A microfluidic chip and its design method and application

Abstract

The present invention discloses a microfluidic chip and its design method and application. The microfluidic chip includes a main channel, a capture unit and a narrow channel; the main channel is used to transmit cells and cell culture medium, and a plurality of capture units are arranged on the side wall of the main channel facing each other and at intervals, and the capture units are used to capture and culture single cells; the narrow channel is used to connect two capture units, form a communication path between single cells after capture, and prevent the captured single cells from flowing into the opposite main channel. The present invention is simple to manufacture and easy to operate, can achieve the capture and culture of two single cells, and conduct research on cell-to-cell interactions, and is suitable for exploring cell heterogeneity and disease pathogenesis. It can be used to carry out research on the mechanism of tumor occurrence and development, provide a basis and new research ideas for constructing an effective in vitro tumor pathogenesis model, and provide certain theoretical support for the occurrence and development of tumors.

Description

Technical Field

[0001] The invention relates to a microfluidic chip and a design method and application thereof, which are used for single cell capture and culture in two types of cells and the study of cell-cell interaction. Background Art

[0002] A single cell is the basic unit of life, and traditional cell research is always conducted on a group of cells. The results obtained are often the average of a group of cells, which conceals the information differences between individuals. In proteomics research, it is found that the same cell behaves and responds completely differently in different periods and environments. In tumor cells and stem cells, the heterogeneity of cells is most significant. The micro-total analysis system, also known as a microfluidic chip or a lab on a chip (LOC), refers to a chip of a few square centimeters (or even smaller) that integrates or basically integrates the basic operation units involved in the fields of chemistry and biology, such as sample preparation, reaction, separation, detection, cell culture, sorting, and lysis. The microchannel forms a network, and the controllable fluid runs through the system. It is a technical platform used to replace various functions of conventional chemical or biological laboratories. Microfluidic chip technology can achieve one-click closed operation from single cell capture, identification and detection, with high efficiency and small error. Therefore, microfluidic chip technology has become a powerful tool for single cell analysis.

[0003] Multicellular systems rely on cell-to-cell interactions to coordinate cell signals and regulate cell functions. Understanding the mechanisms and processes of cell-to-cell interactions is crucial for many physiological and pathological processes, such as embryogenesis, tissue regeneration, stem cell differentiation, and cancer metastasis. There is often a close interaction between cells of different types. This interaction is homotypic or heterotypic, and depends on the distance between cells. It can occur through a variety of mechanisms, such as direct physical contact, diffusion of soluble secreted factors, electrical signal conduction, and cell-surrounding matrix. For example, in diseased tissues, cell-to-cell interactions are disturbed, which plays a vital role in the occurrence and development of the disease. For example, tumor budding is an important pathological feature of highly invasive tumor behavior and is closely related to tumor recurrence and metastasis. In the tumor microenvironment, vascular endothelial cells migrate into the tumor under the action of factors such as VEGF secreted by tumor cells, thereby preparing conditions for the nutrient supply and invasion and metastasis of the tumor. Literature Chung Woosung.; Eum HM; Lee HO; Lee KM; Lee H.B.; Kim KT; Ryu HS; Kim S.; Lee JE; Park YH Nature Communications, 2017, 8. A total of 515 cells from 11 breast cancer patients were collected. Through single-cell transcriptome analysis, cancer cells showed common characteristics within the tumor and exhibited intratumor heterogeneity in breast cancer subtypes and important cancer-related pathways.

[0004] Developing single-cell capture, manipulation, and analysis methods to study cell heterogeneity is of great significance for studying cell growth, differentiation, disease diagnosis, and monitoring. Although microfluidic chips are a good platform for single-cell capture, culture, and analysis, there is currently no research using microfluidic technology to achieve the separation and capture of multiple cells or the interaction between multiple cells. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a microfluidic chip and its design method and application. The microfluidic chip has a miniaturized structure, low cost, and high applicability. It can capture and culture single cells and study the interaction between two cells, and can reveal certain cell-to-cell interaction mechanisms. It solves the problem of separating and capturing two single cells in the above-mentioned background technology, and also solves the problem of studying the interaction between single cells of multiple cells.

