Microfluidic exosome mixer, application and operation method

Abstract

The invention discloses a tumor microenvironment exosome mixer based on a micro-fluidic chip, application and an operation method, and a micro-fluidic operation platform and an operation method for improving the balling rate of single-cell tumor balls. Different types of tumor microenvironment exosomes are mixed to simulate a tumor microenvironment in vitro, co-culture with single tumor cells with different sizes and deformability is carried out, and the influence of the tumor microenvironment exosomes on the biological activity of the tumor stem cells and the heterogeneity of the tumor stem cells are researched. Compared with a traditional method for constructing and simulating the tumor microenvironment by utilizing tumor microenvironment cells, the method disclosed by the invention is simple to operate and high in condition controllability, so that the method has good scientific research and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to a microfluidic chip mixing operation platform capable of concentrating and mixing exosomes derived from cells, and more particularly to a multifunctional microfluidic exosome mixer capable of collecting and mixing exosomes from different types of cells and simulating the in-vivo growth microenvironment of tumor stem cells, and an operation method thereof. Background Art

[0002] The tumor microenvironment has a crucial impact on the growth, metastasis, drug resistance, etc. of tumor cells, especially tumor stem cells. At present, most of the research on the tumor cell microenvironment uses animal models or the transwell cell co-culture method, that is, different cells are co-cultured. Although these two methods are currently widely used in the research of the tumor microenvironment, they also have their own disadvantages. Since there are great differences between animals and humans, using animal models cannot truly simulate the human tumor microenvironment. In addition, with the in-depth research on animal welfare in recent years, there are also animal ethics and welfare risks in using animal models to study the human tumor microenvironment; using in-vitro co-culture techniques (such as microfluidic techniques, transwell techniques, etc.) with multiple tumor microenvironment cells and tumor cells, although these methods can avoid the disadvantages of experimental animals, they have high requirements for cell culture techniques, and because the culture conditions required for different cells to grow (such as cell culture medium, serum, growth factors, etc.) are different, therefore, co-culturing different types of cells and enabling them to simulate the in-vivo tumor microenvironment requires overcoming many problems in cell co-culture.

[0003] At the same time, exosomes are vesicles secreted by cells with a diameter of about 20 - 200 nm. Research shows that exosomes play a key role in cell signal transduction and tumor cell growth. And using exosomes derived from multiple tumor microenvironment cells to simulate the in-vivo tumor microenvironment has the following advantages: 1. Simple operation, compared with cell co-culture techniques, co-culturing exosomes with tumor cells is simpler and easier to operate; 2. Strong condition controllability, due to the heterogeneity of cells and the different amounts of exosomes secreted by different cells, it is impossible to precisely control the impact of exosomes on tumor cell growth using the cell co-culture method. Directly using exosomes can effectively avoid this problem and achieve controllability of the type and quantity of exosomes; 3. The components in exosomes are simpler than cells, so it is beneficial to more quickly and accurately screen key biological factors (such as proteins, microRNAs, etc.) that affect tumor cell growth.

[0004] Based on the above problems, how to design a cell-derived exosome mixer to simulate the in-vivo tumor cell microenvironment and study the mechanisms and key factors of in-vivo tumor cell growth, metastasis, and drug resistance has become an urgent problem that needs to be solved by those skilled in the art currently. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a microfluidic exosome mixer, application, and operation method, mainly for a microfluidic operation platform and its operation method for improving the spheroid formation rate of single-cell tumor spheres.

[0006] The present invention simulates the tumor microenvironment in vitro by mixing different types of tumor microenvironment exosomes, co-cultures with single tumor cells of different sizes and deformabilities, and studies the effects of tumor microenvironment exosomes on the biological activities of tumor stem cells and the heterogeneity of tumor stem cells. Compared with the traditional method of constructing and simulating the tumor microenvironment using tumor microenvironment cells, the present invention has simple operation, strong controllability of conditions, and better prospects for scientific research and clinical applications.

[0007] One of the objectives of the present invention is a microfluidic exosome mixer, including: a concentration gradient generation module and a capture array module;

[0008] Among them, the concentration gradient generation module includes: an inlet end and several levels of concentration gradient channels;

[0009] Each level of the concentration gradient channels is connected in series;

[0010] At least two inlet ends are provided, and each inlet end is respectively connected to the first-level concentration gradient channel;

[0011] Several mixers are provided in the concentration gradient channels, and the mixers are connected in parallel;

[0012] The outlet end of the (n + 1)-th level concentration gradient channel at the end is connected to the capture array module, where n is a positive integer.

