Name: measurement of the compressibility of cell and nucleus based on acoustofluidic microdevice
Uploaded: 2022-07-14T15:00:00
Description: Watch this Scientific Journal Video about Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice at JoVE.com

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).

1. Fabricating and assembly of the acoustofluidic microdevice
<ol>
	<li>Fabrication of the microfluidic chip.
	<ol>
		<li>Design a single-channel chip with only one inlet and outlet as shown in Figure 1. For measuring cells, keep the rectangular cross-section of the microchannel at 740 &#181;m wide and 100 &#181;m deep. For measuring cell nucleus, change the width and depth of the microchannel to 250 &#181;m and 100 &#181;m, respectively.</li>
		<li>Prepare the microchanne...

<ol>
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T., Su, H. -. W., Bruus, H., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iso-acoustic+focusing+of+cells+for+size-insensitive+acousto-mechanical+phenotyping.">Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.</a> Nature Communications. 7 (1), 11556 (2016).</li><li>Cushing, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+characterization+of+microbead+and+cell+suspensions+by+speed+of+sound+measurements+of+neutrally+buoyant+samples.">Ultrasound characterization of microbead and cell suspensions by speed of sound measurements of neutrally buoyant samples.</a> Analytical Chemistry. 89 (17), 8917-8923 (2017).</li><li>Riaud, A., Wang, W., Thai, A. L. 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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 measurement...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin(1x)</td>
			<td>GIBCO</td>
			<td>15050-065</td>
			<td></td>
		</tr>
		<tr>
			<td>502 glue</td>
			<td>Evo-bond</td>
			<td></td>
			<td>cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>lung adenocarcinoma</td>
		</tr>
		<tr>
			<td>Cytonucleoprotein and cytoplasmic protein extraction kit</td>
			<td>Beyotime</td>
			<td>P0027</td>
			<td>Contains cytoplasmic protein extraction reagents A and B</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium (DMEM)&#160;</td>
			<td>corning</td>
			<td>10-013-CVRC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Srum(FBS)</td>
			<td>AUSGENEX</td>
			<td>FBS500-S</td>
			<td></td>
		</tr>
		<tr>
			<td>HCT116</td>
			<td>ATCC</td>
			<td>CCL247</td>
			<td>colorectal carcinoma</td>
		</tr>
		<tr>
			<td>Heat-resistant glass</td>
			<td>Pyrex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium&#160;</td>
			<td>GIBCO</td>
			<td>11415-064</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF-7</td>
			<td>ATCC</td>
			<td>HTB-22&#160;</td>
			<td>breast Adenocarcinoma</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>HTB-26&#160;</td>
			<td>breast Adenocarcinoma</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>corning</td>
			<td>10-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>corning</td>
			<td>21-040-cvc</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Beyotime</td>
			<td>ST506</td>
			<td>100mM</td>
		</tr>
		<tr>
			<td>Polybead Polystyrene Red Dyed Microsphere&#160;</td>
			<td>polysciences</td>
			<td>15714</td>
			<td>The diameter of microshpere is 6.00&#181;m</td>
		</tr>
		<tr>
			<td>propidium iodide(PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184Silicone ELASTOMER</td>
			<td>Dow-Corning</td>
			<td>1673921</td>
			<td>Contains prepolymers and curing agents</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Beyotime</td>
			<td>C0011</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of the compressibility of cell and nucleus based on acoustofluidic microdevice

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.

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 measureme...

Watch this Scientific Journal Video about Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice at JoVE.com

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice

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 ...

