Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.
Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.
Cell mechanical properties play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity1,2. To gain an in-depth understanding of the role of cell mechanical properties in the above process, accurate measurement of cellular mechanics is critical, and the measurement should not cause damage to the cells for subsequent culture and analysis. The measurement process should be as fast as possible, otherwise cell viability may be affected if cells are removed from the cultivation environment for a long time.
Existing cell mechanics measurement methods face some limitations. Some methods, such as magnetic twisting cytometry, magnetic tweezers and particle-tracking microrheology, cause cell damage due to the introduction of particles into cells3,4,5. Methods that measure by contact with cells, such as atomic force microscope (AFM), micropipette aspiration, micro-constriction, and parallel-plate technique, are also prone to cell damage and the throughput is difficult to increase6,7,8. In addition, ionizing radiation will flatten cells and increase their adhesion9; it is therefore necessary to measure whole cell mechanics in suspension.
In response to the above challenges, a cell mechanics measurement system based on acoustofluidic method10,11,12,13,14 has been developed. The channel width is matched to the acoustic half wavelength, thus creating a standing wave node at the midline of the microchannel. Under the action of acoustic radiation force, the cells or standard beads can move to the acoustic pressure node. Since the physical properties of the standard beads (size, density, and compressibility) are known, the acoustic energy density can be determined. Then, the cell compressibility can be obtained by recording the motion trajectories of cells in the acoustic field. Non-destructive high-throughput measurement of cells in suspension state can be achieved. This paper will introduce the design of the microfluidic chip, the establishment of the system and the measurement steps. Measurement of various types of tumor cells has been carried out to verify the accuracy of the method. The application scope of this method had been extended to subcellular structures (such as nucleus) by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. In addition, the changes in cell compressibility after drug-induced EMT or X-ray irradiation with different doses were investigated. The results demonstrate the broad applicability of this method as a powerful tool for studying the correlation between biochemical changes and cellular mechanical properties.
1. Fabricating and assembly of the acoustofluidic microdevice
2. Sample preparation
3. Measuring the compressibility of cell and nucleus
4. Data processing
Here, the work presented a protocol for the construction of a fast and non-destructive cell compressibility measuring system based on acoustofluidic microdevice and demonstrated its advantages for measuring cell and nucleus under different situations. Figure 1 shows the schematic of the microfluidic channel. The components and assembly of the acoustofluidic microdevice are shown in Figure 2. Figure 3 shows the setup of the measurement system. Under the action of the acoustic field force, cells and particles will move toward the midline of the microchannel, as shown in Figure 4A. Measurement of the size and position of cells or particles is shown in Figure 5. The calculation of energy density and cell compressibility by fitting is shown in Figure 6. In order to extend the application scope of this device to cellular organelles, especially the nucleus, high purity and intact nuclei were isolated from cells (Figure 7A). By adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel accordingly, the motion trajectory of the nucleus was obtained (Figure 4B) and the measurement of nucleus compressibility was achieved (Figure 7B).
Based on this system, the compressibility of different types of cancer cells was measured and compared (Figure 8A). In addition, the changes in cell compressibility after drug-induced EMT or X-ray irradiation with different doses were investigated (Figure 8B,C). For EMT induction, growth factor such as transforming growth factor beta (TGFβ), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were used. For irradiation, the dose rate was 153 mU/min, and the doses used were 1, 2, 4, and 8 Gy. These results demonstrate the broad applicability of this method as a powerful tool for studying the correlation between biochemical changes and cellular mechanical properties. Most importantly, this method was a non-destruction measurement. The measured cells were collected and seeded in a cell culture dish, and it was found that there was no significant difference in cell proliferation and viability compared to the untreated group after 48 h of culture, as shown in Figure 9, indicating that the measurement has no effect on cell proliferation and survival.
Figure 1: Schematic diagram of the chip microchannel. This image shows the structure and dimensions of the chip microchannel with a single inlet and outlet. The unit is mm. Please click here to view a larger version of this figure.
Figure 2: Assembly photo for the acoustofluidic microdevice. (A) Front and back photos of the chip. (B) This image shows the inlet PTFE catheter with attached 19 G dispensing needle and stainless-steel needle. (C) This image shows the piezoelectric ceramic with wires welded. (D) This image shows the assembly of the acoustofluidic microdevice with base for fixing. Please click here to view a larger version of this figure.