[0006] The technical solution adopted by the present invention to solve the technical problem is: for single cell capture and research on cell-to-cell interaction, a microfluidic chip is provided, which is formed by bonding a polydimethylsiloxane sheet and a substrate engraved with channels, wherein the channels include a main channel, a capture unit and a narrow channel;

[0007] The main channel is used to transfer cells and cell culture medium, and includes a first main channel and a second main channel arranged side by side; a plurality of capture units are arranged on the side walls of the first main channel and the second main channel facing each other and at intervals, and the capture units are used to capture and culture single cells; the narrow channel is used to connect the capture units of the first main channel with the capture units at corresponding positions of the second main channel, so as to form a communication path between the captured single cells and prevent the captured single cells from flowing into the opposite main channel.

[0008] In a preferred embodiment of the present invention, the width of the capture unit decreases from the main channel to the narrow channel, and when the cells on one side enter the capture unit, the narrow channel acts as a valve.

[0009] In a preferred embodiment of the present invention, the cross-sectional shape of the capture unit is a semi-ellipse or a triangle. The present invention adopts a method for the relationship between fluid streamlines and captured cell positions, and the direction of the streamlines can guide the position of the cells in the capture unit, thereby affecting the next cell flow. Without changing the length and width of the main channel, the width and length of the gap, and the length and width of the capture unit, the shape of the capture unit is changed to check the effect on the cell capture position.

[0010] In a preferred embodiment of the present invention, the width of the main channel is 20 to 40 μm, the spacing distance between the capture units on the same main channel is 100 to 200 μm, the entrance size of the capture unit is 20 to 35 μm, the width of the narrow channel is 2 to 10 μm, the length of the narrow channel is 5 to 10 μm, and the height of the channel is 20 to 40 μm.

[0011] In a preferred embodiment of the present invention, the main channel width is 30 μm, the spacing distance between capture units on the same main channel is 150 μm, the entrance size of the capture unit is 25 μm, the narrow channel width is 5 μm, the narrow channel length is 6 μm, and the height of the channel is 25 μm.

[0012] The present invention also provides a design method for the above-mentioned microfluidic chip, comprising the following steps:

[0013] 1) Using the principle of "path with minimum flow resistance", the flow of fluid in the channel usually occurs at a low Reynolds coefficient (Re). For laminar flow in a rectangular channel without a drop or obstacles, the resistance calculation formula is:

[0014]

[0015] Among them, μ is the viscosity of the fluid, Q is the flow rate, Q = V × A, V is the flow velocity, A is the cross-sectional area of ​​the channel, L, w, h are the length, width and height of the channel respectively, and α is a dimensionless parameter related to the aspect ratio.

[0016]

[0017]

[0018] 2) Calculate the pressure drop △P:

[0019] For the drag of the capture unit, it is approximately calculated as a rectangular drag;

[0020] Pressure drop of fluid passing through the first main channel and the narrow channel, where P0 represents the narrow channel and P1 represents the first main channel

[0021]

[0022] The pressure drop of the fluid passing through the narrow channel and the second main channel, where P2 represents the pressure drop of the second main channel

[0023]

[0024] 3) Since the height of the narrow channel is greater than its width, swap the height h and width w in formula ④ to obtain the following formula

[0025]

[0026] 4) When the pressure drop of the first main channel and the second main channel is the same,

[0027]

[0028] Q 捕获单元 =Q 窄通道 ⑧;

[0029]

[0030] 5) Substitute the numerical calculations and design the channel dimensions according to the size of the cells to be captured.

[0031] The present invention also provides application of the above-mentioned microfluidic chip in single cell capture.

[0032] In a preferred embodiment of the present invention, the application is single cell capture of two types of cells. Single cell capture of the first type of cells is performed in the first main channel, the captured cells are cultured and adhered to the wall, and then single cell capture of the second type of cells is performed in the second main channel.

