Microfluidic device and method for analyzing mechanical properties of cells by using microfluidic device

Abstract

The invention discloses a microfluidic device and a method for analyzing mechanical properties of cells by using the microfluidic device, the microfluidic device comprises a plurality of microchannels, each microchannel is provided with a normal area through which to-be-tested simulated cells easily pass and a narrow area through which the to-be-tested simulated cells difficultly pass, and the size of the microchannels accords with the size range of a microartery. The measuring and analyzing device is simple in structure, and the measuring and analyzing method is easy to execute.

Description

Technical Field

[0001] The present invention relates to the field of measurement technology, and in particular to a microfluidic device and a method for analyzing cell mechanical properties using the microfluidic device. Background Art

[0002] The metastasis of cancer cells in the body is the main cause of death in cancer patients. During the metastatic spread, tumor cells carrying oncogenic driver mutations first destroy the basement membrane and invade deeper tissue layers, then infiltrate into proximal blood vessels or lymphatic vessels, and spread to adjacent spaces through transendothelial migration, and finally extravasate to distant organs. Therefore, it is of great significance to study the factors that affect the metastatic efficiency of cancer cells, and the mechanical properties of cancer cells are effective biomarkers for studying their metastatic ability.

[0003] In the prior art, there are many methods for measuring cell mechanical properties, such as atomic force microscopy (AFM) extrusion, parallel plate technology, micropipette aspiration, magnetic tweezers or optical stretching, etc. These existing single-cell biomechanical measurement technologies require complex instruments and skilled operations. Therefore, in practical applications, a simple and easy-to-operate measuring device and method are urgently needed. Summary of the invention

[0004] The present invention provides a microfluidic device and a method for analyzing cell mechanical properties using the microfluidic device. Not only is the structure of the measuring and analyzing device simple, but the measuring and analyzing method is also easy to execute.

[0005] The present invention provides a microfluidic device, comprising a plurality of microchannels, each of which is provided with a normal region through which simulated cells to be tested can easily pass and a narrow region through which simulated cells to be tested cannot easily pass, and the size of the microchannels conforms to the size range of arterioles.

[0006] The present invention also provides a method for analyzing cell mechanical properties using the above-mentioned microfluidic device, comprising:

[0007] Step 1, controlling the pressure difference between the inlet and outlet of a plurality of microchannels in the microfluidic device by adjusting the liquid flow rate;

[0008] Step 2: adjusting the diameter of the simulated cells to be tested within a preset diameter range, and detecting the first critical surface tension of the simulated cells to be tested with different diameters that can pass through the narrow area.

[0009] The present invention realizes high-throughput detection by setting up multiple microchannels, which is very suitable for rare cell analysis in blood samples; and through microchannels, cell blood vessels are simulated to analyze the mechanical properties of cells, predict the influencing factors of cell transendothelial migration, and provide an efficient detection and prevention verification method for the treatment of cancer cell metastasis. Therefore, the present invention uses a microfluidic device to verify the relationship between the efficiency of cancer cell metastasis and cell surface tension in a narrow area; the critical surface tension values ​​of different cells are obtained by simulation, which can be used as a reference for clinical prognosis; and it is verified that the scheme of using drugs that can reduce cell surface tension to inhibit cancer cell metastasis is feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0011] Figure 1 is a microscopic image of a microchannel array made in an embodiment of the present invention;

[0012] Figure 2 a is a schematic diagram of the process of uveal melanoma cells passing through arteriolar occlusion;

[0013] Figure 2 b is Figure 1 Schematic diagram of the dimensions of a single microchannel designed in;

[0014] Figure 3 a Schematic diagram of the simulation results of pressure distribution around the narrow microchannel array at different inlet flow rates;

[0015] Figure 3 b is a graph showing the relationship between the pressure difference and flow rate between the inlet and outlet of a single narrow microchannel;

[0016] Figure 4 It is a schematic diagram of simulation results of different diameters and surface tensions in an embodiment of the present invention;

[0017] Figure 5 a is a working schematic diagram of the microfluidic cytometer provided by the present invention;

[0018] Figure 5 b is Figure 5 Representative images of measurements in a;

[0019] Figure 6 a Image of MP46 cells treated with cytochalasin passing through a narrow area at a driving flow rate of 40 μL / min;

[0020] Figure 6 b is a schematic diagram showing the comparison of the average surface tension values ​​of untreated MP46 cells and cytochalasin-treated MP46 cells;

[0021] Figure 6 c: Image of cytochalasin-treated MP46 cells floating along a narrow microchannel array. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Existing single-cell biomechanical measurement techniques require complex instruments and skilled operations. In addition, most of these methods are single-parameter evaluations with low accuracy and throughput. As an emerging technology, microfluidics is suitable for various biological applications, including clinical liquid biopsy and cell mechanical property examination. The structure of the microfluidic device can be customized, and high-throughput detection can be achieved by increasing the number of microchannels, which is more suitable for analyzing rare cells in blood samples. Based on the use of microfluidic channels to measure cell surface tension, the present invention further designs a microfluidic device to simulate cancer cells passing through narrow blood vessels, and combines the finite element method to demonstrate the effect of surface tension on the transendothelial migration of cancer cells. This structure is not only simple in structure, but also convenient and easy to operate.

