Cell electrotaxis migration monitoring microfluidic chip, preparation method and monitoring method thereof

Abstract

The application provides a cell electrotaxis migration monitoring microfluidic chip, a preparation method and a monitoring method. The microfluidic chip is bonded by two layers of PDMS channel structures, and specifically comprises a plurality of electric field channels, two fluid channels and two narrow channels. The electric field channels are respectively connected with rectangular buffer pools on two sides. An electric field stimulation hole is opened above the buffer pool. The fluid channels are respectively provided with sample inlet holes and sample outlet holes which are in communication with the outside. The narrow channels are used for intercepting suspended cells, and the two narrow channels are at a certain angle. The overall size of the chip is small. The chip is used for monitoring tumor cell electrotaxis migration under the action of an electric field. The chip has the characteristics of simple operation, small sample consumption, stable electric field formation and the like. The long-time monitoring of cell migration and cell interaction under different distances in the electric field microenvironment of tumors can be realized. Furthermore, the cell electrotaxis migration participated by multiple cells can be comprehensively considered. The chip has a good application prospect in the field of tumor cell biology.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of cell biology and microfluidics, and particularly relates to a cell electrotaxis monitoring microfluidic chip, a preparation method thereof and a monitoring method. BACKGROUND

[0002] At present, malignant tumors as an important global public health problem, seriously threaten human life and health and quality of life, although the diagnosis and treatment means continue to improve, but the strong invasion, high metastasis rate is still the main reason leading to tumor treatment failure and patient death. In the development of malignant tumors, the tumor cells can invade the blood and lymph, and then migrate to the new tissue site to form metastatic tumor. Studies have shown that the development and metastasis of tumor depend on the complex microenvironment of tumor cells (mainly including adjacent non-tumor cells, growth factors, chemotactic factors and extracellular matrix), like "seed and soil" as a whole, tumor cells and their microenvironment are interdependent and mutually restricted.

[0003] Traditional methods for studying cell migration are mainly based on scratch test, Boyden chamber and Transwell chamber, etc., and the consideration of tumor microenvironment factors is relatively single, and the data obtained are often from the population response of a large number of cells, which is easy to ignore the specific performance of a small number of cells. With the development and maturity of micro-nano processing technology, microfluidic chip has the advantages of network format microchannel design, flexible combination of various unit operation technologies, etc., and can accurately control microfluids in time and space, which shows unique application prospect in tumor microenvironment simulation and cell migration research.

[0004] Electrophysiologists have detected endogenous direct current electric field (dcEF) (several to several hundred mV / mm) widely existing around various tumor tissues, and this electric field comes from the trans-epithelial potential (TEP) generated by the change of tumor cell surface charge. Studies have found that this electric field can enhance the activity of tumor cells and induce cells to exhibit a tendency to migrate in a certain direction, and the electrotaxis ability of tumor cells with high metastatic potential is significantly higher than that of tumor cells with low metastatic potential. In recent years, with the help of the advantages of microfluidic chip, some direct current electric field simulation devices have been designed and developed, which makes the research of cell electrotaxis more convenient and easy to operate, and to a certain extent, the tumor electric field microenvironment is reproduced.

[0005] The (electrotaxis) migration of tumor cells is not only related to the heterogeneity of tumor cells, but also affected by non-tumor cells (stromal cells) in the microenvironment, such as endothelial cells, fibroblasts, macrophages and the like. In the process of tumor metastasis, tumor cells secrete factors into the tumor microenvironment, interact with local or distant stromal cells through paracrine, and create a suitable microenvironment for the growth and directional metastasis of tumor cells. At the same time, stromal cells can produce chemotactic factors, growth factors and matrix degradation enzymes, promote angiogenesis, participate in extracellular matrix (ECM) remodeling, immune escape and tumor cell epithelial-mesenchymal transition, and have important promoting effects on tumor invasion and metastasis. Based on this, in the study of the electrotaxis migration of tumor cells, the influence of stromal cells cannot be ignored, and in-depth study of the effects of stromal cells and tumor cells in the electric field microenvironment has important significance for elucidating the role of cell microenvironment in tumor progression and / or metastasis. However, the current tumor cell electrotaxis migration model mainly focuses on tumor cells themselves, and cannot monitor the electrotaxis migration of tumor cells under the condition of co-culture of multiple cells.

[0006] There is no related report on studying the electrotaxis migration of tumor cells in combination with the stromal cell microenvironment around the tumor in the prior art. SUMMARY

[0007] The purpose of the present application is to provide a cell electrotaxis migration monitoring microfluidic chip, a preparation method and a monitoring method thereof, which are used for simulating the electrotaxis migration of tumor cells under complex in-vivo environment.