[0013] Preferably, the mixer is a checkerboard mixer, and the mixer is provided with an outlet end;

[0014] The spacing of the checkerboard channels provided in the checkerboard mixer is 50 μm, and the aspect ratio of the checkerboard channels is 1:4;

[0015] Furthermore, the spacing of the checkerboard channels provided in the checkerboard mixer is 50 μm, and the width of the checkerboard channels is 200 μm and the height is 50 μm;

[0016] The beneficial effects of sampling the above technical solutions at least include: Such a design can produce a good exosome mixing effect, and the best mixing effect for exosomes with an average diameter of 100 nm is achieved when using a width of 200 μm and a height of 50 μm.

[0017] Preferably, the number of mixers provided in the (n + 1)-th stage concentration gradient channel is at least one more than that in the n-th stage concentration gradient channel.

[0018] Moreover, the first stage concentration gradient channel is provided with at least three mixers.

[0019] Preferably, 15 ≥ n ≥ 4.

[0020] The beneficial effects of adopting the above technical solutions at least include: Since too many levels will generate greater fluid resistance and ultimately lead to a poor mixing effect, therefore, the concentration gradient channel has a better mixing effect under the above-defined conditions.

[0021] Preferably, the capture array module is provided with several rows of microstructural obstacle filtration layers, and each layer of the microstructural obstacle filtration layer is provided with several microstructural obstacle filters arranged in parallel.

[0022] Preferably, the first gap G1 of the microstructural obstacle filter is 2 μm wider than the second gap G2, and the microstructural obstacle filter is a cylindrical microporous structure with a micropore diameter of 150 μm.

[0023] The beneficial effects of the above technical solutions at least include: The cylindrical microporous structure designed in the present invention fits the shape of the cell sphere better than the conventionally used cuboid microporous structure design, which can promote cell spheroid formation.

[0024] The second object of the present invention is to provide the application of the above microfluidic exosome mixer in simulating the tumor microenvironment.

[0025] The third object of the present invention is to provide the application of the above microfluidic exosome mixer in studying the mechanism and influencing factors of the biological activity of tumor microenvironment exosomes on tumor stem cells.

[0026] The fourth object of the present invention is to provide an operation method for improving the single-cell tumor spheroid formation rate, which is operated by using the above microfluidic exosome mixer.

[0027] Preferably, the flow rate at the inlet end is 15 μL / min.

[0028] The beneficial effects of adopting the above technical solutions at least include: The ECG mixer adopted in the present invention can achieve rapid and efficient mixing, and can maintain a stable mixing effect even at different flow rates.

[0029] In summary, the technical effects that the present invention can produce at least include:

[0030] 1) The present invention designs a concentration gradient module (CMP):

[0031] a. Efficient mixing: The CMP mixer can achieve fast and efficient mixing, and maintain a stable mixing effect even at different flow rates;

[0032] b. Linear concentration gradient: Through multi-stage dilution, a linear concentration gradient can be generated, which is convenient for subsequent single-cell drug screening;

[0033] c. Modular design: The CMP module can be used independently and generate more linear concentration gradients according to the increment of n + 1; it can also be used in combination with other modules such as the capture array module to achieve multiple functions;

[0034] 2) The present invention designs a capture array module (MMOF):

[0035] a. Efficient single-cell capture: The MMOF array can achieve high-efficiency single-cell capture, avoiding the problems of cell aggregation and experimental errors in traditional methods;

[0036] b. High throughput: The MMOF module can capture multiple cells simultaneously, and group them according to cell size and deformability. This design can also improve the single-cell capture throughput through simple integration;

[0037] c. Adjustable: Different types of cells can optimize the single-cell capture efficiency by adjusting experimental conditions (such as cell density and driving flow rate);

[0038] d. Modular design: The MMOF module can be used in combination with other modules such as CMP to achieve functions such as Exo-assisted STS formation and drug screening;

[0039] e. Compared with squares and regular hexagons, the design of the F cylindrical microporous structure fits the shape of cell spheres better and can promote cell spheroid formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0041] Figure 1 The drawings are schematic diagrams of the operation of the microfluidic exosome mixer provided in Embodiment 1 of the present invention;

[0042] Figure 2The attached drawing is an unfolded physical diagram of the microfluidic exosome mixer provided in Embodiment 1 of the present invention;

