Microfluidic device and method for analyzing cell mechanical properties using a microfluidic device

Abstract

The application discloses a micro-fluidic device and a method for analyzing cell mechanical properties by using the micro-fluidic device. The micro-fluidic device comprises a plurality of micro-channels, each of which is provided with a normal region through which simulated cells to be measured can easily pass and a narrow region through which the simulated cells to be measured are not easy to pass, and the size of the micro-channels meets the size range of micro-arteries. The application has the advantages of simple structure of a measuring and analyzing device and easy execution of a measuring and analyzing method.

Description

Technical Field

[0001] This invention relates to the field of measurement technology, and in particular to a microfluidic device and a method for analyzing the mechanical properties of cells using a microfluidic device. Background Technology

[0002] Metastasis of cancer cells is a leading cause of death in cancer patients. During metastasis, tumor cells carrying oncogenic driver mutations first breach 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, finally extravasating to distant organs. Therefore, research into factors influencing the efficiency of cancer cell metastasis is of great significance, and the mechanical properties of cancer cells are an effective biomarker for studying their metastatic ability.

[0003] Existing technologies include various methods for measuring cellular biomechanical properties, such as atomic force microscopy (AFM) compression, parallel plate techniques, micropipette aspiration, magnetic tweezers, or optical stretching. However, these existing single-cell biomechanical measurement techniques require complex instruments and skilled operation. Therefore, in practical applications, there is an urgent need for a simple and easy-to-operate measurement device and method. Summary of the Invention

[0004] This invention provides a microfluidic device and a method for analyzing cell mechanical properties using the microfluidic device. The measurement and analysis device has a simple structure, and the measurement and analysis method is easy to implement.

[0005] The present invention provides a microfluidic device comprising multiple microchannels, each microchannel having a normal region through which the simulated cell to be tested can easily pass and a narrow region through which it cannot easily pass, the size of the microchannel conforming to the size range of microarteries.

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

[0007] Step 1: Control the pressure difference between the inlet and outlet of multiple microchannels in the microfluidic device by adjusting the liquid flow rate;

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

[0009] This invention achieves high-throughput detection through multiple microchannels, making it ideal for analyzing rare cells in blood samples. Furthermore, by simulating cellular blood vessels using microchannels, it analyzes cellular mechanical properties and predicts factors influencing cell transendothelial migration, providing an efficient detection and prevention method for treating metastatic cancer. Therefore, this invention utilizes a microfluidic device to verify the relationship between cancer cell metastasis efficiency and cell surface tension in confined areas; it obtains critical surface tension values ​​for different cells through simulation, which can serve as a reference for clinical prognosis; and it verifies the feasibility of using drugs that reduce cell surface tension to inhibit cancer cell metastasis. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 The image shows a microscopic image of the microchannel array fabricated in an embodiment of the present invention.

[0012] Figure 2 a is a schematic diagram of the process by which uveal melanoma cells pass through small artery occlusion.

[0013] Figure 2 b is Figure 1 A schematic diagram of the dimensions of a single microchannel in the design;

[0014] Figure 3 A schematic diagram of the simulation results of the pressure distribution around the narrow microchannel array under different inlet flow velocities;

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

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

[0017] Figure 5 a is a schematic diagram of the operation of the microfluidic cell analyzer provided by the present invention;

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

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

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

[0021] Figure 6 c is an image of MP46 cells treated with cytochalasin floating along a narrow microchannel array. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] Existing single-cell biomechanical measurement techniques require complex instruments and skilled operation. Furthermore, these methods are mostly single-parameter assessments with low accuracy and throughput. Microfluidics, as an emerging technology, is suitable for various biological applications, including clinical liquid biopsy and cell biomechanical property testing. The structure of microfluidic devices can be custom-designed, and high-throughput detection can be achieved by increasing the number of microchannels, making them more suitable for analyzing rare cells in blood samples. This invention, based on the measurement of cell surface tension using microfluidic channels, further designs a microfluidic device to simulate cancer cells passing through narrow blood vessels, and uses 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 design but also convenient and easy to operate.

