Cell migration micro-fluidic chip integrated with multi-channel array and preparation method of cell migration micro-fluidic chip

Abstract

The invention relates to a cell migration micro-fluidic chip integrated with a multi-channel array and a preparation method of the cell migration micro-fluidic chip. The cell migration micro-fluidic chip comprises a cell culture unit and a cell migration unit, the cell culture unit comprises a cell loading unit and a culture solution loading unit which are arranged in parallel; the cell migration unit comprises a plurality of microchannel arrays; the micro-channel array is positioned on two sides of a cell culture channel in the cell loading unit and is vertical to the cell culture channel; the height of the cell culture channel is greater than that of the micro-channel array. Through the cell migration micro-fluidic chip, the migration state of cells can be dynamically observed in real time, and different physical and chemical stimulation factors can be added into the chip to intervene cell migration, so that a new platform is provided for researching a wound repair mechanism and screening drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and particularly relates to a cell migration microfluidic chip integrated with a multi-channel array and a preparation method thereof. Background Art

[0002] Cell migration is one of the basic biological behaviors of cells, and plays an important role in organogenesis, tissue repair, immune response, and cancer metastasis. Among them, wound repair is a dynamic process involving a series of complex biological behaviors. In addition to important wound repair-related cells and cytokines, the speed of cell migration also plays a vital role in accelerating wound healing. Regulating the local physical and chemical microenvironment of cells can affect the migration speed of wound cells by changing cytoskeleton-related proteins, including physiological and biochemical factors such as extracellular matrix, growth factors, enzymes, and reactive oxygen species. Therefore, screening physical and chemical stimulation factors that can promote cell migration will help the development of drugs for wound repair and significantly accelerate the healing of wounds.

[0003] Currently, the commonly used methods for studying cell migration include scratch experiments and Transwell experiments. The scratch experiment is simple to operate, but it will damage the cells when making the scratch, and the migration of individual cells cannot be observed. The Transwell experiment can quantitatively detect the number of cell migration, but it cannot dynamically monitor the cell migration process in real time, nor can it distinguish between migrating and proliferating cells. Microfluidic chip technology can accurately manipulate fluids in micron-scale channels, integrate various units of biochemical analysis, and complete the entire process of analysis and detection. The use of microfluidic chips can realize real-time dynamic monitoring of cell migration status at different times without damaging cells, and combine image processing methods to quantitatively analyze the migration speed of individual cells. Therefore, the construction of an in vitro test platform for cell migration based on microfluidic chip technology is helpful to screen drugs that promote wound repair. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a cell migration microfluidic chip with an integrated multi-channel array and a preparation method thereof. The cell migration microfluidic chip can be used to dynamically observe the migration state of cells in real time, and different physical and chemical stimulation factors can be added into the chip to intervene in cell migration, providing a new platform for studying wound repair mechanisms and drug screening.

[0005] The present invention provides a cell migration microfluidic chip with an integrated multi-channel array, wherein the cell migration microfluidic chip comprises a cell culture unit and a cell migration unit; the cell culture unit comprises a cell loading unit and a culture fluid loading unit arranged in parallel; the cell migration unit comprises a plurality of microchannel arrays; the microchannel arrays are located on both sides of a cell culture channel in the cell loading unit and are perpendicular to the cell culture channel (for limiting the path of cell migration); the height of the cell culture channel is greater than the height of the microchannel array.

[0006] Preferably, the cell loading unit comprises a cell injection port, a cell culture channel and a cell outflow port, which are used for injecting cells and defining the initial position of cell migration.

[0007] Preferably, the culture fluid loading unit comprises a culture fluid inlet, a fluid flow channel and a culture fluid outlet, for adding cell culture fluid.

[0008] Preferably, the height of the liquid flow channel is the same as the height of the cell culture channel.

[0009] Preferably, the microchannel array is composed of a plurality of straight channels, and the straight channels are symmetrically distributed on both sides of the cell culture channel. Cells can migrate toward the microchannels on both sides at will, thereby increasing the throughput of the cell migration experiment.

[0010] Preferably, the widths of the plurality of microchannel arrays are the same or different.