[0043] Figure 3 The attached drawing is a test diagram of the feasibility of the structure of the microfluidic exosome mixer in Embodiment 1 of the present invention; among them,

[0044] Figure 3 (A) The attached drawing is a comparison diagram of the concentrations of green and red fluorescently labeled nanoparticles generated by the mixer at different flow rates with the standard curve;

[0045] Figure 3 (B) The attached drawing is a comparison diagram of the concentrations of green and red fluorescently labeled nanoparticles generated by the mixer at different flow rates with the standard curve;

[0046] Figure 3 (C) The attached drawing is a fluorescence image of different outlets of the mixer module;

[0047] Figure 3 (D) Fluorescence images of different units in the single-cell tumor sphere module under the condition of a flow rate of 15 μL / min;

[0048] Figure 4 The attached drawing is a bright-field image, separation, and analysis flow schematic diagram of HUVEC and NIH / 3T3 cells cultured before exosome extraction;

[0049] Among them, Figure 4 (A) The attached drawing is a bright-field image of HUVEC and NIH / 3T3 cells cultured before exosome extraction, and the scale bar is marked in the image;

[0050] Figure 4 (B) A photo of the exosome separation device and a schematic diagram describing its structure. The device consists of a vacuum pump, a 240 mL solution filtering bottle, and a nested funnel-shaped device.

[0051] Figure 4 (C) Transmission electron microscope (TEM) image of HUVEC exosomes, particle size distribution diagram (7.6×10 8 mL -1 ) and Western blot analysis (CD63 and CD81 expression).

[0052] Figure 4 (D) TEM image of NIH / 3T3 cell exosomes, particle size distribution diagram (3.2×10 8 mL -1 ) and Western blot analysis (CD63 and CD81 expression); Figure 4 (C) and Figure 4 The scale bars in (D) are both 150 nm.

[0053] Figure 4 (E) Schematic diagram of the process for collecting, isolating, and analyzing exosomes from HUVEC and NIH / 3T3 cell culture media;

[0054] Figure 5 The accompanying figure is a schematic diagram of the sphere formation rate when a single glioma cell is co-cultured with exosomes derived from HUVEC or NIH / 3T3 cells at different concentrations;

[0055] Figure 6 The accompanying figure is a schematic diagram of the sphere size when a single glioma cell is co-cultured with exosomes derived from HUVEC or NIH / 3T3 cells at different concentrations;

[0056] Figure 7 The accompanying figure shows the observation of the formation rate and sphere rate of single primary glioblastoma cell-derived tumor spheres under single culture and co-culture conditions with extracted exosomes, presenting images of single-cell source tumor spheres derived from single primary glioblastoma cells;

[0057] Among them, Figure 7 (A) White light images of single-cell source tumor spheres on day 0 and day 10 in monoculture (Mono);

[0058] Figure 7 (B) White light images of single-cell source tumor spheres on day 0 and day 10 in co-culture with mixed exosomes from the outlet O7 of the ECG module (HUVEC@Ex);

[0059] Figure 7 (C) White light images of single-cell source tumor spheres on day 0 and day 10 in co-culture with mixed exosomes from the outlet O1 of the ECG module (NIH / 3T3 cell@Ex);

[0060] Figure 7 (D) White light images of single-cell source tumor spheres on day 0 and day 10 in co-culture with mixed exosomes from the outlet O3 of the ECG module (Mixed-Ex);

[0061] Figure 7 In the figures (A - D), the size of the single-cell sorting microstructure is the size of microstructure G2, and the scale bar in (A - D) is 40 μm;

[0062] Figure 8 The accompanying figure is a white light image of single-cell source tumor spheres on day 10 in co-culture with mixed exosomes from the outlets O2, O4, O5, and O6 of the ECG module. The size of the single-cell sorting microstructure in the figure is the size of microstructure G2, and the scale bar is 40 μm;

[0063] Figure 9The attached figure shows the spheroid formation rate of individual primary glioblastoma cells in the chamber after 10 days of co-culture with exosomes mixed by a microfluidic exosome mixer. The control group is the single-cell-derived tumor spheroids cultured alone.

[0064] Figure 10 The attached figure shows the spheroid size of individual primary glioblastoma cells in the chamber after 10 days of co-culture with exosomes mixed by a microfluidic exosome mixer. The control group is the single-cell-derived tumor spheroids cultured alone.

