Single-cell microfluidic impedance deformability cytometry

Abstract

The complex structural and molecular features of a cell lead to a set of specific electrical and mechanical properties which can serve as intrinsic biophysical markers that enable different cell populations to be characterised and distinguished. This project has developed a novel microfluidic technique to simultaneously measure the electrical and mechanical properties of single cells at high throughput. It exploits single-cell impedance cytometry to measure single-cell electrical properties along different geometric axes to provide deformability data. Cells flowing along a microchannel are deformed to different degrees by the shear force created by a viscoelastic fluid. Elongation of the cell modifies the impedance signal which is measured by specific electrode configurations. Multiple electrode arrays are used to measure the impedance of individual deformed cells. The electrical signals are used to determine the deformability, size and dielectric properties of individual cells at about one hundred cells per second. The device design was first optimised by numerical modelling. Different shaped models representing cells with different mechanical properties were simulated in order to obtain the frequency-dependent dielectric properties. These were compared with theoretical calculations from Maxwell’s Mixture theory. These results were used to determine the shape-dependent impedance parameters that are processed to give a deformability index. The influence of particle trajectory on the impedance was also analyzed, and the electrode array design was optimized. Rigid polystyrene beads were used for preliminary experimental verification of the system, demonstrating agreement with simulations. The velocity-impedance distribution of beads in different suspending buffers confirms that viscoelastic fluids achieve excellent focusing. The deformability and electrical properties of HL60 cells were then measured with different chemical and biochemical treatments, including glutaraldehyde fixation, and exposure to different osmolality-suspending media to change size, stiffness and dielectric properties. Cells were also measured following drug-induced cytoskeleton destabilisation. The cytometer was used to measure lung fibroblasts treated with TGFβ. This mediates fibroblast activation, differentiating them into stiffer myofibroblasts. Finally, the role of the Hypoxia Inducing Factor (HIF) on fibroblasts was investigated. The electrical and mechanical properties of Factor Inhibiting HIF (FIH) knock-out cells were measured to examine this effect. This novel cytometer allows the simultaneous analysis of multiple bio-physical parameters of individual cells, specifically their dielectric and mechanical properties. The device delivers high throughput analysis with a small sample volume, enabling the detection and characterization of rare cell populations. Compared with the state-of-the art it does not require a high-speed camera and microscope, reducing the complexity and time required to process large image files. It provides real-time data on the effect of external stimuli or drugs on cells and allows the study of subcellular changes caused by external stimuli. It has the potential to provide new insights into the mechanistic phenotypic changes of cells caused by chronic and degenerative diseases. In the future, this technique could be combined with real-time analysis for single-cell sorting.

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Identifiers

ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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