[0024] To make the technical solution of the present invention clearer, embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0025] An embodiment of the present invention provides a microfluidic device including a plurality of microchannels, each of which is provided with a normal region through which the simulated cells to be tested can easily pass and a narrow region through which the cells cannot easily pass, and the size of the microchannels conforms to the size range of arterioles.

[0026] Figure 1 is a microscopic image of a microchannel array made in an embodiment of the present invention; Figure 2 a is a schematic diagram of the process of uveal melanoma cells passing through the arteriolar occlusion. Left: just entering the narrow area; middle: successfully passing through the narrow area; right: trapped in the narrow area; Figure 2 b is Figure 1 Schematic diagram of the dimensions of a single microchannel designed for simulation Figure 2 The structure of the arteriolar channel in a; Figure 1 and Figure 2As shown, the cross-sectional size of the normal area of ​​each microchannel in the microchannel array (multiple microchannels) is 40 μm×40 μm, and the cross-sectional size of the narrow area is 10 μm×40 μm.

[0027] The above-mentioned microchannel array can be prepared by photolithography technology. Specifically, it includes: first, spin-coating SU-8 photoresist (SU-82025, Microchem) on a silicon wafer to form a 40μm SU-8 layer, placing a mask for exposure, and developing to complete the mold. A mixture of polydimethylsiloxane (PDMS) (Sylgard-184, Dow Corning) and a curing agent with a weight ratio of 10:1 is cast onto the mold. Place it in a vacuum chamber for 30 minutes to remove bubbles, and bake it in an oven at 85°C for 2 hours to cure the PDMS. After peeling it off from the manufactured mold, holes are punched at the inlet and outlet of the device. The PDMS layer is then bonded to a glass slide using oxygen plasma treatment. Finally, bake it in a drying oven for 10 minutes to solidify the boundaries. The final microfluidic device contains a set of narrow microchannels with dimensional parameters such as Figure 2 The microscopic image is shown in b. Figure 1 shown.

[0028] In order to simulate the pressure of the liquid entering the microchannel, finite element calculation can be performed on the microchannel of the microfluidic device obtained above, and the relationship between the inlet flow rate of the microfluidic device and the pressure difference of a single narrow microchannel is obtained, that is, the pressure distribution of the mainstream area of ​​the microfluidic device at different flow rates. Figure 3 a Schematic diagram of the simulation results of pressure distribution around the narrow microchannel array at different inlet flow rates, unit: Pa; Figure 3 b is a graph showing the relationship between the pressure difference and flow rate between the inlet and outlet of a single narrow microchannel. Figure 3 As shown in a, the pressure distribution changes with the flow rate. A series of simulations were performed at different flow rates, and the results are shown in Figure 3 As shown in b. Figure 3 It can be clearly seen in b that there is a linear correlation between the pressure difference and the flow rate. Therefore, in practical applications, the pressure difference in narrow microchannels can be controlled by adjusting the liquid flow rate.

[0029] Surface tension is a physical phenomenon in which molecules on the surface of a liquid attract each other to minimize their area. In order to understand the effect of cell surface tension on the movement of cells along narrow areas, in an embodiment of the present invention, based on the microchannel designed above, the 'Laminar Two-Phase Flow, Moving Mesh' module of COMSOL Multiphysics5.6 was used for numerical simulation to study the effect of cell tension and cell diameter on the movement of cancer cells along narrow areas. Taking into account the heterogeneity of cancer cells, cells of different diameters and surface tensions are selected in the present invention. The cell diameter ranges from 12μm to 22μm. The surrounding liquid is regarded as blood, with a mass density of 1050kg / m3 and a dynamic viscosity of 5.73mPa·s. The cell mass density and dynamic viscosity are defined as 1020kg / m3 and 30mPa·s, respectively. The inlet and outlet pressure difference is set to 98.5Pa, which is a reasonable value for arterioles (67~1333Pa). Figure 4 Schematic diagram of simulation results for different diameters and surface tensions in an embodiment of the present invention. Figure 4 As shown, the simulation results show that cells of the same size and different surface tensions will lead to different behaviors when squeezed in narrow blood vessel areas. Cells with the same tension value will also show different behaviors when the diameter is different. These results show that both cell diameter and cell tension are important parameters that affect the metastatic process of cancer cells.