[0065] Figure 11 The attached figure is a quantitative analysis chart of the spheroid formation rate (A) and spheroid size (B) of tumor spheroids derived from individual primary glioblastoma cells on the 10th day. Detailed implementation manners

[0066] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0067] Example 1

[0068] In this example, a microfluidic exosome mixer is designed, and it is an ECG mixer based on a Chinese character-shaped pipeline, which can be used to mix two different exosomes and generate a series of concentration gradients. The design diagram of the ECG mixer is as Figure 2 shown, with a channel width of 200 μm and a height of 50 μm. From the center to the periphery, the ECG mixer has five levels with a channel spacing of 50 μm, and uses centrifugal force to generate secondary flow to ensure efficient mixing.

[0069] As Figure 1 shown, the ECG mixer generates 7 gradient concentrations of exosomes through a five-step dilution process and flows out from 7 outlets, which are connected to a single tumor cell long-term culture module for subsequent experiments, as Figure 2 .

[0070] Figure 1 is a schematic diagram of the operation of the microfluidic exosome mixer. The microfluidic exosome mixer includes two independent functional modules. The first module is the exosome concentration gradient generation module, that is, the concentration gradient generation module, which realizes the mixing of two exosome types through a compact Chinese character-shaped mixer (ECG).

[0071] The second module is the capture array module, that is, the single-cell tumor spheroid formation capture array module, which consists of multiple microstructural arrays and is dedicated to culturing, observing, and evaluating the formation of single-cell-derived tumor spheroids downstream.

[0072] Meanwhile, in Figure 2 among Figure 2 (A) Physical photo of the microfluidic exosome mixture. Figure 2 (B) Schematic diagram of the hui - shaped pattern pipeline. Figure 2 (C) Schematic diagram of the single - cell tumor spheroid formation capture array module; among which, the first gap (G1) of the micro - structure obstacle filter is 2 μm wider than the second gap (G2), and there are a total of 39×5 rows of structures from the input end to the output end of the device, that is, 39*5 rows of micro - structure obstacle filters; the diameter of each cylindrical micropore is 150 μm for cell culture.

[0073] As Figure 3 (A) - Figure 3 (D) shows, in order to test the feasibility of the microfluidic exosome mixer structure, two kinds of 100 - nm - sized fluorescent microspheres labeled with different fluorescences were used and poured in at different flow rates at a particle concentration of 5×10^8 particles per milliliter for mixing tests. It was found that under the condition of a flow rate of 15 μL / min, the fluorescence images of different units in the single - cell tumor spheroid module were closest to the standard curve.

[0074] Figure 3 (A) - Figure 3 For the verification of the performance of the gradient dilution module in the microfluidic exosome mixer in (A) - (D), in Figure 3 (A) and Figure 3 (B) are respectively the concentrations of green and red fluorescence - labeled nanoparticles generated by the mixer at different flow rates (from 1 μL / min to 20 μL / min, and the nanoparticle concentration is 5×10 8 particles per milliliter), which are compared with the theoretical values. Figure 3 (C) Fluorescence images of different outlets of the mixer module. Figure 3 (D) Fluorescence images of different units in the single - cell tumor spheroid module under the condition of a flow rate of 15 μL / min. Figure 3 (C) and Figure 3 The scale bars in (D) are 400 μm.

[0075] As Figure 4 shown, exosomes derived from fibroblast NIH / 3T3 and exosomes from human umbilical vein endothelial cell HUVEC were extracted by the method mentioned in the patent with the declared patent number 202311273906.6, publication number: CN117305106A, and patent name: A Device and Application for Enrichment and Extraction of Tumor Microenvironment Exosomes.

[0076] Figure 4 (A) Bright - field images of HUVEC and NIH / 3T3 cells before exosome extraction, and the scale bar is marked in the image.

[0077] Figure 4(B) Photograph of the exosome isolation device and schematic diagram describing its structure, which consists of a vacuum pump, a 240 mL solution filtration flask, and a nested funnel-shaped device.

[0078] Figure 4 (C) Transmission electron microscope (TEM) image of HUVEC exosomes, particle size distribution diagram (7.6×10 8 mL -1 ), and Western blot analysis (CD63 and CD81 expression).

[0079] Figure 4 (D) TEM image of NIH / 3T3 cell exosomes, particle size distribution diagram (3.2×10 8 mL -1 ), and Western blot analysis (CD63 and CD81 expression); Figure 4 (C) and Figure 4 The scale bars in (D) are both 150 nm.

