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

Summary

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.

Abstract

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.

Introduction

Cell mechanical properties play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity^1,2. 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....

Protocol

1. Fabricating and assembly of the acoustofluidic microdevice

1. Fabrication of the microfluidic chip.
   1. 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 µm wide and 100 µm deep. For measuring cell nucleus, change the width and depth of the microchannel to 250 µm and 100 µm, respectively.
   2. Prepare the microchannel on silicon wafer via reactive ion etching. Seal the top of the microchannel with a piece of transparent heat-resistant glass by anodic bond....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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