of cell exosomes, comprising: L1. Prepare a matrix solution with appropriate pH and nutrient conditions suitable for cell growth: add 112 μL of HO 2 O, 4 μL 1 mol / L NaOH, 20 μL 8% NaHCO 3 , 100 μL of DMEM, mix immediately; L2. Add 20 μL of 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel of the microfluidic chip model; L3. After the gel solidifies, connect the circulation pump system to the culture medium inlet and start the circulation pump until the culture medium just reaches the inlet; L4. Use a medical needle to take 200 μL of endothelial cell suspension and add it into the microfluidic chip model from the culture fluid outlet. Then connect the culture fluid outlet to the circulation pump system, start the circulation pump until the culture fluid completely covers the entire cell culture channel, and place the microfluidic chip body into a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall. L5. Start the circulation pump system to culture the cells in a fixed shear force system.

[0013] It can be seen from the above technical solution that the present invention provides a microfluidic chip model structure for increasing the production of cell exosomes. Compared with the prior art, the present invention has the following advantages: 1. The present invention can simulate the mechanical stimulation of the liquid environment in the body on cells by setting up a cell culture channel in the microfluidic chip model, thereby facilitating the increase of exosome production, simplifying the operation process and improving experimental efficiency.

[0014] 2. The present invention can better control the effect of shear force on cells by designing a specific structural layout between the cell culture layer and the collagen gel layer, such as a spiral or zigzag cell culture channel, thereby optimizing the cell growth environment and significantly improving the production efficiency and quality of exosomes.

[0015] 3. The present invention uses a circulating pump system to achieve the flow and circulation of the culture medium, and cultures cells under different shear force conditions, which can accurately control the intensity of mechanical stimulation applied to the cells, thereby comparing the influence mechanism of different shear forces on cell morphology, orientation and exosome production, and facilitating data analysis and processing of exosome secretion.

[0016] 4. The present invention uses PDMS as the main material of the microfluidic chip and glass as the bottom substrate material, which can ensure the transparency of the chip while providing good biocompatibility, thereby facilitating the observation of cell growth conditions and supporting long-term and stable cell culture experiments.

[0017] 5. The present invention not only increases the yield of exosomes, but also shows obvious advantages in ease of operation and efficiency compared with traditional methods, and has practical application value. Especially under the spiral structure, the yield of exosomes reaches the highest level, which further verifies the effectiveness and practicality of the present invention.

[0018] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present invention, nor is it intended to limit the scope of the present invention. Other features of the present invention will become easy to understand through the following description. Of course, it is not necessary to achieve all of the advantages described above simultaneously for any product implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 It is a schematic diagram of the bonding structure of the microfluidic chip model of the present invention; Figure 2 It is a three-dimensional schematic diagram of the fork-shaped structure of the microfluidic chip model of the present invention; Figure 3 It is a three-dimensional schematic diagram of the spiral structure of the microfluidic chip model of the present invention; Figure 4 It is a three-dimensional schematic diagram of the broken-line structure of the microfluidic chip model of the present invention; Figure 5 It is a schematic diagram of the cell dynamic culture system of the present invention; Figure 6 It is a schematic diagram comparing the simulation results of the fork-type and spiral-type models, where the upper figure is the simulation result of the fork-type model fluid, and the lower figure is the simulation result of the spiral-type model fluid; Figure 7 A schematic diagram showing the comparison of the results of the cells of the present invention after being cultured for 24 hours under different shear force conditions in a microfluidic chip model; Figure 8 It is a schematic diagram comparing the results of the cells of the present invention after being cultured for 48 hours under different shear force conditions in the microfluidic chip model; Fig. 9 It is a schematic diagram comparing the exosome production of cells of the present invention under different shear force environments in the spiral microfluidic chip model; Fig.10It is a schematic diagram comparing the results of using nanoparticle tracking analysis technology for exosomes of cells in different shear force environments in the spiral microfluidic chip model of the present invention; Fig.11 A schematic diagram showing a comparison of transmission electron microscopy results of exosomes extracted from cells of the present invention under different shear force environments in a spiral microfluidic chip model; Fig.12 A schematic diagram showing the comparison of flow cytometry results of exosomes extracted from cells of the present invention under different shear force environments in a spiral microfluidic chip model; Fig.13 A schematic diagram showing the comparison of miRNA expression of cells under different shear stress environments in the spiral microfluidic chip model of the present invention; Fig.14 It is a schematic diagram comparing the results of exosome protein concentration of the cells of the present invention under different microfluidic chip model structures and culture flask conditions; Fig.15 A schematic diagram showing the comparison of exosome concentrations of different types of cells cultured under different shear force environments in the model of the present invention; In the figure: 1. Culture medium inlet; 2. Culture medium outlet; 3. Cell culture channel; 4. Microfluidic chip body; 5. Bottom substrate. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] In the embodiment, see Figures 1 to 15 .