[0080] Figure 4 (E) Schematic diagram of the process for collecting, isolating, and analyzing exosomes from HUVEC and NIH / 3T3 cell culture media.

[0081] Two different types of exosomes (7.6×10 8 mL -1 exosomes from HUVEC and 4.0×10 8 mL -1 exosomes from NIH / 3T3 cells) mixed using a microfluidic exosome mixer were co-cultured with single glioma cells, and it was found that co-culturing two different exosomes with single glioma cells resulted in a higher spheroid formation rate and larger spheroids compared to culturing alone and co-culturing with single types of exosomes.

[0082] Single glioblastoma cell-derived tumor spheres were treated with DMEM containing different doses of isolated HUVEC or NIH / 3T3 cell-derived exosomes. These doses corresponded to exosomes harvested from 60, 120, 180, 240, 300, 360, and 420 mL of HUVEC or NIH / 3T3 cell culture media collected by the exosome extraction device. In Figure 5 and Figure 6 , for HUVEC-derived exosomes, the size and formation rate of tumor spheres increased with the increase in the volume of isolated cell culture media, ranging from 60, 120, and 180 mL. This trend continued until the maximum efficiency was reached at 240 mL (i.e., 7.6×10 8 per milliliter). Subsequently, when the cell culture media volume was in the range of 360 to 420 mL, there was no significant difference in the sphere size and formation rate.

[0083] For exosomes derived from NIH / 3T3 cells, as the volume of the isolated cell culture medium increased, the tumor sphere formation rate and size showed an increasing trend, ranging from 60, 120, 180 to 240 mL. This trend continued until the maximum efficiency was reached at 300 mL (i.e., the exosomes were distributed at approximately 4.0×10 8 per milliliter). Similarly, when the cell culture medium volume was 300 to 420 mL, there was no significant difference in the sphere size and formation rate. Therefore, we selected exosomes derived from NIH / 3T3 cells with a density of 4.0×10 8 per milliliter, and exosomes derived from HUVECs with a density of 7.6×10 8 per milliliter as the conditions for the next step of exosome mixing to simulate the tumor microenvironment ( Figure 5 and 6 ).

[0084] Figure 5 Sphere formation rate of single glioma cells co-cultured with exosomes derived from HUVECs or NIH / 3T3 cells at different concentrations. Error bars represent the standard deviation of 10 parallel experiments.

[0085] Figure 6 Sphere size of single glioma cells co-cultured with exosomes derived from HUVECs or NIH / 3T3 cells at different concentrations. Error bars represent the standard deviation of 10 parallel experiments.

[0086] Two different exosome concentrates (exosomes derived from NIH / 3T3 cells at 4.0×10 8 per milliliter, and exosomes derived from HUVECs with a density of 7.6×10 8 per milliliter) were introduced into the microfluidic exosome mixer shown at a flow rate of 15 μL / min from two inlets. And the seven outlets were sequentially connected to an independent single-cell tumor sphere module chip for co-culture with single cells. After 14 days of culture, the size and sphere formation rate of single-cell-derived tumor spheres were compared. As shown in Figure 1 , Figure 7 , 8 and 9, it was found that the exosome mixtures at the O1 (i.e., exosomes derived from 3T3 cells) and O7 (i.e., exosomes derived from HUVEC cells) outlets ( Figure 7 B and 7C) had a higher sphere formation rate and sphere size after co-culture with single cells compared to single-cell-derived tumor spheres cultured alone ( Figure 7 A). As further shown in the figure, it was found that when the two different exosomes were mixed, the O2 - O6 outlets could exhibit a higher sphere formation rate and sphere size ( Figure 7 D and Figure 8 ), and the exosome mixture at the O3 outlet had the highest sphere formation rate and sphere size when co-cultured with single cells ( Figure 9), that is, the concentration of 3T3 cell-derived exosomes was 2.61×10 8 per milliliter, and the concentration of HUVEC cell-derived exosomes was 2.46×10 8 per milliliter. In addition, as shown in Figure ( Figure 11 ), as the cell size and deformability changed, that is, the spheroid formation rate and spheroid area of smaller and / or more deformable subpopulations were larger.

[0087] Figure 7 The formation rate and spheroid rate of single primary glioblastoma cell-derived tumor spheroids observed under single culture and co-culture conditions with extracted exosomes (A-D) show images of single cell-derived tumor spheroids derived from single primary glioblastoma cells.

[0088] Figure 7 (A) White light images of single cell-derived tumor spheroids on day 0 and day 10 in monoculture (Mono).

