Hydrodynamic stretching of single cells for large population mechanical phenotyping

Abstract

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells∕s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.flow cytometry | high-throughput | cytology | mechanophenotype

Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device

“…It is interesting to note that although at least one previous study (13) has successfully captured changes in stiffness and correlated these differences to phenotype, the authors did not report changes in stiffness when cells were treated with cytoskeletal depolymerization drugs. The authors hypothesized that this may be because the high strain rates in their system (xU/D~2 Â 10 5 s À1 ) effectively fluidize the cytoskeleton and are instead dominated by the viscous properties of the cytosol and chromatin.…”

“…A different high-throughput device from Di Carlo and colleagues (15) extended cells asymmetrically with pinching sheathing flows so that the leading edge of the cell experienced higher shearing stresses than the trailing edge. The device was operated at strain rates (xU/D~1 Â 10 5 s À1 , though this is a less accurate estimate, because the flow is not pure extensional flow) similar to those in their cross-slot device in (13), but deformed the cells less. The high-strain-rate cross-slot device in (13) deformed cells to strains of ε~0.32 for control and depolymerization-drug-treated cells, whereas the pinched-flow stretching device in (15) deformed control cells to ε~0.15 and treated cells to ε~0.2-0.3.…”

“…Therefore, the effects of fluid inertia can be assumed to be negligible compared to viscous fluid forces and omitted from our model. In contrast, the Reynolds number in the cross-slot devices of Di Carlo and colleagues (13,15) were finite at operating conditions (Re > 40) and therefore fluid inertia would need to be included in the modeling of cell deformation in their system.…”

“…By elongating cells at the stagnation point of extensional flow or pinching cells with two sheathing flows, they successfully developed population ''signatures'' based on distributions of cell deformability versus size. These population signatures responded in expected ways to cytoskeletal drugs in the pinched-flow sheathing device for which strain rates and imposed cell strains were not too large (15) (the expected effects of cytoskeletal depolymerization drugs were not detected in the high-strain-rate, highstrain-extensional-flow device (13)) and enabled prediction of disease state from clinical samples (13,14). Nonetheless, this work did not present an analytical route for extracting cell constitutive model parameters, instead requiring numerical solutions due to the high inertial component of the flow.…”

“…To address these issues, microfluidic tools have recently been explored as a strategy to measure cellular structural and mechanical properties with a rapidity that may be better suited to drug discovery and clinical application (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Although these approaches have indeed massively improved measurement throughput and reduced operator skill/bias issues relative to traditional measurements, the extraction of cell mechanical properties (e.g., elastic modulus) remains challenging, primarily due to complex viscous forces that severely complicate analysis of deformations.…”