[0008] The purpose of the present application is achieved as follows:

[0009] A cell electrotaxis migration monitoring microfluidic chip, which is internally provided with a plurality of parallel electric field channels, and two fluid channels and two narrow channels intersecting and communicating with the electric field channels; the two narrow channels are located between the two fluid channels, and the two narrow channels are oppositely arranged with the two fluid channels; the included angle between the two narrow channels is 8-15°, and the narrow channels are used for intercepting suspended cells; the electric field channel between the two narrow channels is a co-culture area, and different cells entering from the two fluid channels can adhere to the wall and enter the co-culture area from the two narrow channels under the action of an electric field.

[0010] Preferably, the two ends of the electric field channel are respectively connected with buffer pools, and the electric field stimulation holes are opened above the buffer pools.

[0011] Preferably, the two ends of the fluid channel are respectively provided with sample inlet holes and sample outlet holes.

[0012] Preferably, the height of the fluid channel and the electric field channel is the same, and is 35-40 μm; the height of the narrow channel is set to 8-10 μm.

[0013] Preferably, the two narrow channels form an included angle of 10°.

[0014] Preferably, the lengths of the co-culture regions on the different electric field channels are different, and the lengths of the co-culture regions range from 380 to 1630 μm.

[0015] The application also provides a preparation method of the cell electrotaxis monitoring microfluidic chip, which is formed by aligning the upper and lower two-layer PDMS channel structures and bonding by oxygen plasma, and the specific preparation method is as follows:

[0016] S1, printing the upper and lower two-layer PDMS channel structure patterns by using a laser to obtain a mask;

[0017] S2, placing the mask on a printed circuit board, and after ultraviolet exposure and development, placing the printed circuit board in a FeCl3 solution for etching, and after etching, rinsing with running water and air-drying to obtain the upper and lower two-layer chip templates;

[0018] S3, mixing and stirring the PDMS prepolymer and the crosslinking agent uniformly, pouring on the printed circuit board, placing in an oven for baking and curing after removing bubbles;

[0019] S4, peeling off the PDMS from the template after solidification and cooling, and punching holes at the corresponding positions of the upper-layer PDMS channel structure;

[0020] S5, performing surface modification on the upper and lower two-layer PDMS channel structures by using oxygen plasma, and then aligning and bonding the upper and lower two-layer channels under a microscope to obtain the microfluidic chip.

[0021] Preferably, in step S4, the punched holes include electric field stimulation holes, sample inlet holes and sample outlet holes.

[0022] The application also provides a cell electrotaxis monitoring method, which adopts the above-mentioned cell electrotaxis monitoring microfluidic chip, and the specific method is as follows:

[0023] (1) culturing two different cells;

[0024] (2) placing the microfluidic chip in a culture dish after sterilization and surface treatment for standby use: first, soaking the chip channel and buffer pool with alcohol, and sterilizing by ultraviolet irradiation; then, rinsing the channel and buffer pool with PBS buffer solution to remove alcohol residues, injecting 20 μg / mL fibronectin into the chip, and incubating at 37°C for 2 h to enhance the cell adhesion ability;

[0025] (3) inoculating cells: first, absorb the fibronectin, then inject a cell suspension cultured in step (1) into one of the fluid channels, and add complete culture medium at the same side of the electric field stimulation hole, so that the cells flow through the electric field channels in parallel, and the suspended cells are intercepted at the narrow channel near the co-culture area on one side;

[0026] (4) moisturize the chip with sterile water around, incubate in a 37℃, 5% CO2 incubator, and after the cells in step (3) adhere, inoculate another cell cultured in step (1) in another fluid channel;

[0027] (5) after the two cells adhere completely, insert platinum wire electrodes into the two electric field stimulation holes through salt bridges, apply voltage, form a direct current electric field in the electric field channel, and under the electric field stimulation, the two cells can adhere to the co-culture area.

[0028] Preferably, in step (4), Cell Tracker fluorescent probe can be used for labeling before inoculating another cell.

[0029] The present application has the following beneficial effects:

[0030] The present application simulates the direct current electric field around the tumor, considers the microenvironment factors of different distance stromal cells on tumor cells, and constructs a tumor cell electrotaxis monitoring microfluidic chip based on a multi-cell co-culture environment. The chip is used to monitor tumor cell electrotaxis under the action of electric field, which has the characteristics of simple operation, small sample amount, stable electric field formation, small overall size, etc. The long-term monitoring of cell migration under tumor electric field microenvironment and cell interaction under different distances can be realized, and the comprehensive consideration of tumor cell electrotaxis involving multiple cells can be realized. The present application has good application prospect in the field of tumor cell biology. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the overall structure of the microfluidic chip of the present application.