Figure 3: Setup of the cell compressibility measuring system. This image shows the setup of the measuring system consisting of signal generator, syringe pumper, the acoustofluidic microdevice, microscope, and CCD camera. This figure has been modified from10. Please click here to view a larger version of this figure.
Figure 4: The movement of cells and nuclei. This figure shows the movement of cells (A) and cell nuclei (B) to the midline of the microchannel under the action of the acoustic field force. Scale bar = 250 µm. Please click here to view a larger version of this figure.
Figure 5: Measurement of the size and position of a cell or particle using ImageJ software. This figure shows the button click for obtaining parameters such as area and centroid in the ImageJ software. The pixel coordinates of the four corners of the microchannel were recorded as (0, y1), (0, y2), (x3, y3), (x4, y4). The pixel width was represented as H. Please click here to view a larger version of this figure.
Figure 6: The calculation of energy density and cell compressibility by fitting. (A) Mean squared error (LS) values at different energy densities. The acoustic energy density was obtained from the best fitting result. The unit of energy density is J/m3. (B) This figure shows the fitting of the numerical solution and the measured motion trajectory for the particle. (C) Mean squared error (LS) values at different cell compressibility. The cell compressibility was obtained from the best fitting result. (D) This figure shows the fitting of the numerical solution and the measured motion trajectory for the cell. The unit of cell compressibility is 10-10 Pa-1. Please click here to view a larger version of this figure.
Figure 7: Nuclei extraction and compressibility measurement. (A) This image shows the cell nuclei extracted from A549 cells viewed with a 40x objective. Scale bar = 20 µm. (B) This image shows the measured compressibility comparison of A549 cells (N = 38) and their nuclei (N = 45). The unit of cell compressibility is 10-10 Pa-1. The length of the box represents the standard deviation. Student's t-test and chi-square analysis were used to assess significance. **P < 0.01. Please click here to view a larger version of this figure.
Figure 8: The compressibility of different types of cancer cells and cells after drug-induced EMT or X-ray irradiation. (A) The cell compressibility of various types of cancer cells. HCT116 (N = 36), A549 (N = 68), and MDA-MB-231 (N = 32) were the cell lines of colorectal carcinoma, lung adenocarcinoma, and breast adenocarcinoma, respectively. (B) The cell compressibility of MCF7 and A549 before and after induction of EMT. (C) Cell compressibility measured at 3 h after irradiation with different doses. The unit of cell compressibility is 10-10 Pa-1. The end bars of the boxplot represent the 10% and 90% values, respectively. The length of the box represents the standard deviation. The middle horizontal line represents the median value. ns means no statistical significance. Student's t-test and chi-square analysis were used to assess significance. *P < 0.05, **P < 0.01. Part of this figure has been modified from18,19. Please click here to view a larger version of this figure.
Figure 9: Cell proliferation and viability after compressibility measurement. This figure shows the A549 cell proliferation (A) and viability (B) after compressibility measurement. Control groups are untreated cells. Test groups are the cells grown for 48 h after compressibility measurement. Each experiment has at least three replicates. For cell viability, at least 300 cells were counted. Student's t-test and chi-square analysis were used to assess significance. ns indicates not statistically significant. Please click here to view a larger version of this figure.
Commonly used cell mechanics measurement methods are AFM, micropipette aspiration, microfluidics methods, parallel-plate technique, optical tweezers, optical stretcher, and acoustic methods20. Microfluidics methods can work with three approaches: micro-constriction, extensional flow, and shear flow. Among them, optical stretcher, optical tweezers, acoustic methods, extensional flow, and shear flow approaches are non-contact measurements. In contrast to contact measurements, non-contact measurements can effectively avoid the cell damage problem caused by contact or local deformation. Optical tweezers are very sensitive to the measurement environment21. Perturbations in optical conditions can cause significant differences in measurement results, and high laser power during measurement may also cause cell damage. Besides, optical stretcher and optical tweezers generally have low throughput20,22. Microfluidics methods such as extensional flow and shear flow cannot measure micromechanics of tumor cells directly23. Compared with other methods, acoustic methods have the advantages of non-destructive, high-throughput and wide applicability in the measurement of cell mechanics.