[0022] In the first embodiment, if Figure 1 The microfluidic chip model structure for improving the yield of exosomes proposed in the embodiment of the present invention aims to solve the problems that the traditional collection of exosomes is very time-consuming and labor-intensive, and the steps are complicated and inefficient.

[0023] The microfluidic chip model structure for increasing the production of cell exosomes can simulate the liquid environment in the body, produce mechanical stimulation to the cells, thereby increasing the production of exosomes, and is easy to operate and highly efficient. It is composed of a microfluidic chip made of PDMS and a bottom substrate 5 made of glass, and includes a cell culture channel 3 with a culture fluid inlet 1 and a culture fluid outlet 2; The cell culture channel 3 includes an upper culture fluid flow layer, a middle cell culture layer, and a lower collagen gel layer; The culture solution inlet 1 is connected to the circulation pump system to realize the flow of the culture solution, and the culture solution outlet 2 is connected to the circulation pump system to realize the circulation of the culture solution; The culture fluid inlet 1 and the culture fluid outlet 2 are connected to a circulation pump system for circulating the culture fluid, which can be used to cooperate with the cell culture channel 3 to apply mechanical force stimulation to the cells in the cell culture layer to promote the production of exosomes.

[0024] This model uses a circulating pump system to achieve the flow and circulation of the culture medium and culture cells under different shear force conditions. It can accurately control the intensity of mechanical stimulation applied to the cells, thereby comparing the impact of different shear forces on cell morphology, orientation and exosome production, facilitating data analysis and processing of exosome secretion.

[0025] What needs to be explained at this point is that the main body of the microfluidic chip of this model can be made of glass, acrylic board, 3D printing and other materials in addition to PDMS, and similarly, the bottom substrate is not limited to glass material.

[0026] It can also be added that the present application also uses PDMS as the material of the microfluidic chip body 4 and glass as the material of the bottom substrate 5, which can ensure the transparency of the chip while providing good biocompatibility, thereby facilitating the observation of cell growth conditions and supporting long-term and stable cell culture experiments.

[0027] In the experimental comparative example, Figure 7 As shown, based on the comparison of the results after cells were cultured in the PDMS chip under 0 dyn / cm2 shear force (left) and 20 dyn / cm2 shear force (right) for 24 hours, it is obvious that compared with the 0 dyn / cm2 shear force, the cell morphology and cell orientation cultured under the 20 dyn / cm2 shear force environment for 24 hours have changed significantly.

[0028] In the experimental comparative example, Figure 8 As shown, based on the comparison of the results after cells were cultured in the PDMS chip under 0 dyn / cm2 shear force (left) and 20 dyn / cm2 shear force (right) for 48 hours, it is obvious that compared with the 0 dyn / cm2 shear force, the cell morphology and cell orientation cultured under the 20 dyn / cm2 shear force environment for 48 hours have changed significantly.

[0029] In the experimental comparative example, Fig. 9As shown in the figure, based on the comparison of exosome production in cells under three shear stress environments of 0 dyn / cm2, 10 dyn / cm2, and 20 dyn / cm2, it is obvious that within a certain limit, the exosome production is proportional to the shear stress, and the exosome production of cells increases significantly in the shear stress environment of 20 dyn / cm2.