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

[0025] This invention provides a microfluidic device comprising multiple microchannels, each microchannel having a normal region through which the simulated cell to be tested can easily pass and a narrow region through which it cannot easily pass, the size of the microchannel conforming to the size range of microarteries.

[0026] Figure 1 The image shows a microscopic image of the microchannel array fabricated in an embodiment of the present invention. Figure 2 A diagram illustrates the process by which uveal melanoma cells pass through a small artery occlusion. Left: just entering the narrow region; Middle: successfully passing through the narrow region; Right: trapped in the narrow region. Figure 2 b is Figure 1 A schematic diagram of the individual microchannel dimensions designed in the image is used to simulate... Figure 2 The arteriolar pathway structure in a; such as Figure 1 and Figure 2As shown, the cross-sectional dimensions of the normal region of each microchannel in the microchannel array (multiple microchannels) are 40μm×40μm, and the cross-sectional dimensions of the narrow region are 10μm×40μm.

[0027] The aforementioned microchannel array can be fabricated using photolithography. Specifically, the process involves: first, spin-coating SU-8 photoresist (SU-82025, Microchem) onto a silicon wafer to form a 40μm SU-8 layer; then, exposure and development using a mask to complete the mold fabrication. Next, a mixture of polydimethylsiloxane (PDMS) (Sylgard-184, Dow Corning) and a curing agent (weight ratio 10:1) is poured into the mold. The mixture is placed in a vacuum chamber for 30 minutes to remove air bubbles and then baked in an oven at 85°C for 2 hours to cure the PDMS. After peeling from the fabricated mold, holes are drilled at the device inlet and outlet. Then, oxygen plasma treatment is used to bond the PDMS layer to a glass slide. Finally, baking in a drying oven for 10 minutes solidifies the boundaries. The resulting microfluidic device contains a set of narrow microchannels with the following dimensional parameters: Figure 2 As shown in b, its microscopic image is as follows Figure 1 As shown.

[0028] To simulate the pressure of the liquid entering the microchannel, finite element analysis was performed on the microchannel of the microfluidic device obtained above, and the relationship between the inlet velocity of the microfluidic device and the pressure difference of a single narrow microchannel was obtained, that is, the pressure distribution in the mainstream region of the microfluidic device at different flow velocities. Figure 3 A schematic diagram of the simulation results of pressure distribution around a narrow microchannel array under different inlet flow velocities, unit: Pa; Figure 3 b is a graph showing the relationship between the pressure difference and flow velocity between the inlet and outlet of a single narrow microchannel. (e.g.) Figure 3 As shown in Figure a, the pressure distribution changes with flow velocity. A series of simulations were performed at different flow velocities, and the results are as follows. Figure 3 As shown in b. From Figure 3 As can be clearly seen in b, there is a linear correlation between pressure difference and 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 where molecules on a liquid surface attract each other to minimize their surface area. To understand the effect of cell surface tension on cell movement along narrow regions, in this embodiment of the invention, based on the microchannel designed above, numerical simulations were performed using the 'Laminar Two-Phase Flow, Moving Mesh' module of COMSOL Multiphysics 5.6 to study the influence of cell tension and cell diameter on the movement of cancer cells along narrow regions. Considering the heterogeneity of cancer cells, cells of different diameters and surface tensions were selected in this invention. The cell diameter ranged from 12 μm to 22 μm. The surrounding liquid was considered as blood, with a mass density of 1050 kg / m³ and a dynamic viscosity of 5.73 mPa·s. The cell mass density and dynamic viscosity were defined as 1020 kg / m³ and 30 mPa·s, respectively. The inlet and outlet pressure difference was set to 98.5 Pa, which is a reasonable value (67–1333 Pa) for arterioles. Figure 4 This is a schematic diagram illustrating simulation results for different diameters and surface tensions in an embodiment of the present invention, such as... Figure 4 As shown, simulation results indicate that cells of the same size but different surface tensions exhibit different behaviors when compressed in narrow vascular regions. Cells with the same surface tension also show different behaviors depending on their diameter. These results suggest that cell diameter and cell tension are important parameters affecting the metastasis process of cancer cells.