[0032] Figure 2 It is a schematic diagram of suspended cell interception and adherent cell electrotaxis.

[0033] Figure 3 It is a physical map of the microfluidic chip provided by the present application.

[0034] Figure 4 It is an electric field distribution simulation diagram of the microfluidic chip provided by the present application.

[0035] Figure 5 It is the migration behavior of tumor-associated fibroblasts CAFs and lung cancer cells A549 under direct current electric field. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, further detailed descriptions are provided below with reference to specific embodiments. The specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0037] Example 1: A microfluidic chip for monitoring cell electrotaxis and migration.

[0038] like Figure 1 As shown, the cell electrotactic migration monitoring microfluidic chip provided by this invention is a PDMS structure containing internal channels, made of PDMS material. The PDMS structure has interconnected electric field channels A, fluid channels B, and a narrow channel C. Specifically, the channels within the PDMS structure include: several parallel and oppositely arranged electric field channels A (10 shown in the figure), two fluid channels B that intersect and communicate with the electric field channels, and a narrow channel C adjacent to and connected to the two fluid channels. Rectangular buffer pools D are connected to both ends of the electric field channels A, and two electric field stimulation holes 1 and 2 are respectively opened on the top of the buffer pools D. Each fluid channel B intersects and communicates with all electric field channels A. One end of two fluid channels B intersects outside the electric field channels A, and the intersecting end connects to the sample outlet 5. The other ends of two fluid channels B are connected to sample inlets 3 and 4 respectively on the other side of the electric field channels A. Sample inlets 3 and 4 and sample outlet 5 are all connected to the outside. In this embodiment of the invention, the two fluid channels B intersect at a 10° angle. One fluid channel B can be positioned so that it intersects the electric field channel A at an 85° angle, and the other fluid channel B intersects the electric field channel A at a 95° angle. Two narrow channels C are positioned between the two fluid channels B and are adjacent to and parallel to their corresponding fluid channels; that is, narrow channel C and the adjacent fluid channel B are parallel and connected (connected at the bottom, because narrow channel C is lower in height). These narrow channels are used to trap suspended cells. Figure 2 , Figure 2 The example illustrates a narrow channel C with a height of 10 μm, while the fluid channel B and electric field channel A have the same height of 37 μm. Typically, the height of the narrow channel C is set to 8–10 μm to facilitate the retention of suspended cells, while the heights of the fluid channel B and electric field channel A are 35–40 μm to meet the normal growth requirements of adherent cells.

[0039] Because fluid channel B and electric field channel A are at the same height, suspended cells in fluid channel B can flow into each electric field channel A. However, because the narrow channel C is lower in height, suspended cells are trapped at narrow channel C and cannot enter the electric field channel A between the two narrow channels C. The electric field channel A between the two narrow channels C constitutes the co-culture region, such as... Figure 1The two narrow channels C are designed to have a certain angle, usually 8-15°, preferably 10°, so that each electric field channel A is divided into co-culture regions of different lengths by the two narrow channels C. In the embodiment, the shortest co-culture region is 380 μm x 400 μm (length x width), and the longest co-culture region is 1630 μm x 400 μm (length x width), so that the influence of different distances of stromal cells on the electrotaxis of tumor cells can be detected. Figure 2 In the embodiment, the co-culture region has a length of 800 μm x 400 μm (length x width). The height of the co-culture region is the height of the electric field channel A. When a certain voltage is applied, a uniform and stable direct current electric field microenvironment can be formed in each region.

[0040] In the research of tumor cell electrotaxis by using the microfluidic chip, the direct current electric field stimulation is as follows: two platinum wire electrodes (diameter 0.5 mm, length 37 mm) are connected to a direct current power supply through external wires, one end of the platinum wire is embedded in a salt bridge (agar saturated potassium chloride), and is placed in the electric field stimulation holes 1 and 2 of the microfluidic chip, respectively, to form a loop with the culture solution in the microchannel. A constant applied voltage can provide a uniform and stable direct current electric field microenvironment for the cells in the microchannel (as shown in FIG. 2). By adjusting the size of the applied voltage, an electric field stimulation of different intensities can be obtained. Figure 4

[0041] Embodiment 2: A preparation method of a cell electrotaxis monitoring microfluidic chip.