[0011] Preferably, the height of the microchannel array is controlled within 10 μm; the height of the cell culture channel is 20-200 μm. This height design limits the cells in the cell migration unit microchannel to pass only in a single form, preventing the cells from freely diffusing into the microchannel when the cell suspension is injected, but allows the adherent cells to actively migrate into the microchannel, so as to intuitively study the migration behavior of a single cell in the microchannel, and at the same time provide a convenient liquid flow channel for perfusing the cell suspension.

[0012] Furthermore, in the present invention, the height of the cell culture channel is preferably 50 μm.

[0013] The inlet and outlet corresponding to the cell culture unit are both circular with a diameter of 1-6 mm, preferably 2 mm, and the width of the cell culture unit channel (including the cell culture channel and the liquid flow channel) is 0.4-1.5 mm, preferably 0.5 mm.

[0014] The height of the microchannel array is 2-10 μm, preferably 10 μm.

[0015] The width of the microchannel array is 5-100 μm, the spacing between microchannels of the same width is 20-50 μm, preferably 30 μm, and the number of straight channels on one side of the microchannel array of the same width can be 20-60, preferably 40.

[0016] The present invention also provides a method for preparing a cell migration microfluidic chip integrated with a multi-channel array, comprising the following steps:

[0017] (1) Using standard soft lithography technology to make a template for the microfluidic chip pattern, including spin coating of photoresist, pre-baking, UV exposure, post-baking, development and film solidification;

[0018] (2) mixing polydimethylsiloxane (PDMS) prepolymer and a cross-linking agent in a mass ratio of (8-12):1, pouring the resulting mixture onto the surface of a silicon plate with a chip pattern, and then removing bubbles with a vacuum pump. After the bubbles are completely removed, placing the plate in an oven until the PDMS is cured;

[0019] (3) Peel off the cured PDMS chip from the surface of the silicon plate, and use a puncher to make the inlet and outlet corresponding to the channel as required;

[0020] (4) Plasma treatment allows the PDMS chip to be irreversibly bonded to the clean glass slide through chemical bonds to seal the microfluidic chip.

[0021] The present invention also provides an application of a cell migration microfluidic chip integrated with a multi-channel array in the study of wound repair mechanism.

[0022] The present invention also provides an application of a cell migration microfluidic chip integrated with a multi-channel array in wound repair drug screening.

[0023] The operation flow of the cell migration microfluidic chip integrated with a multi-channel array of the present invention when in use is as follows:

[0024] (1) Add 20-100 uL of Fibronectin solution to the prepared microfluidic chip and incubate at room temperature for 1 hour to modify the inner wall of the microfluidic chip channel with Fibronectin protein and enhance cell adhesion in the chip channel;

[0025] (2) aspirating the Fibronectin solution in the PDMS chip, and then adding phosphate buffer to the chip to rinse the microfluidic chip channel;

[0026] (3) Prepare a cell suspension of target cells with a cell density of 2×10 6 -8×10 6 cells / mL, preferably 6×10 6cells / mL; (4) using a pipette to inject the cell suspension into the cell culture channel from the injection port of the central channel of the cell culture unit of the microfluidic chip; (5) after the flow of the cell suspension is stable, add the cell culture solution to the injection ports of the two side channels of the cell culture unit of the microfluidic chip;

[0027] (6) When the cells to be inoculated on the chip adhere to the wall and grow to the microchannel of the cell migration unit of the microfluidic chip, the migration of the cells in the microchannel can be observed under a microscope. The dynamic position of each cell migration in several microchannels can be recorded in real time by combining with a live cell dynamic imaging microscope, and the change of the cell migration rate can be quantitatively analyzed based on this.

[0028] Furthermore, the target cells may be normal tissue cells or disease-related cells.

[0029] Beneficial Effects

[0030] (1) The microfluidic chip of the present invention can be used for the study of cell migration of normal tissue cells or disease-related cells under the intervention of different biochemical factors or drugs.

[0031] (2) The microfluidic chip of the present invention can be integrated with a live cell dynamic imaging system to place the cell migration movement area under a microscope to observe the cell migration movement trajectory, thereby achieving real-time dynamic observation of cell biological behavior.

[0032] (3) The microfluidic chip of the present invention can observe the interference effect of different biochemical factors or drugs on cell migration. By adding different biochemical factors or drugs to the cell culture unit of the microfluidic chip, high-throughput screening of biochemical factors / drugs that promote cell migration or wound repair can be achieved.