[0030] To determine the critical surface tension for different cell diameters, simulations were performed for cell diameters ranging from 12 μm to 22 μm. The simulation results show that when the cell diameter is between 12 μm and 16 μm, the cell diameter significantly influences the movement of cancer cells within narrow blood vessels (cross-sectional dimensions: 10 μm × 40 μm). If the cell diameter is greater than 16 μm, cell surface tension becomes the dominant parameter. These results suggest that numerical simulation may be helpful in predicting cancer metastasis prognosis.

[0031] Based on the above simulation, this embodiment of the invention provides a method for analyzing cell mechanical properties using the aforementioned 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 property analysis, the liquid flow rate can be predetermined 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: Adjust the diameter of the simulated cell to be tested within a preset diameter range, and detect the first critical surface tension that simulated cells of different diameters can pass through the narrow region.

[0035] The preset diameter range is 12μm to 22μm. For a specific cell diameter, the cell surface tension is adjusted to obtain the first critical surface tension corresponding to the simulated cell at that specific cell diameter. By observing the first critical surface tension at a specific cell diameter, the influence of surface tension on the passage of a specific cell through a narrow region can be determined. That is, based on the first critical surface tension obtained by the above detection, the influence of cell surface tension in blood vessels on the cell transfer efficiency in narrow blood vessels can be further predicted.

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

[0037] To verify the crucial role of surface tension at the cell fluid interface in the hematogenous metastasis of cancer cells, this invention uses a microfluidic cytometer to quantify the surface tension of cancer cells. Figure 5 a is a schematic diagram of the operation of the microfluidic cell analyzer provided by the present invention. Figure 5 b is Figure 5 Based on the representative images measured in a, and the above analysis, a microfluidic cell analyzer for measuring cellular mechanical properties can be fabricated using a multi-microchannel array of microfluidic devices, such as... Figure 5 As shown in Figure a, the microfluidic cell analyzer can acquire the forces exerted on deformable cells in a high-throughput manner. Representative images of the measurements are shown below. Figure 5 As shown in b. In practical applications, an injection pump can be used to drive the cancer cell suspension into a microfluidic cell analyzer, with the inlet flow rate adjusted to 20 μL / min to achieve a pressure difference of approximately 50 Pa between the inlet and outlet of the confined microchannel. By considering the force balance of the captured cells, the compressive force of the cells is calculated, and the surface tension can be obtained through calculation. After obtaining the surface tension value of a single cell, the transit behavior of cancer cells in narrow blood vessels can be predicted. Finally, microfluidic experiments are used to verify whether cancer cells can pass through the narrow channels simulated by the microfluidic device.

[0038] In practical applications, based on the predicted results of cell surface tension, cells to be validated can be selected and divided into two equal groups: a first group and a second group. The surface tension of the first group of cells is treated, and the percentage of cells that failed to pass through the narrowed blood vessel is compared between the treated first group and the untreated second group. For example, if the cells to be validated are uveal melanoma cells, cytochalasin can be used to treat the surface tension of uveal melanoma cells; the effect of cytochalasin treatment on the transendothelial migration of uveal melanoma cells within blood vessels can be compared.

[0039] The following explanation uses uveal malignant melanoma as an example. Uveal malignant melanoma is the most common type of malignant intraocular tumor in adults. It is highly malignant, with the posterior pole of the eye being a common site. It is prone to metastasis via the bloodstream and has a poor prognosis. Therefore, this invention selected human uveal melanoma cells (MP46) for experiments.

[0040] To make the experimental results more apparent, MP46 cells were treated with cytochalasin D in this embodiment. Cytochalasin D is a cell permeability inhibitor of actin polymerization and is also used in cancer treatment.

