Pinched-flow hydrodynamic stretching of single-cells

Abstract

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.

Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device

“…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.…”

“…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.…”

“…In an attempt to achieve high-throughput mechanical measurements within a simpler geometry, Di Carlo and colleagues developed higher-Reynolds-number (Re > 40) microfluidic systems that measure cell deformability with throughput ranging from 1000 cells/s (14) to 65,000 cells/s (15). 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.…”

“…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.…”

High‐throughput linear optical stretcher for mechanical characterization of blood cells

“…One approach is the microfluidic cell deformability cytometer which are inexpensive to prototype, use small sample volumes (nanoliters), and employ laminar flow characteristics (24) allowing for predictable and controllable flow. For example, physical constrictions (25)(26)(27)(28) or inertial focusing flow (29)(30)(31) have been used to create contact or shear forces (> 1 nN (30)) capable of significantly deforming flowing cells. Large strains (>10% deformation) however, can damage cells and should be avoided when cell isolation and viability post-analysis are of interest.…”