[0030] In the second embodiment, if Figure 2 As shown, the microfluidic chip model structure for improving the production of extracellular vesicles obtained by bonding in the first embodiment is adopted. At this time, the cell culture channel 3 is divided into two upper and lower channels at the culture fluid inlet 1, and is re-merged into one channel at the culture fluid outlet 2.

[0031] Furthermore, the specific method for making and using the microfluidic chip model structure for increasing the production of extracellular vesicles includes: S1. Use computer-aided design (CAD) to draw the microfluidic chip template, then make the PDMS chip, and then bond the PDMS chip to the glass slide.

[0032] S2. Prepare a matrix solution suitable for cell growth and having appropriate pH and nutrient conditions: take out the centrifuge tube and place it on ice, add collagen type I to the centrifuge tube placed in an ice bath, add NaOH to adjust the pH to 7.4, and then mix immediately; S3. Add 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel 3 of the microfluidic chip model; S4. After the gel is solidified, a circulation pump system is connected to the culture medium inlet 1, and the circulation pump is started until the culture medium just reaches the inlet; S5. Use a medical needle to take 200 μL of endothelial cell suspension and add it into the microfluidic chip model from the culture fluid outlet 2. Then connect the culture fluid outlet 2 to the circulation pump system, start the circulation pump until the culture fluid completely covers the entire cell culture channel 3, and put the microfluidic chip body 4 into a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall; S6. Start the circulation pump system to culture the cells in a fixed shear force system.

[0033] In the third embodiment, Figure 3 As shown, the microfluidic chip model structure for improving the production of extracellular vesicles obtained by bonding in the first embodiment is adopted. At this time, the cell culture channel 3 is divided into two upper and lower channels at the culture fluid inlet 1, and is re-merged into one channel at the culture fluid outlet 2.

[0034] Furthermore, the specific method for making and using the microfluidic chip model structure for increasing the production of extracellular vesicles includes: S1. Use computer-aided design (CAD) to draw the microfluidic chip template, then make the PDMS chip, and then bond the PDMS chip to the glass slide.

[0035] S2. Prepare a matrix solution with appropriate pH and nutrient conditions suitable for cell growth: add 112 μL of HO 2 O, 4 μL 1 mol / L NaOH, 20 μL 8% NaHCO 3 , 100 μL of DMEM, mix immediately; S3. Add 20 μL of 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel 3 of the microfluidic chip model; S4. After the gel is solidified, a circulation pump system is connected to the culture medium inlet 1, and the circulation pump is started until the culture medium just reaches the inlet; S5. Use a medical needle to take 200 μL of endothelial cell suspension and add it into the microfluidic chip model from the culture fluid outlet 2. Then connect the culture fluid outlet 2 to the circulation pump system, start the circulation pump until the culture fluid completely covers the entire cell culture channel 3, and put the microfluidic chip body 4 into a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall; S6. Start the circulation pump system to culture the cells in a fixed shear force system.

[0036] At this time, the microfluidic chip model structure comparison for increasing the production of extracellular vesicles proposed in the second embodiment and the third embodiment is summarized as follows: Figure 6 As shown, by comparing the spiral and fork models, it can be seen that the shear force distribution of the spiral type is more uniform and stable. At the same time, the spiral type has a higher space utilization rate than the fork type (for the same size of design area, the spiral type has a larger area) and the spatial layout of the spiral type is more in line with the cell environment than the fork type. Therefore, the spiral type is a more superior microfluidic chip model than the zigzag type.

[0037] In the fourth embodiment, if Figure 4 As shown, the microfluidic chip model structure for improving the production of extracellular vesicles obtained by bonding in the first embodiment is adopted. At this time, the cell culture channel 3 is in a broken line shape, and the number of cell culture channels 3 is set to 2 and arranged in parallel, and obviously, the culture fluid inlet 1 and the culture fluid outlet 2 are also 2.

[0038] Furthermore, the specific method for making and using the microfluidic chip model structure for increasing the production of extracellular vesicles includes: S1. Use computer-aided design (CAD) to draw the microfluidic chip template, then make the PDMS chip, and then bond the PDMS chip to the glass slide.