[0033] (4) The microfluidic chip of the present invention is easy to operate and can simultaneously construct multiple independent and repetitive cell migration research systems, thereby improving experimental efficiency, reducing the amount of cells and reagents used, and lowering experimental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The overall structure schematic diagram of the cell migration microfluidic chip integrated with a multi-channel array of the present invention;

[0035] Figure 2 is a cross-sectional view of a cell culture unit and a cell migration unit of the present invention;

[0036] Figure 3 The migration of human skin keratinocytes in the microchannel array of the cell migration unit in the cell migration microfluidic chip of the present invention;

[0037] Among them, 1 is a cell injection port; 2 is a cell culture channel; 3 is a cell outflow port; 4 is a culture fluid injection port; 5 is a liquid flow channel; 6 is a culture fluid outflow port; 7 is a microchannel array; 8 is a polydimethylsiloxane (PDMS) substrate; and 9 is a glass slide. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0039] Example 1

[0040] This embodiment provides a cell migration microfluidic chip with an integrated multi-channel array, such as Figure 1 As shown, the cell migration microfluidic chip includes a cell culture unit and a cell migration unit; the cell culture unit includes a cell loading unit and a culture fluid loading unit arranged in parallel; the cell migration unit includes a plurality of microchannel arrays; the microchannel arrays are located on both sides of the cell culture channel in the cell loading unit and are perpendicular to the cell culture channel; the height of the cell culture channel is greater than the height of the microchannel array. The cell loading unit includes a cell injection port, a cell culture channel and a cell flow outlet. The culture fluid loading unit includes a culture fluid injection port, a liquid flow channel and a culture fluid flow outlet. The height of the liquid flow channel is the same as the height of the cell culture channel. The microchannel array is composed of a plurality of straight channels, and the straight channels are symmetrically distributed on both sides of the cell culture channel. The widths of the plurality of microchannel arrays are the same or different.

[0041] like Figure 2 As shown, this embodiment adopts a nested microfluidic channel structure with two layers of different heights, wherein the height of the microchannel in the cell migration unit is controlled within 10 μm, and the height of the cell culture unit is greater than the height of the microchannel in the cell migration unit. This height design limits the cells in the microchannel of the cell migration unit to pass through only in a single form, preventing the cells from freely diffusing into the microchannel when the cell suspension is injected, but allows the adherent cells to actively migrate into the microchannel, so as to intuitively study the migration behavior of a single cell in the microchannel, and at the same time provide a convenient liquid flow channel for perfusing the cell suspension.

[0042] More specifically, the height of the cell culture unit channel of the cell migration microfluidic chip of this embodiment is 50μm; the inlet and outlet corresponding to the channel in the cell culture unit are circular with a diameter of 2mm, and the width of the cell culture unit channel is 0.5mm; the height of the cell migration unit channel is 10μm, and the widths of the microchannels are 10μm, 20μm, 30μm, and 50μm, respectively. The spacing between microchannels of the same width is 30μm, and the number of single-sided straight channels in the microchannel array of the same width is 40.

[0043] The present embodiment provides a method for preparing a cell migration microfluidic chip with an integrated multi-channel array, and the specific steps are as follows: (1) using standard soft lithography technology to prepare a template for the microfluidic chip pattern, including spin coating of photoresist, pre-baking, ultraviolet exposure, post-baking, development and film solidification;

[0044] (2) Mixing polydimethylsiloxane (PDMS) prepolymer and cross-linking agent in a mass ratio of 10:1, pouring PDMS onto the surface of a silicon plate with a chip pattern, and then removing bubbles with a vacuum pump. After the bubbles are completely removed, placing the prepolymer in an oven until the PDMS is cured; the prepolymer may also be polyvinylsiloxane, polyalkylenesiloxane or octamethylcyclotetrasiloxane; the cross-linking agent may be polyvinylsiloxane, methylhydrogenpolysiloxane or toluene.

[0045] (3) Peel off the cured PDMS chip from the surface of the silicon plate, and use a puncher to make the inlet and outlet corresponding to the channel as required;

[0046] (4) Plasma treatment allows the PDMS chip to be irreversibly bonded to the clean glass slide through chemical bonds to seal the microfluidic chip.