[0041] Figure 6 a. An image of MP46 cells treated with cytochalasin passing through a narrow region at a driving flow rate of 40 μL / min; Figure 6 b is a schematic diagram comparing the average surface tension values ​​of untreated MP46 cells and cytochalasin-treated MP46 cells; Figure 6 c is an image of MP46 cells treated with cytochalasin floating along a narrow microchannel array. (See image.) Figure 6 As shown, untreated MP46 cells exhibited significantly higher surface tension values ​​than those treated with cytochalasin. These results suggest that the MP46 cytoskeleton may be disrupted by cytochalasin, hindering transendothelial migration. Comparison with the obtained critical cell tension values ​​revealed a lower percentage of cells (around 40%) located in the "capture" domain, unable to pass through narrow channels, a significant reduction compared to the results for untreated MP46 cells (98.1%). These results indicate that cytochalasin treatment reduces the likelihood of MP46 cells migrating across the endothelium. To validate this prediction, microfluidic experiments were also conducted in this embodiment of the invention, such as... Figure 6 As shown in Figure c, the time-lapse image shows that after cytochalasin treatment, MP46 cells are flushed out of the narrowed vascular region under the same inlet pressure. Microfluidic experiments demonstrate that the measurement method provided by this invention can reasonably predict the likelihood of MP46 cell transendothelial migration in narrowed vessels, providing a new method for the prognosis of uveal melanoma.

[0042] The microfluidic device and cell mechanical analysis method provided in this invention achieve high-throughput detection through the use of multiple microchannels, making it highly suitable for the analysis of rare cells in blood samples. Furthermore, by simulating cellular blood vessels through microchannels to analyze cellular mechanical properties and predict factors influencing cell migration across the endothelium, this invention provides an efficient detection and prevention verification method for the treatment of metastatic cancer. Therefore, this invention utilizes a microfluidic device to verify the relationship between cancer cell metastasis efficiency and cell surface tension in narrow regions; it obtains critical surface tension values ​​for different cells through simulation, which can serve as a reference for clinical prognosis; and it verifies the feasibility of using drugs that reduce cell surface tension to inhibit cancer cell metastasis.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions 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 method for analyzing the mechanical properties of cells using a microfluidic device, characterized in that, include: Step 1: Control the pressure difference between the inlet and outlet of multiple microchannels in the microfluidic device by adjusting the liquid flow rate; wherein, the microfluidic device includes multiple microchannels, each microchannel having a normal region through which the simulated cells to be tested can easily pass and a narrow region through which they cannot easily pass. The size of the microchannels conforms to the size range of microarteries to simulate cancer cells passing through narrow blood vessels, and numerical simulation is used to assist in the prognosis of cancer metastasis. Step 2: Adjust the diameter of the simulated cell to be tested within a preset diameter range, and detect the first critical surface tension that simulated cells of different diameters can pass through the narrow region; Step 3: Based on the first critical surface tension obtained from the detection, predict the effect of cell surface tension on the cell transfer efficiency in narrow blood vessels; and based on the prediction results, select cells to be verified, divide them into equal groups of first and second groups of cells, treat the surface tension of the first group of cells, and compare the percentage of cells in the first group of cells after surface tension treatment with that in the second group of cells after surface tension untreatment that did not pass through the narrow blood vessels.

2. The method according to claim 1, 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 with cytochalasin. The effects of cytochalasin treatment on the transendothelial migration of human uveal melanoma cells within blood vessels were compared.

3. The method according to any one of claims 1-2, characterized in that, The preset diameter range is 12µm~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 under the specific cell diameter.

4. The method according to claim 1, characterized in that, Also includes: For a specific cell surface tension, the maximum diameter that the simulated cell can pass through a narrow region is detected; the effect of cell diameter on cell transendothelial migration is analyzed.

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

6. The method according to claim 1, characterized in that, The multiple microchannels were fabricated using photolithography.

7. The method according to claim 1, characterized in that, The cross-sectional dimensions of the normal region are 40μm × 40μm, and the cross-sectional dimensions of the narrow region are 10μm × 40μm.

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