[0039] S2. Prepare a matrix solution with appropriate pH and nutrient conditions suitable for cell growth: add 112 μL of HO 2 O, 4 μL 1 mol / L NaOH, 20 μL 8% NaHCO 3 , 100 μL of DMEM, mix immediately; S3. Add 20 μL of 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel 3 of the microfluidic chip model; S4. After the gel is solidified, a circulation pump system is connected to the culture medium inlet 1, and the circulation pump is started until the culture medium just reaches the inlet; S5. Use a medical needle to take 200 μL of endothelial cell suspension and add it into the microfluidic chip model from the culture fluid outlet 2. Then connect the culture fluid outlet 2 to the circulation pump system, start the circulation pump until the culture fluid completely covers the entire cell culture channel 3, and put the microfluidic chip body 4 into a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall; S6. Start the circulation pump system to culture the cells in a fixed shear force system.

[0040] Further, such as Figure 5 As shown, the circulation pump system, the microfluidic chip model and the culture fluid constitute a cell dynamic culture system. At this time, the cell dynamic culture system has three parts: a peristaltic pump, a microfluidic chip and a culture fluid. The cells are cultured in the culture fluid circulation system. The peristaltic pump provides power for the culture fluid circulation system. The microfluidic chip is the cell culture environment. The culture fluid provides sufficient culture fluid. The three together constitute the cell dynamic culture system of the present invention.

[0041] It should be noted that in order to increase the production of exosomes, the present application adopts dynamic cell culture. Compared with the static culture used in the prior art, it enables cells to grow in a three-dimensional environment that is closer to the body, and can target exosome production, enhance cell activity and exosome secretion, and greatly improve the efficiency of exosome production during cell culture.

[0042] Based on the above example, the experiment was performed and 0 dyn / cm 2 The environment is a culture bottle environment, so it can be understood that 0dyn / cm 2 The environment is a culture bottle environment, with: In the specific test comparison process, Fig.10 As shown, the results of nanoparticle tracking analysis of exosomes in cells under two shear stress environments of 0 dyn / cm2 and 20 dyn / cm2 in a spiral microfluidic chip model showed that the exosomes produced by cells under the shear stress environment of 20 dyn / cm2 were several times that under the shear stress environment of 0 dyn / cm2. In the specific test comparison process, Fig.11 As shown, by comparing the transmission electron microscopy results of exosomes extracted from cells in a spiral microfluidic chip model under two shear stress environments of 0 dyn / cm2 and 20 dyn / cm2, it can be seen that the number of exosomes extracted under the shear stress environment of 20 dyn / cm2 (right picture) is significantly higher than that under the shear stress environment of 0 dyn / cm2 (left picture); In the specific test comparison process, Fig.12 As shown, the results of flow cytometry were compared by extracting exosomes from cells under two shear stress environments of 0 dyn / cm2 and 20 dyn / cm2 in a spiral microfluidic chip model. It was found that the number of exosomes produced by cells under the shear stress environment of 20 dyn / cm2 (red) was higher than that under the shear stress environment of 0 dyn / cm2 (blue). In the specific test comparison process, Fig.13 As shown, by comparing the miRNA expression of cells in the spiral microfluidic chip model under two shear stress environments of 0dyn / cm2 and 20dyn / cm2, it is obvious that the expression of cell miRNA increases under high shear stress, indicating that the secretion of cell exosomes increases; In the specific test comparison process, Fig.14 As shown in the figure, by comparing the results of exosome protein concentrations of cells under different microfluidic chip model structures and culture flask conditions, it was found that the spiral exosome production was the highest, which was several times that of the culture flask, followed by the zigzag and fork types, which were 2-3 times that of the culture flask; In the specific test comparison process, Fig.15 As shown in the figure, by comparing the exosome concentrations of different types of cells cultured in the model under two shear force environments of 0 dyn / cm2 and 20 dyn / cm2, it was found that the exosome production of the five types of cells (endothelial cells, epithelial cells, CT26 cells, 4T1 cells, LLC cells) in the two shear force environments of 20 dyn / cm2 was several times that of those cultured under 0 dyn / cm2.