The measurement method based on acoustofluidic microdevice designed in this paper uses non-contact acoustic radiation force, and the greatest benefit is that it does not damage cells, which was confirmed by the experimental results shown here (Figure 9). Another advantage of this method is the high throughput, which can be adjusted by changing the cell concentration. Currently, a measurement speed of about 50-70 cells/min can be achieved on the premise of ensuring the accuracy of subsequent data analysis. Because the cells are measured in suspension, the measurement of non-adherent cells can be also achieved, which expands its scope of application. Moreover, measurement of the cell compressibility after irradiation was achieved in this paper, and the relationship between cell compressibility and irradiation dose was revealed (Figure 8). In addition, the measurement of cells or nucleus with different stiffness can also be adapted by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. As shown in Figure 7, measurement of the compressibility of the nucleus was achieved.
Here, only the movement of cells along the length and width of the microfluidic channel was considered. The cells were considered to be in suspension because the density of the cells is close to that of the fluid. If there was cell settlement or friction with the bottom, it would be found in the subsequent trajectory fitting process. Such data will be eliminated. In order to validate the proposed method, two kinds of polystyrene beads with different diameter (6 µm and 10 µm) were tested. The beads with 6 µm diameter were used as control. The compressibility of beads with 10 µm diameter was obtained as 2.37 ± 0.11 x 10-10 Pa-1, consistent with the value (2.1-2.4 x 10-10 Pa-1) reported in another research15,24.
Based on this method, the compressibility of cells and nuclei was measured. Since nucleus plays an important role in mechanosensing and mechanotransduction, measurement of the mechanical properties of nucleus can help to better understand how they respond to external stimulus such as ionizing radiation-induced DNA damage (Figure 8). Furthermore, experiments showed that cell compressibility can serve as a new indicator for monitoring the EMT process (Figure 8), showing its application prospects in cancer diagnosis. Since the acoustic radiation force is related to cell size, cell density, and compressibility, this method can also be used for cell isolation such as the separation of circulating tumor cells (CTCs) or their clusters from the blood sample. CTCs and their clusters play an important role in the early detection of metastatic tumors and the monitoring of radiotherapy efficacy25,26. Therefore, this method has great clinical application prospects in cancer early screening and efficacy evaluation.
It is worth noting that the key factor affecting the measurement of this method is the matching of the resonant frequency with the width of the microchannel, resulting in the appearance of a standing wave node at the midline of the microchannel. At the same time, attention should be paid to the pasting of piezoelectric ceramics. Besides, in order to accurately identify and track the trajectory of cells or particles and avoid the problem of cell aggregation due to adhesion, the throughput should not be too high. How changes in other parameters (such as cell size, cell density, etc.) affect the measurement of compressibility has been studied in previous work18. Moreover, when measuring cell nuclei, due to their softness, a large acoustic radiation driving force is required to make them move smoothly to the midline of the microchannel, thus requiring a fast camera to accurately capture the trajectories.
The authors have nothing to disclose.
This study was supported by the National Natural Science Foundation of China (Grant numbers 12075330 and U1932165) and the Natural Science Foundation of Guangdong Province, China (Grant number 2020A1515010270).
0.25% trypsin(1x) | GIBCO | 15050-065 | |
502 glue | Evo-bond | cyanoacrylate glue | |
A549 | ATCC | CCL-185 | lung adenocarcinoma |
Cytonucleoprotein and cytoplasmic protein extraction kit | Beyotime | P0027 | Contains cytoplasmic protein extraction reagents A and B |
Dulbecco’s modified Eagle medium (DMEM) | corning | 10-013-CVRC | |
Fetal Bovine Srum(FBS) | AUSGENEX | FBS500-S | |
HCT116 | ATCC | CCL247 | colorectal carcinoma |
Heat-resistant glass | Pyrex | ||
Leibovitz’s L-15 medium | GIBCO | 11415-064 | |
MCF-7 | ATCC | HTB-22 | breast Adenocarcinoma |
MDA-MB-231 | ATCC | HTB-26 | breast Adenocarcinoma |
Minimum Essential Medium (MEM) | corning | 10-010-CV | |
Penicillin-Streptomycin | GIBCO | 15140-122 | |
Phosphate buffer | corning | 21-040-cvc | |
PMSF | Beyotime | ST506 | 100mM |
Polybead Polystyrene Red Dyed Microsphere | polysciences | 15714 | The diameter of microshpere is 6.00µm |
propidium iodide(PI) | Sigma-Aldrich | P4170 | |
SYLGARD 184Silicone ELASTOMER | Dow-Corning | 1673921 | Contains prepolymers and curing agents |
Trypan Blue | Beyotime | C0011 |