[0033] The present invention adopts a method of simulating cell movement by three-dimensional modeling: using the particle tracking module in COMSOL software, a cell model is constructed to simulate its flow in a microchannel, and it is proposed that the captured cells on one side need to be cultured to make them adhere to the wall before the cells on the other side can be captured. When the cells are suspended, the volume of the cells is equivalent to a microvalve, and most of the fluid flows over the cells, which is equivalent to the height of the narrow channel changing. After the cells captured on one side were cultured and attached to the wall, H 有效高度 Increase, at this time The cells on the other side can be captured, providing a more reliable basis for chip production.

[0034] In a preferred embodiment of the present invention, the application is to study cell-cell interactions, single cells are captured in both the first main channel and the second main channel, and cytokines produced by the cells are transmitted to each other through diffusion, thereby realizing the study of cell-cell interactions.

[0035] In a preferred embodiment of the present invention, the particle tracking module in COMSOL software is used to perform three-dimensional modeling of the microfluidic chip to simulate cell movement; or the rare species transfer module in COMSOL software is used to simulate the signal transmission between cells captured by the microfluidic chip.

[0036] The microfluidic chip for single cell capture and cell-to-cell interaction research of the present invention has shown some significant advantages, including: simple manufacture, easy operation, good biocompatibility of the materials used, cell culture conditions close to the in vivo microenvironment, high single cell capture efficiency, and intercellular signal transmission, providing a good device for studying cell-to-cell interactions. The microfluidic chip can be used to study the mechanism of tumor budding, provide a basis and new research ideas for constructing an effective in vitro tumor budding model, and provide certain theoretical support for the occurrence and development of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Diagram of microfluidic chip fabrication.

[0038] Figure 2 A is the structure diagram of the microfluidic chip, B is the actual schematic diagram of the microfluidic chip, C, D and E are the mixing diagrams after the two dyes are introduced.

[0039] Figure 3 Schematic diagram of single-cell capture in microfluidic chip and schematic diagram of simulation structure.

[0040] Figure 4 (a) Velocity distribution diagram of different main channel lengths of 100μm, (b) 150μm, and (c) 200μm, and (d) relationship between channel length ratio and Q.

[0041] Figure 5 (a) Triangle, (b) semi-ellipse, (c) rectangle, (d) circle. From top to bottom, they are the velocity distribution diagram, velocity streamline diagram, and pressure distribution diagram of the capture unit.

[0042] Figure 6 (a), (b), (c) Channel entrance velocity optimization diagram and (d) Relationship diagram of the pressure on the captured cells on the other side after one cell is captured.

[0043] Figure 7 Optimization of the inlet width of the single-sided capture unit, (a) velocity streamline diagram of the capture unit with an inlet velocity of 5 μm / s, and (b) simulation of cell movement using the particle tracing module in COMSOL.

[0044] Figure 8 Three-dimensional simulation captures the velocity distribution of the fluid on the other side when cells are suspended, (a) three-dimensional velocity distribution of the narrow channel, (b) yz cross-sectional velocity distribution of the narrow channel.

[0045] Fig. 9 Three-dimensional simulation of particle movement on the other side when cells adhere to the wall, (a) three-dimensional velocity distribution diagram of narrow channel, (b) particle flow position distribution diagram.

[0046] Fig.10 Simulate the diffusion diagram of substances interacting between cells. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0048] Example 1

[0049] like Figure 1 The microfluidic chip of this embodiment is manufactured by a soft lithography method, and includes the following steps:

[0050] Mold making: The silicon wafer was heated to boiling in a hydrochloric acid solution for 30 minutes, washed with ultrapure water, and dried with nitrogen. A layer of SU-8 2015 negative photoresist was thrown on the surface with a glue spreader at a speed of 2500 rpm. The wafer was baked at 65 for 10 minutes and 95 m for 4 minutes. The wafer was exposed 3 times with a UV exposure machine for 30 seconds each time. After development, the wafer was dried with nitrogen to obtain the cell capture pattern on the silicon wafer.