[0043] Based on the comparative experiments of the above different embodiments, the comparison of the exosome protein concentrations of cells in the above-mentioned microfluidic chip models of different structural types and culture flask conditions, compared with the static environment, the cells cultured in the microfluidic chip model have a higher exosome protein yield, among which the "spiral type" has the highest yield, and the kit used is the BCA protein concentration test kit.

[0044] Therefore, the present application can better control the effect of shear force on cells by designing a specific structural layout between the cell culture layer and the collagen gel layer, such as a spiral or zigzag cell culture channel 3, thereby optimizing the cell growth environment and significantly improving the production efficiency and quality of exosomes.

[0045] In addition, this model structure not only increases the yield of exosomes, but also shows obvious advantages in ease of operation and efficiency compared to traditional methods, and has practical application value. Especially under the spiral structure, the yield of exosomes reaches the highest level, which further verifies the effectiveness and practicality of the present invention.

[0046] In summary, this model and its usage method can increase the production of exosomes. Compared with traditional methods, it has the advantages of simple operation and high efficiency, and has practical application effects.

[0047] It is understandable that the system provided by the embodiment of the present invention corresponds to the method provided by the embodiment of the present invention, and the explanation, examples and beneficial effects of the relevant contents can refer to the corresponding parts in the above method.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0049] In addition, it should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back...), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0050] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, in the embodiments of the present invention, "multiple" refers to more than two. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in the field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

Claims

1. A microfluidic chip model structure for increasing the production of exosomes, characterized in that: It is composed of a microfluidic chip body (4) and a bottom substrate (5) bonded together, and includes a cell culture channel (3) with a culture fluid inlet (1) and a culture fluid outlet (2); The cell culture channel (3) comprises an upper culture fluid flow layer, a middle cell culture layer and a lower collagen gel layer; The culture fluid inlet (1) and the culture fluid outlet (2) are both connected to a circulation pump system for circulating the culture fluid, and are used to cooperate with the cell culture channel (3) to apply mechanical force stimulation to the cells in the cell culture layer to promote the production of exosomes.

2. The microfluidic chip model structure for increasing the production of extracellular vesicles according to claim 1, characterized in that: The cell culture channel (3) is separated into two upper and lower channels at the culture fluid inlet (1), and is reunited into one channel at the culture fluid outlet (2).

3. The microfluidic chip model structure for increasing the production of exosomes according to claim 1, characterized in that: The cell culture channel (3) is spiral-shaped, and the culture fluid outlet (2) is located at the center of the spiral.

4. The microfluidic chip model structure for increasing the production of exosomes according to claim 1, characterized in that: The cell culture channel (3) is in the shape of a broken line, and the number of the cell culture channels (3) is set to 2.

5. A method for using a microfluidic chip model, for using the microfluidic chip model structure for increasing the production of cell exosomes according to any one of claims 1 to 4, characterized in that: include: S1. Prepare the specified matrix solution; S2. Add 10× PBS or 10× culture medium, mix well and immediately add to the cell culture channel (3) of the microfluidic chip model; S3. After the gel is solidified, a circulation pump system is connected to the culture medium inlet (1), and the circulation pump is started until the culture medium just reaches the inlet; S4. Use a medical needle to take 200 μL of endothelial cell suspension from the culture medium outlet (2) and add it to the microfluidic chip model. Then connect the culture medium outlet (2) to the circulation pump system, start the circulation pump until the culture medium completely covers the entire cell culture channel, and place the microfluidic chip model in a carbon dioxide incubator and wait for 4 hours for the cells to adhere to the wall. S5. Start the circulation pump system to culture the cells in a fixed shear force system.

6. The method for using the microfluidic chip model as claimed in claim 5, characterized in that: The preparation process of the matrix solution in step S1 includes: taking out a centrifuge tube and placing it on ice, adding collagen type I to the centrifuge tube placed in an ice bath, adding NaOH to adjust the pH to 7.4, and then immediately mixing.

7. The method for using the microfluidic chip model as claimed in claim 5, characterized in that: The preparation process of the matrix solution specified in step S1 includes: adding 112 μL of H2O, 4 μL of 1 mol / L NaOH, 20 μL of 8% NaHCO3, and 100 μL of DMEM, and mixing immediately.

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