[0042] In the present application, the microfluidic chip is formed by aligning the upper and lower PDMS channel structures and then bonding them by oxygen plasma. The material is transparent to light and air and has good sealing performance. Figure 2 In the embodiment, the upper PDMS channel is above the dashed line, and the lower PDMS channel is below the dashed line. The height of the upper PDMS channel can be set to 27-30 μm, and the height of the lower PDMS channel can be set to 8-10 μm. After the upper and lower layers are bonded, the internal channels of the microfluidic chip form the electric field channels A, the fluid channels B and the narrow channels C described in embodiment 1. The height of the narrow channel C is the height of the lower PDMS channel, and the height of the electric field channel A and the fluid channel B is the sum of the height of the upper PDMS channel and the height of the lower PDMS channel. The fluid channel B and the narrow channel C are cross-connected with the electric field channel A, and the narrow channel C can well retain the newly inoculated suspended cells at the same point. The two narrow channels C are designed to have a certain angle, so that the lengths of the co-culture regions E are different, thereby simulating the microenvironment in which two types of cells coexist at different distances.

[0043] The preparation method of the cell electrotaxis monitoring microfluidic chip provided in the embodiment is as follows:

[0044] ​1. Using high-resolution laser printing of the upper and lower PDMS channel structure pattern, a photomask is obtained.

[0045] 2. Place the mask on the printed circuit board (PCB board), after UV exposure and development, place the PCB board in FeCl3 solution for etching (the height of the channel is determined by the concentration of the etching solution and the etching time), after etching, rinse with running water and dry, to obtain the upper and lower chip templates.

[0046] 3. Stir the PDMS prepolymer and crosslinking agent uniformly at a mass ratio of 10:1, pour onto the PCB board, remove bubbles, and bake in an oven at 65°C for 3h.

[0047] 4. After the PDMS is cooled and solidified, it is carefully peeled off from the template, and according to the design requirements, holes are punched in the corresponding positions of the upper PDMS channel. All the holes are circular with a diameter of 2mm.

[0048] 5. Place the two layers of PDMS channel structure layer upwards, treat the structure surface with oxygen plasma for 30s, then align the channels under a microscope and bond them to form a closed microchannel, thus obtaining a microfluidic chip. The formed microfluidic chip has a glass slide as the substrate, the thickness of the upper and lower PDMS is 1mm, and the overall size of the microfluidic chip is 2.5cm×2.5cm (as shown in Figure 3

[0049] Example 3, a method for monitoring cell electrotaxis migration under co-culture conditions.

[0050] This example is a method for monitoring cell electrotaxis migration of tumor-associated fibroblasts (CAFs) and lung cancer cells (A549) under co-culture conditions using a microfluidic chip, which includes the following steps:

[0051] (1) Use MEM containing 10% fetal bovine serum (FBS) and 1% double-antibiotic (penicillin and streptomycin) as the culture medium to culture human lung cancer cells A549 and tumor-associated fibroblasts CAFs.

[0052] (2) After sterilization and surface treatment of the prepared microfluidic chip channel, place it in a culture dish for standby. First, soak the chip channel and buffer pool with 75% medical alcohol, and sterilize it under UV irradiation for 30min; second, rinse the channel and buffer pool with PBS buffer solution to remove alcohol residues, inject 20μg / mL fibronectin (Fn) into the chip, and incubate at 37°C for 2h to enhance the adhesion ability of cells.

[0053] (3) Before inoculating cells, discard Fn, and inoculate 1×10 6 ​A549 cell suspension (5 μL volume) was injected from inlet 4 into the fluidic channel, while complete culture medium (10 μL) was added at the same side of electric field stimulation hole 2, and the speed of microfluidic was controlled, so that the cells flowed slowly and in parallel through each electric field channel A. Under the microscope, the results showed that the suspended cells were trapped in the narrow channel C near the co-culture area on one side.

[0054] (4) The chip was moisturized with sterile water around the chip, and was incubated in a 37 °C, 5% CO2 incubator. After the A549 cells adhered, CAFs cells were inoculated at inlet 3 on the other side. In order to better distinguish the two kinds of cells, the second kind of cells could be labeled with Cell Tracker fluorescent probe before inoculation.

[0055] (5) After the two kinds of cells adhered completely, platinum wire electrodes were inserted into electric field stimulation holes 1 and 2 through salt bridges, and a certain intensity of voltage was applied to form a direct current electric field in the channel. The position and morphological changes of the cells were recorded in real time by taking pictures under the microscope, so that electrotaxis migration of the two kinds of cells under different spacing co-culture conditions could be realized (as shown in Figure 2 ). Under the electric field stimulation, the migration of the two kinds of cells was as shown in Figure 5 .