[0051] Chip production: Add trifluoro (1H, 1H, 2H, 2H perfluorooctyl) silane and mold to a drying vessel, keep the vacuum for 2 hours to make the mold surface hydrophobic, mix polydimethylsiloxane (PDMS) prepolymer and initiator at a mass ratio of 10:1 and pour into the mold, remove bubbles and place at 75 for 2 hours. Peel off the polymerized PDMS sheet, cut into shape, punch holes with a flat-head syringe needle, bond the PDMS to the glass after plasma treatment, and the chip is successfully produced.

[0052] like Figure 2 and Figure 3 The microfluidic chip is formed by bonding a polydimethylsiloxane sheet and a substrate engraved with channels, wherein the channels include a main channel, a capture unit and a narrow channel;

[0053] The main channel is used to transfer cells and cell culture medium, and includes a first main channel and a second main channel arranged side by side; the two main channels are arranged in parallel or in a bent manner on the chip to form a capture array;

[0054] A plurality of capture units are arranged on the side walls of the first main channel and the second main channel facing each other and at intervals, and the capture units are used for capturing and culturing single cells; the narrow channel is used to connect the capture units of the first main channel with the capture units at corresponding positions of the second main channel, so as to form a communication path between the captured single cells and prevent the captured single cells from flowing into the opposite main channel.

[0055] The width of the capture unit decreases from the main channel to the narrow channel. The width of the main channel is 30 μm, the spacing between the capture units on the same main channel is 150 μm, the inlet size of the capture unit is 25 μm, the width of the narrow channel is 5 μm, the length of the narrow channel is 6 μm, and the height of the microfluidic chip is 25 μm.

[0056] Embodiments 2 to 4

[0057] The design method of the size of each part of the microfluidic chip includes the following steps:

[0058] 1) Using the principle of "path with minimum flow resistance", the flow of fluid in the channel usually occurs at a low Reynolds coefficient (Re). For laminar flow in a rectangular channel without a drop or obstacles, the resistance calculation formula is:

[0059]

[0060] Among them, μ is the viscosity of the fluid, Q is the flow rate, Q = V × A, V is the flow velocity, A is the cross-sectional area of ​​the channel, L, w, h are the length, width and height of the channel respectively, and α is a dimensionless parameter related to the aspect ratio.

[0061]

[0062]

[0063] 2) Calculate the pressure drop △P:

[0064] For the drag of the capture unit, it is approximately calculated as a rectangular drag;

[0065] Pressure drop of fluid passing through the first main channel and the narrow channel, where P0 represents the narrow channel and P1 represents the first main channel

[0066]

[0067] The pressure drop of the fluid passing through the narrow channel and the second main channel, where P2 represents the pressure drop of the second main channel

[0068]

[0069] 3) Since the height of the narrow channel is greater than its width, swap the height h and width w in formula ④ to obtain the following formula

[0070]

[0071] 4) When the pressure drop of the first main channel and the second main channel is the same,

[0072]

[0073] Q 捕获单元 =Q 窄通道 ⑧;

[0074]

[0075] 5) Substitute the numerical calculations and design the channel dimensions according to the size of the cells to be captured.

[0076] Examples 2 to 4 investigated three cases of 100 μm, 150 μm and 200 μm respectively, and used COMSOL software to simulate fluid flow;

[0077] like Figure 3 The positions of inlet 1 and inlet 2 are shown. The flow rate of the fluid in inlet 1 is set to 1 μL.min -1 , V = 0.0267ms -1 , L 入口长度，层流 Inlet length for development of laminar flow. Water as the simulated material.

[0078] ρ 水 =1000kg / m 3 , μ 水 =1.01×10 -3 Pa·s, The change in color in the channel indicates the magnitude of the velocity. Inlet 2 is set to outlet and the pressure is 0. The results of COMSOL simulation are as follows: Figure 4 As shown, Q 窄通道 / Q 主通道 As the main channel length increases, Q 窄通道 / Q 主通道 >1, but at the same time the pressure inside the chip will increase, and the pressure that the chip bonded with PDMS and glass can withstand is limited.

[0079] Embodiments 5 to 8

[0080] Water was used as the simulation material, the main channel length between the capture units was 150 μm, the capture unit inlet width and length remained unchanged, and the shape of the capture unit was changed. The shapes of the capture units in Examples 3 to 8 were triangle, semi-ellipse, square and circle, respectively.