[0030] In order to detect the critical surface tension of different diameters, simulations were performed for cell diameters ranging from 12μm to 22μm. According to the simulation results, it is shown that when the cell diameter ranges from 12μm to 16μm, the cell diameter can greatly affect the movement of cancer cells in narrow blood vessels (cross-sectional dimensions: 10μm×40μm). If the cell diameter is greater than 16μm, the cell surface tension becomes the dominant parameter. These results show that numerical simulation is expected to assist in the prognosis of cancer metastasis.

[0031] Based on the above simulation, an embodiment of the present invention provides a method for analyzing cell mechanical properties using the above microfluidic device. The method of this embodiment includes:

[0032] Step 1: Control the pressure difference between the inlet and outlet of multiple microchannels in the microfluidic device by adjusting the liquid flow rate.

[0033] Based on the above analysis, it can be seen that the inlet and outlet pressure difference of the microfluidic device can be controlled by the liquid flow rate. Therefore, when performing cell mechanical properties analysis, the liquid flow rate can be determined in advance based on the relationship between the inlet flow rate of each microchannel of the microfluidic device and the pressure difference between the inlet and outlet of the single microchannel calculated by finite element analysis.

[0034] Step 2: adjusting the diameter of the simulated cells to be tested within a preset diameter range, and detecting the first critical surface tension of the simulated cells to be tested with different diameters that can pass through the narrow area.

[0035] Among them, the preset diameter range is 12μm to 22μm; for a specific cell diameter, the surface tension of the cell is adjusted to obtain the first critical surface tension corresponding to the simulated cell to be tested under the specific cell diameter, and the effect of surface tension on the passage of specific cells through the narrow area can be determined by observing the first critical surface tension under the specific cell diameter. That is, based on the first critical surface tension obtained by the above detection, the effect of cell surface tension in blood vessels on the efficiency of cell migration in narrow blood vessels can be further predicted.

[0036] For a specific cell surface tension, the influence of cell diameter on cell transendothelial migration can be analyzed and verified by detecting the maximum diameter of the simulated cell that can pass through the narrow area.

[0037] In order to verify that the surface tension of the cell-fluid interface plays a vital role in the blood metastasis of cancer cells, the present invention uses a microfluidic cytometer to quantify the surface tension of cancer cells. Figure 5 a is a working schematic diagram of the microfluidic cytometer provided by the present invention, Figure 5 b is Figure 5 Representative images of the measurements in a. Based on the above analysis and multiple microchannel arrays of microfluidic devices, a microfluidic cytometer for measuring cell mechanical properties can be prepared, such as Figure 5 As shown in a, the microfluidic cytometer can obtain the forces experienced by deformed cells in a high-throughput manner. Representative images of the measurements are shown in Figure 5 As shown in b. In practical applications, a syringe pump can be used to drive the cancer cell suspension into the microfluidic cytometer, and the inlet flow rate can be adjusted to 20 μL / min to achieve a pressure difference of about 50 Pa between the inlet and outlet of the restrictive microchannel. Through the captured cells, the cell compression force is calculated considering the force balance of the captured cells, and the surface tension can be obtained by calculation. After obtaining the single-cell surface tension value, the transit behavior of cancer cells in narrow blood vessels can be predicted. Finally, microfluidic experiments are performed to verify whether cancer cells can pass through the narrow channels simulated by the microfluidic device.

[0038] In a specific application, based on the above-mentioned prediction results of cell surface tension, cells to be verified can be selected and divided into a first group of cells and a second group of cells in equal parts, the surface tension of the first group of cells can be treated, and the proportion of cells that fail to pass through narrow blood vessels can be compared between the first group of cells after surface tension treatment and the second group of cells without surface tension treatment. For example, if the cells to be verified are human uveal melanoma cells, the surface tension of human uveal melanoma cells can be treated with cytochalasin, and the effects of cytochalasin treatment or not on the transendothelial migration of human uveal melanoma cells in blood vessels can be compared.

[0039] The following is an example of uveal melanoma. Uveal melanoma is the most common malignant intraocular tumor in adults. Uveal melanoma is highly malignant, and the posterior pole of the eye is a common site. It is easy to metastasize through the blood flow and has a poor prognosis. Therefore, the present invention selected human uveal melanoma cells (MP46) for the experiment.

[0040] In order to make the experimental results more obvious, in this example, MP46 cells were treated with cytochalasin D. Cytochalasin D is a cell-permeable inhibitor of actin polymerization and is also used in cancer treatment.