[0047] Example 2

[0048] In this example, the cell migration microfluidic chip with integrated multi-channel array described in Example 1 is used to explore the migration behavior of human skin keratinocytes. The operation process of using the chip is as follows:

[0049] (1) Add 50 uL of Fibronectin solution to the prepared microfluidic chip and incubate at room temperature for 1 hour to modify the inner wall of the microfluidic chip channel with Fibronectin protein and enhance cell adhesion in the chip channel;

[0050] (2) aspirating the Fibronectin solution in the PDMS chip, and then adding phosphate buffer to the chip to rinse the microfluidic chip channel;

[0051] (3) Prepare a cell suspension of human skin keratinocytes with a cell density of 6×10 6 cells / mL;

[0052] (4) using a pipette to inject the cell suspension into the cell culture channel from the injection port of the central channel of the cell culture unit of the microfluidic chip;

[0053] (5) After the liquid flow of the cell suspension is stabilized, adding the cell culture solution to the injection ports of the two side channels of the cell culture unit of the microfluidic chip;

[0054] (6) The cells to be inoculated on the chip adhere to the wall and grow to the microchannel of the cell migration unit of the microfluidic chip, such as Figure 3 As shown, the migration of cells in the microchannels can be observed under a microscope. Combined with the live cell dynamic imaging microscope, the dynamic position of each cell migration in several microchannels can be recorded in real time, and the changes in the cell migration rate can be quantitatively analyzed based on this.

[0055] The microfluidic chip integrated multi-channel array of the present invention is applied to the study of cell migration, can realize real-time dynamic imaging of the cell migration state, and can add different physical and chemical stimulation factors into the chip to intervene in cell migration and perform drug effect testing and screening.

Claims

1. A cell migration microfluidic chip with integrated multi-channel array, characterized in that: The cell migration microfluidic chip includes a cell culture unit and a cell migration unit; the cell culture unit includes a cell loading unit and a culture fluid loading unit arranged in parallel; the cell migration unit includes a plurality of microchannel arrays; the microchannel arrays are located on both sides of the cell culture channel in the cell loading unit and are perpendicular to the cell culture channel; the height of the cell culture channel is greater than the height of the microchannel array.

2. The cell migration microfluidic chip according to claim 1, characterized in that: The cell loading unit comprises a cell injection port, a cell culture channel and a cell outflow port.

3. The cell migration microfluidic chip according to claim 1, characterized in that: The culture solution loading unit comprises a culture solution inlet, a liquid flow channel and a culture solution outlet.

4. The cell migration microfluidic chip according to claim 3, characterized in that: The height of the liquid flow channel is the same as that of the cell culture channel.

5. The cell migration microfluidic chip according to claim 1, characterized in that: The microchannel array consists of a plurality of straight channels, and the straight channels are symmetrically distributed on both sides of the cell culture channel.

6. The cell migration microfluidic chip according to claim 1, characterized in that: The widths of the plurality of micro-channel arrays are the same or different.

7. The cell migration microfluidic chip according to claim 1, characterized in that: The height of the microchannel array is controlled within 10 μm; the height of the cell culture channel is 20-200 μm.

8. A method for preparing a cell migration microfluidic chip with an integrated multi-channel array as claimed in any one of claims 1 to 7, comprising the following steps: (1) Using standard soft lithography technology to make a template for the microfluidic chip pattern, including spin coating of photoresist, pre-baking, UV exposure, post-baking, development and film solidification; (2) mixing polydimethylsiloxane (PDMS) prepolymer and a cross-linking agent in a mass ratio of (8-12):1, pouring the resulting mixture onto the surface of a silicon plate with a chip pattern, and then removing bubbles with a vacuum pump. After the bubbles are completely removed, placing the plate in an oven until the PDMS is cured; (3) Peel off the cured PDMS chip from the surface of the silicon plate, and use a puncher to make the inlet and outlet corresponding to the channel as required; (4) Plasma treatment allows the PDMS chip to be irreversibly bonded to the clean glass slide through chemical bonds to seal the microfluidic chip.

9. Use of a cell migration microfluidic chip with an integrated multi-channel array as claimed in any one of claims 1 to 7 in the study of wound repair mechanism.

10. Use of the cell migration microfluidic chip with integrated multi-channel array as claimed in any one of claims 1 to 7 in wound repair drug screening.

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