[0081] like Figure 5 As shown in the figure, the four shapes of capture units have no effect on the flow resistance, but the fluid streamlines passing through the capture unit are different. When the capture unit is triangular and semi-elliptical, the fluid is more aggregated, which can reduce the aggregation of cells in the capture unit. The cells occupy a larger area ratio. The circular and rectangular capture units have more nonlinear fluid in the capture unit, and the pressure diagram shows that the circular and rectangular capture units are more likely to generate shear force at the connection between the main channel and the capture unit.

[0082] 1. Single cell capture analysis of two types of cells

[0083] The cell enters the capture unit, like a valve closing the entrance of the capture unit, and most of the fluid will not pass through, resulting in Q 窄通道 / Q 主通道 <1. The next cell will enter the second capture unit, and so on, to achieve single-side single cell capture. When the second channel is captured, in order to reduce the resistance of the second main channel liquid entering the capture unit, we open the entrance of the first main channel, set the speed to 0μm / s, and the pressure to 0pa. Due to the presence of the capture unit cell in the first main channel, the resistance of the liquid flow in the second main channel is increased. By calculating Q 窄通道 / Q 主通道 = between 0.23 and 0.25.

[0084] like Figure 6 As shown in Figure 1, it is not feasible to capture single cells in the second main channel by increasing the inlet velocity of the second main channel. In addition, the pressure on the sides of the captured cells will continue to increase. When other conditions such as flow rate and outlet pressure remain unchanged, the width of the inlet of the cell flow capture unit is increased, as shown in Figure 1. Figure 7 As shown, the result Q gap / Qbypass <1, and it will also cause nonlinear aggregation of the fluid. According to the formula, the factor that determines Q gap / Q bypass The length and width of the narrow channel are mainly affected. The width of the entrance of the cell into the capture unit has little effect on the flow rate.

[0085] like Figure 6 and Figure 7 , when one side of the cell is captured, by changing the speed of the other side entrance, the width of the capture cell entrance cannot achieve Q 窄通道 / Q 主通道 >1, and a three-dimensional simulation was performed using COMSOL software. The height of the channel is an important parameter for stretching with the two-dimensional figure as the working plane. H must be greater than the diameter of a single cell, but if H is too large, multiple cells will aggregate in the capture unit (Φ<H<1.4Φ), resulting in one capture unit and one cell. Therefore, we set the height of the entire channel to 25μm for simulation. According to the habit of cell adherence flow, we set the cells captured in the first main channel to be 1μm from the bottom of the channel and the cell diameter to 16μm. In a three-dimensional environment, the cell flow in the second main channel is simulated, and the inlet velocity of the second main channel is set to 5μm / s, and the inlet velocity of the first main channel is set to 0μm / s. Figure 8 As shown in the figure, the fluid in the narrow channel gathers at a position higher than the cell diameter, where most of the fluid flows. When the cell is suspended, the volume of the cell is equivalent to a microvalve, and most of the fluid flows over the cell, which is equivalent to a change in the height of the narrow channel, H 有效高度 =H 窄通道 -R 细胞 <H 窄通道 , Q 窄通道 / Q 主通道 =0.598<1, according to Figure 6 The results of the two-dimensional simulation show that changing the inlet velocity cannot effectively improve the capture efficiency. On the contrary, it will increase the shear force on the cells on the other side. The increase in velocity will also cause the captured cells to rush back into the main channel.

[0086] The increase in the flow resistance of the capture unit is mainly due to the reduction in the effective height of the solution flowing in the narrow channel after the cells are captured on one side. The cells captured on one side are placed in a CO2 incubator and cultured at 37°C for a period of time. Living cells will grow by adhering to the wall, which reduces the height of the cells. The height of the cells after adhering to the wall is 5μm for simulation. Fig. 9 As shown in the figure, after the cells grow attached to the wall, the effective height of the narrow channel solution flow increases compared with the suspension cells. 窄通道 / Q 主通道>1, the flow through the narrow channel is much greater than the flow through the main channel. Cell capture can be achieved, and the morphology of dead cells does not change. By appropriately increasing the entrance speed of the second main channel, the dead cells will be flushed back into the main channel, and the cells captured on the first main channel side can be screened for life or death.