[0041] Figure 6 a Image of MP46 cells treated with cytochalasin passing through a narrow area at a driving flow rate of 40 μL / min; Figure 6 b is a schematic diagram showing the comparison of the average surface tension values ​​of untreated MP46 cells and cytochalasin-treated MP46 cells; Figure 6 c is an image of cytochalasin-treated MP46 cells floating along a narrow microchannel array. Figure 6 As shown, it can be found that untreated MP46 cells have significantly higher surface tension values ​​than those treated with cytochalasin. These results indicate that the MP46 cytoskeleton may be destroyed by cytochalasin, making it difficult to migrate across the endothelium. Comparison with the obtained critical value of cell tension revealed that a lower percentage of cells (more than 40%) were located in the "capture" domain, i.e., unable to pass through the narrow channel, which was significantly lower than the result of untreated MP46 cells (98.1%). The results indicate that after cytochalasin treatment, the possibility of MP46 cells migrating across the endothelium is reduced. In order to verify this prediction, microfluidic experiments were also conducted in the present embodiment, such as Figure 6 c. The time-lapse image shows that after cytochalasin treatment, under the same inlet pressure, MP46 cells will be flushed out of the narrow blood vessel area. Microfluidic experiments prove that the measurement method provided by the present invention can reasonably predict the possibility of transendothelial migration of MP46 cells in narrow blood vessels, providing a new method for the prognosis of uveal melanoma.

[0042] The microfluidic device provided by the embodiment of the present invention and the method for cell mechanics analysis using the same achieve high-throughput detection by setting up multiple microchannels, which is very suitable for rare cell analysis in blood samples; and the microchannels simulate cell blood vessels to analyze the cell mechanical properties, predict the influencing factors of cell transendothelial migration, and provide an efficient detection and prevention verification method for the treatment of cancer cell metastasis diseases. Therefore, the present invention uses a microfluidic device to verify the relationship between the efficiency of cancer cell metastasis and cell surface tension in a narrow area; the critical surface tension values ​​of different cells are obtained by simulation, which can be used as a reference for clinical prognosis; and it is verified that the scheme of using drugs that can reduce cell surface tension to inhibit cancer cell metastasis is feasible.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic device, characterized in that: It comprises a plurality of microchannels, each of which is provided with a normal area for the simulated cells to be tested to pass easily and a narrow area for the cells to pass hardly, and the size of the microchannel is in accordance with the size range of the arteriole.

2. The device according to claim 1, characterized in that The multiple microchannels are prepared by using photolithography technology.

3. The device according to any one of claims 1 to 2, characterized in that: The cross-sectional size of the normal area is 40 μm×40 μm, and the cross-sectional size of the narrow area is 10 μm×40 μm.

4. A method for analyzing cell mechanical properties using the microfluidic device according to any one of claims 1 to 3, characterized in that: include: Step 1, controlling the pressure difference between the inlet and outlet of a plurality of microchannels in the microfluidic device by adjusting the liquid flow rate; Step 2: adjusting the diameter of the simulated cells to be tested within a preset diameter range, and detecting the first critical surface tension of the simulated cells to be tested with different diameters that can pass through the narrow area.

5. The method according to claim 4, characterized in that Also includes: Step 3: Predict the effect of the surface tension of cells in blood vessels on the migration efficiency of cells in narrow blood vessels based on the first critical surface tension obtained by detection.

6. The method according to claim 5, characterized in that According to the prediction results in step three, cells to be verified are selected and divided into a first group of cells and a second group of cells in equal parts. The surface tension of the first group of cells is treated, and the proportion of cells that have not passed through the narrowed blood vessels is compared between the first group of cells after surface tension treatment and the second group of cells without surface tension treatment.

7. The method according to claim 6, characterized in that The cells to be verified are human uveal melanoma cells, and the surface tension of the human uveal melanoma cells is treated by using cytochalasin; To compare the effects of cytochalasin treatment or not on the transendothelial migration of human uveal melanoma cells in blood vessels.

8. The method according to any one of claims 4 to 7, characterized in that: The preset diameter range is 12 μm to 22 μm; for a specific cell diameter, the surface tension of the cell is adjusted to obtain the first critical surface tension corresponding to the simulated cell to be tested under the specific cell diameter.

9. The method according to claim 4, characterized in that Also includes: For a specific cell surface tension, the maximum diameter of the simulated cell that can pass through the narrow area is detected; the effect of cell diameter on cell transendothelial migration is analyzed.

10. The method according to claim 4, characterized in that Before the step 1, the method further includes: using finite element analysis to calculate the relationship between the flow rate at the inlet of each microchannel of the microfluidic device and the pressure difference between the inlet and outlet of the single microchannel.

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