[0089] Figure 7 (B) White light images of single cell-derived tumor spheroids on day 0 and day 10 in co-culture with mixed exosomes from the outlet O7 of the ECG module (HUVEC@Ex).

[0090] Figure 7 (C) White light images of single cell-derived tumor spheroids on day 0 and day 10 in co-culture with mixed exosomes from the outlet O1 of the ECG module (NIH / 3T3 cell@Ex).

[0091] Figure 7 (D) White light images of single cell-derived tumor spheroids on day 0 and day 10 in co-culture with mixed exosomes from the outlet O3 of the ECG module (Mixed-Ex).

[0092] Figure 7 (A-D) The size of the single cell sorting microstructure in the figure is the size of microstructure G2, and the scale bar in (A-D) is 40 μm.

[0093] Figure 8 White light image of single cell-derived tumor spheroids on day 10 in co-culture with mixed exosomes from the outlets O2, O4, O5, and O6 of the ECG module. The size of the single cell sorting microstructure in the figure is the size of microstructure G2, and the scale bar is 40 μm.

[0094] Figure 9 It is the spheroid formation rate of single primary glioblastoma cells in the chamber after 10 days of co-culture with exosomes mixed by the microfluidic exosome mixer. The control group was single cell-derived tumor spheroids in monoculture.

[0095] Figure 10The sphere size of individual primary glioblastoma cells in the chamber after 10 days of co-culture with exosomes mixed by the microfluidic exosome mixer. The control group was the single-cell-derived tumor spheres cultured alone.

[0096] Figure 11 Quantitative analysis was performed on the sphere formation rate (A) and sphere size (B) of tumor spheres derived from individual primary glioblastoma cells on the 10th day. Among them, single-cell-derived tumor spheres cultured alone (Mono), single-cell-derived tumor spheres co-cultured with exosomes mixed at outlet O1 (NIH / 3T3 cell@Ex), outlet O7 (HUVEC@Ex), and outlet O3 (Mixed-Ex) were compared. Error bars represent the standard deviation of 10 parallel experiments, and statistical significance was evaluated by ANOVA: **p < 0.01; *p < 0.05; N.S. indicates no significant difference.

[0097] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0098] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the concept or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microfluidic exosome mixer, characterized in that, Comprising: A concentration gradient generation module and a capture array module; Wherein, the concentration gradient generation module comprises: an inlet end and several stages of concentration gradient channels; The concentration gradient channels of each stage are connected in series; There are at least two inlet ends, and each inlet end is respectively connected to the first-stage concentration gradient channel; The concentration gradient channels are provided with several mixers, and the mixers are connected in parallel; The outlet end of the (n + 1)-th stage concentration gradient channel at the end is connected to the capture array module, where n is a positive integer.

2. The microfluidic exosome mixer according to claim 1, characterized in that The mixer is a figure-eight mixer, and the mixer is provided with an outlet end; The spacing of the figure-eight channels provided in the figure-eight mixer is 50 μm, and the aspect ratio of the figure-eight channels is 1:

4.

3. The microfluidic exosome mixer according to claim 1, characterized in that, The number of mixers provided in the (n + 1)-th stage concentration gradient channel is at least one more than that in the n-th stage concentration gradient channel, and, the first-stage concentration gradient channel is provided with at least three mixers.

4. A microfluidic exosome mixer according to claim 1, characterized in that, 15 ≥ n ≥ 4.

5. A microfluidic exosome mixer according to any one of claims 1-4, characterized in that The capture array module is provided with several rows of microstructural barrier filter layers, and each layer of microstructural barrier filter layer is provided with several juxtaposed microstructural barrier filters.

6. The microfluidic exosome mixer according to claim 5, wherein The first gap G1 of the microstructural barrier filter is 2 μm wider than the second gap G2, and the microstructural barrier filter is a cylindrical microporous structure with a micropore diameter of 150 μm.

7. The application of the microfluidic exosome mixer according to claim 6 in simulating the tumor microenvironment.

8. The application of the microfluidic exosome mixer according to claim 6 in studying the mechanism and influencing factors of the biological activity of tumor stem cells by exosomes in the tumor microenvironment.

9. An operation method for improving the spheroid formation rate of single - cell tumors, characterized in that, Operating using the microfluidic exosome mixer according to claim 6.

10. The operation method for improving the spheroid formation rate of single-cell tumors according to claim 9, characterized in that, The flow rate of the inlet end is 15 μL / min.

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