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. Acoustofluidic method can realize fast and non-destructive measurement in suspended states. This technique can be used for mirroring the change of self compressibility during epithelial to mesenchymal transition and separating circulating tumor cells, showing its application prospects in cancer diagnosis.To begin, bind the PDMS block to the chip by punching holes in the PDMS block for inlet and outlet ports with a one millimeter diameter hollow needle. Then put the PDMS blocks and chip with the backside up in a plasma cleaner for one minute. After aligning the holes on the PDMS blocks with the chip inlet and outlet, gently press the PDMS blocks to the chip for 15 seconds, resulting in bonding to occur between the PDMS blocks and the surface of the chip.To connect the PTFE catheter to the chip, cut two pieces of catheter with an inner diameter of 0.8 millimeter and a length of 10 centimeter. Bend a stainless steel needle with an inner diameter of 0.7 millimeter and a length of 1.5 centimeter by 90 degrees into an L shape and connect it to one end of the catheter. Insert the stainless steel needles to the holes of the PDMS blocks.For the inlet, connect a 19 gauge dispensing needle to the other end of the catheter as a connector for a syringe. Afterward, inject deionized water to test the tightness of the overall channel. For a piezoelectric ceramic assembly, select precut piezoelectric ceramic sheets with a width of five millimeter.Then weld wires on both sides of the piezoelectric ceramic at one end. Glue the piezoelectric ceramic to the middle of the back of the chip by placing a drop of cyanoacrylate glue on the piezoelectric ceramic. Smooth the glue with the toothpick and remove the excess glue.Then quickly press it on the chip and continue to press for about one minute. To mount the micro device, cut a piece of PDMS as the base of the micro device and stick one side of the base to the inlet and outlet PDMS blocks and the other side to a transparent glass slide using double-sided tape. Fix the whole microdevice to the microscope stage to keep the chip in one focal plane.To prepare the polystyrene standard particle solutions add 0.05 milliliters of polystyrene particle solution to 10 milliliter of PBS and mix well. Then prepare the cell suspensions by washing the adherent cells at 90%confluency with PBS. Add 500 microliters of 0.25 Trypsin for one to two minutes at room temperature.After removing the Trypsin, add one milliliter complete medium and form a cell suspension by pipetting. Next centrifuge the cell suspension at 100 G for five minutes. Remove the supernatant and resuspend in 0.5 to one milliliter of PBS in order to obtain a cell suspension.Then cells were counted with a hemocytometer and the concentration was about 300 to 500, 000 cells per milliliter. After a preparing the cell nucleus suspension as demonstrated previously, remove the supernatant and add 200 microliters of cytoplasmic protein extraction reagent A per 20 microliters cell palette and mix well. Then vortex the above mixture at 220 G for five seconds and place on ice bath for 10 minutes.Next, add 10 microliters of cytoplasmic protein extraction reagent B to the solution after incubation. After vortexing, place on ice bath for one minute and vortex again at 220 G for five seconds. Then, finally centrifuge at 1000 G for five minutes at four degrees Celsius.Remove the supernatant and resuspend the palette in one milliliter of PBS. Then centrifuge at 1000 G at four degrees Celsius for four minutes. After removing the supernatant, resuspend in 100 microliters of PBS as cell nucleus suspension.Add trypan blue to the above cell nucleus suspension and stain at room temperature for four minutes. Next, count the number of nuclei under the inverted microscope with a 10x objective. Dilute the above cell nucleus suspension with PBS buffer to a concentration of 200 to 300, 000 nucleus per milliliter.Then filter the cell nucleus suspension through a 70 micrometer sieve. To set up the measurement system, turn on the light source of the microscope and open the camera software. Use the 4x objective to find the middle position of the microchannel, that is, the position of the piezoelectric ceramic.After connecting the wires, weld them to the positive and negative terminals of the signal generator output of the piezoelectric ceramic respectively. Then place the syringe on the microinjection pump and connect it to the inlet catheter. To collect the fluid from the microchannel, place a small container at the end of the outlet catheter.To determine measurement parameters, aspirate the polystyrene particle solution with the syringe. Then inject the solution into the chip microchannel while avoiding air bubbles and in chip microchannel to ensure accurate measurement. Set the output of the signal generator to a sign signal with a frequency of one megahertz and peak-to-peak voltage of 10 volts until it's observed that the particles move toward the midline of the microchannel and remain in forward motion along the midline after reaching the midline.After mixing one milliliter cell or nucleus suspension with the standard particle solution at the ratio of one to one, inject it into the microchannel with a syringe to measure cells and nuclei. Start recording with CCD camera when the cells or nuclei enter the field of view and turn on the signal generator. Then rinse the microchannel with deionized water, 75%alcohol, and deionized water in sequence for later use.The movement of the cells and particles was observed under the action of the acoustic field force towards the midline of the microchannel at different time intervals. The calculation of energy, density, and cell compressibility were performed and acoustic energy density and the measured motion trajectory for the particle was obtained from the best fitting result. The high purity and intact nuclei were isolated from cells and nucleus compressibility measurement was achieved by obtaining the motion trajectory of the nucleus.The compressibility of different types of cancer cells and cells after drug-induced EMT or x-ray irradiation with different doses was measured and compared. The measured cells were collected and it was found that there was no significant difference in cell proliferation and viability compared to the untreated group after 48 hours of culture. Indicating that the compressibility measurement has no effect on cell proliferation and survival.The most important thing is to fine tune the frequency of the signal until it's observed that particles move towards the midline of the microchannel.

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A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...