[0056] After a period of direct current electric field stimulation, the migrated cells could be detected in situ by commonly used biological detection methods, including cell dead and live labeling and staining, immunofluorescence staining, etc.

[0057] The different kinds of cells described in the embodiment include but are not limited to lung cancer cells and tumor-related fibroblasts.

Claims

1. A cell electrotaxis migration monitoring microfluidic chip, characterized in that, The microfluidic chip is internally provided with a plurality of parallel electric field channels and two fluid channels and two narrow channels intersecting with the electric field channels; the two narrow channels are located between the two fluid channels and oppositely arranged with the two fluid channels; the two narrow channels form an included angle of 8-15° between them, and the narrow channels are used for intercepting suspended cells; the electric field channel between the two narrow channels is a co-culture area, and different cells entering from the two fluid channels can enter the co-culture area from the two narrow channels under the action of an electric field; the height of the fluid channel and the electric field channel is the same, and is 35-40 μm; the height of the narrow channel is set to 8-10 μm.

2. The cell electrophoretic migration monitoring microfluidic chip according to claim 1, wherein, The two ends of the electric field channel are respectively connected with buffer pools, and an electric field stimulation hole is opened above the buffer pool.

3. The cell electrophoretic migration monitoring microfluidic chip according to claim 1, wherein, Sample inlets and sample outlets are respectively arranged at the two ends of the fluid channel.

4. The cell electrophoretic migration monitoring microfluidic chip according to claim 1, wherein, The included angle between the two narrow channels is 10°.

5. The cell electrophoretic migration monitoring microfluidic chip according to claim 1, wherein, The lengths of the co-culture areas on different electric field channels are different, and the length of the co-culture area ranges from 380 to 1630 μm.

6. The method for preparing a cell electrophoretic migration monitoring microfluidic chip according to claim 1, wherein, The microfluidic chip is formed by aligning the upper and lower two layers of PDMS channel structures and bonding by oxygen plasma, and the specific preparation method is as follows: S1, laser printing upper and lower two layers of PDMS channel structure patterns to obtain a mask; S2, place the mask on a printed circuit board, after ultraviolet exposure and development, place the printed circuit board in a FeCl3 solution for etching, after etching, rinse with running water and dry, to obtain the upper and lower two layers of chip templates; S3, mix and stir the PDMS prepolymer and crosslinking agent uniformly, pour it on the printed circuit board, and place it in an oven after removing bubbles for baking and curing; S4, after the PDMS is cured and cooled, it is peeled off from the template, and holes are punched at the corresponding positions of the upper layer of PDMS channel structure; S5, the upper and lower two layers of PDMS channel structure are surface modified by oxygen plasma, and then aligned and bonded under a microscope to obtain a microfluidic chip.

7. The method for fabricating a microfluidic chip for monitoring cell electrotaxis and migration according to claim 6, characterized in that, In step S4, the punched holes include electric field stimulation holes, sample inlets and sample outlets.

8. A method of monitoring cell electrotaxis, comprising: The cell electrotaxis monitoring microfluidic chip of claim 2 comprises the following steps: (1) culture two different cells; (2) sterilize and surface treat the microfluidic chip, and then place it in a culture dish for standby: first, soak the chip channel and buffer pool with alcohol, and sterilize by ultraviolet irradiation; then, rinse the channel and buffer pool with PBS buffer solution to remove alcohol residues, inject 20 μg / mL of fibronectin into the chip, and incubate at 37°C for 2 h to enhance the adhesion ability of cells; (3) inoculate cells: first, discard the fibronectin, then inject the cell suspension cultured in step (1) into one of the fluid channels, and add complete culture medium at the same side of the electric field stimulation hole, so that the cells flow through the electric field channels in parallel, and the suspended cells are intercepted in the narrow channel close to the co-culture area on one side; (4) add sterile water around the chip for moisturizing, and incubate it in a 37°C, 5% CO2 incubator, after the cells in step (3) adhere, inoculate another kind of cells cultured in step (1) in the other fluid channel; (5) After the two kinds of cells are all adhered, platinum wire electrodes are inserted into the two electric field stimulation holes through salt bridges, and an external voltage is applied to form a direct current electric field in the electric field channel. Under the electric field stimulation, the two kinds of cells can adhere into the co-culture area.

9. The method of monitoring cell electrophoretic migration according to claim 8, wherein, In step (4), the other cell can be labeled with a Cell Tracker fluorescent probe before inoculation.

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