[0087] 2. Analysis of Cell-cell Interactions

[0088] Under the condition that the velocity of the inlet channels on both sides is the same, the diffusion of substances between the cells of the capture unit is simulated. Fig.10 As shown, substances between cells can diffuse effectively, and as the length of the channel increases, the exchange of substances between cells becomes more sufficient. The simulation found that every time the U-shaped channel at the bottom of the channel passes, the direction of the diffusion of cell concentration on both sides will change.

[0089] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A microfluidic chip, formed by bonding a polydimethylsiloxane sheet and a substrate engraved with channels, characterized in that: The channel includes a main channel, a capture unit and a narrow channel; The main channel is used to transfer cells and cell culture medium, and includes a first main channel and a second main channel arranged side by side; a plurality of capture units are arranged on the side walls of the first main channel and the second main channel facing each other and spaced apart, and the capture units are used to capture and culture single cells; the narrow channel is used to connect the capture units of the first main channel with the capture units at corresponding positions of the second main channel, so as to form a communication path between captured single cells and prevent the captured single cells from flowing into the opposite main channel; the width of the capture unit decreases from the main channel to the narrow channel; The cross-sectional shape of the capture unit is semi-elliptical or triangular; the width of the main channel is 20 to 40 μm, the spacing between the capture units on the same main channel is 100 to 200 μm, the entrance size of the capture unit is 20 to 35 μm, the width of the narrow channel is 2 to 10 μm, the length of the narrow channel is 5 to 10 μm, and the height of the channel is 20 to 40 μm.

2. A microfluidic chip according to claim 1, characterized in that: The width of the main channel is 30 μm, the spacing between capture units on the same main channel is 150 μm, the inlet size of the capture unit is 25 μm, the width of the narrow channel is 5 μm, the length of the narrow channel is 6 μm, and the height of the channel is 25 μm.

3. A method for designing a microfluidic chip as claimed in claim 1 or 2, characterized in that: The steps include: 1) Using the principle of "path with minimum flow resistance", the flow of fluid in the channel usually occurs at a low Reynolds coefficient (Re). For laminar flow in a rectangular channel without a drop or obstacles, the resistance calculation formula is: Among them, μ is the viscosity of the fluid, Q is the flow rate, Q = V × A, V is the flow velocity, A is the cross-sectional area of ​​the channel, L, w, h are the length, width and height of the channel respectively, and α is a dimensionless parameter related to the aspect ratio. 2) Calculate the pressure drop △P: For the drag of the capture unit, it is approximately calculated as a rectangular drag; Pressure drop of fluid passing through the first main channel and the narrow channel, where P0 represents the narrow channel and P1 represents the first main channel The pressure drop of the fluid passing through the narrow channel and the second main channel, where P2 represents the pressure drop of the second main channel 3) Since the height of the narrow channel is greater than its width, swap the height h and width w in formula ④ to obtain the following formula 4) When the pressure drop of the first main channel and the second main channel is the same, Q 铺获单元 =Q 窄通道 ⑧; 5) Substitute the numerical calculations and design the channel dimensions according to the size of the cells to be captured.

4. The method for using a microfluidic chip in single cell capture according to claim 1 or 2, characterized in that: The application is single cell capture of two types of cells, single cell capture of the first type of cells is performed in the first main channel, the captured cells are cultured and attached to the wall, and then single cell capture of the second type of cells is performed in the second main channel; The application method is to study the interaction between cells, and single cells are captured in both the first main channel and the second main channel, and the cytokines produced by the cells are transmitted to each other through diffusion, thereby realizing the study of the interaction between cells; The particle tracking module in COMSOL software will be used to perform three-dimensional modeling of the microfluidic chip to simulate cell movement; or the dilute species transfer module in COMSOL software will be used to simulate the signal transmission between cells captured by the microfluidic chip.

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