Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device.

The quantification of cellular mechanical properties is of tremendous interest in biology and medicine. Recent microfluidic technologies that infer cellular mechanical properties based on analysis of cellular deformations during microchannel traversal have dramatically improved throughput over traditional single-cell rheological tools, yet the extraction of material parameters from these measurements remains quite complex due to challenges such as confinement by channel walls and the domination of complex inertial forces. Here, we describe a simple microfluidic platform that uses hydrodynamic forces at low Reynolds number and low confinement to elongate single cells near the stagnation point of a planar extensional flow. In tandem, we present, to our knowledge, a novel analytical framework that enables determination of cellular viscoelastic properties (stiffness and fluidity) from these measurements. We validated our system and analysis by measuring the stiffness of cross-linked dextran microparticles, which yielded reasonable agreement with previously reported values and our micropipette aspiration measurements. We then measured viscoelastic properties of 3T3 fibroblasts and glioblastoma tumor initiating cells. Our system captures the expected changes in elastic modulus induced in 3T3 fibroblasts and tumor initiating cells in response to agents that soften (cytochalasin D) or stiffen (paraformaldehyde) the cytoskeleton. The simplicity of the device coupled with our analytical model allows straightforward measurement of the viscoelastic properties of cells and soft, spherical objects.

Experimental observation of the asymmetric instability of intermediate-reduced-volume vesicles in extensional flow.

Vesicles provide an attractive model system to understand the deformation of living cells in response to mechanical forces. These simple, enclosed lipid bilayer membranes are suitable for complementary theoretical, numerical, and experimental analysis. A recent study [Narsimhan, Spann, Shaqfeh, J. Fluid Mech., 2014, 750, 144] predicted that intermediate-aspect-ratio vesicles extend asymmetrically in extensional flow. Upon infinitesimal perturbation to the vesicle shape, the vesicle stretches into an asymmetric dumbbell with a cylindrical thread separating the two ends. While the symmetric stretching of high-aspect-ratio vesicles in extensional flow has been observed and characterized [Kantsler, Segre, Steinberg, Phys. Rev. Lett., 2008, 101, 048101] as well as recapitulated in numerical simulations by Narsimhan et al., experimental observation of the asymmetric stretching has not been reported. In this work, we present results from microfluidic cross-slot experiments observing this instability, along with careful characterization of the flow field, vesicle shape, and vesicle bending modulus. The onset of this shape transition depends on two non-dimensional parameters: reduced volume (a measure of vesicle asphericity) and capillary number (ratio of viscous to bending forces). We observed that every intermediate-reduced-volume vesicle that extends forms a dumbbell shape that is indeed asymmetric. For the subset of the intermediate-reduced-volume regime we could capture experimentally, we present an experimental phase diagram for asymmetric vesicle stretching that is consistent with the predictions of Narsimhan et al.

Non-contact microfluidic analysis of the stiffness of single large extracellular vesicles from IDH1-mutated glioblastoma cells.

In preparation for leveraging extracellular vesicles (EVs) for disease diagnostics and therapeutics, fundamental research is being done to understand EV biological, chemical, and physical properties. Most published studies have investigated nanoscale EVs and focused on EV biochemical content. There is much less understanding of large microscale EV characteristics and EV mechanical properties. We recently introduced a non-contact microfluidic technique that measures the stiffness of large EVs (>1 µm diameter). This pilot study probes the robustness of the microfluidic technique to distinguish between EV populations by comparing stiffness distributions of large EVs derived from glioblastoma cell lines. EVs derived from cells expressing the IDH1 mutation, a common glioblastoma mutation known to disrupt lipid metabolism, were stiffer than those expressed from wild-type cells in a statistical comparison of sample medians. A supporting lipidomics analysis showed that the IDH1 mutation increased the amount of saturated lipids in EVs. Taken together, these data encourage further investigation into the potential of high-throughput microfluidics to distinguish between large EV populations that differ in biomolecular composition. These findings contribute to the understanding of EV biomechanics, in particular for the less studied microscale EVs.

Non-contact microfluidic mechanical property measurements of single apoptotic bodies.

BACKGROUND: Cells exchange information by secreting micro- and nanosized extracellular vesicles (EVs), ranging from exosomes (30-100 nm) to apoptotic bodies (ABs, 1-5 µm). There is still much to understand about fundamental EV biological, physical, and chemical properties before clinical applications can be developed. EV mechanical properties have only been measured with atomic force microscopy (AFM) with its problematic adhesion and hard substrate effects. To understand EV mechanical behavior in less extreme mechanical conditions relevant to blood flow and many soft tissue environments, a non-contact measurement technique is needed. METHODS: We measured the mechanical properties of single microscale ABs derived from human blood plasma using non-contact microfluidics. EVs were gently stretched in extensional flow, similar to a traditional tensile test, and a linear mechanical model was applied to estimate mechanical stiffnesses from the observed stretching. RESULTS: The effective shear elastic modulus of ABs in non-contact flow conditions is approximately 5.6 ± 0.5 Pa, 7 orders of magnitude lower than previously reported AFM-measured biological exosome stiffnesses and 200 times smaller than suspended cells. CONCLUSIONS: Apoptotic bodies are very soft in fluid environments and exhibit lower effective stiffnesses than suspended cells. By measuring ABs in a natural fluid environment and low-force regime without hard probes and surfaces, we achieved closer agreement with linear mechanical theory and therefore more accurate stiffness measurements. GENERAL SIGNIFICANCE: AFM manufacturers and users should consider implementing new mechanical models to interpret AFM force indentation curves so that accurate extracellular vesicle mechanical properties can be extracted.

Microfluidic Strategies for Understanding the Mechanics of Cells and Cell-Mimetic Systems.

Microfluidic systems are attracting increasing interest for the high-throughput measurement of cellular biophysical properties and for the creation of engineered cellular microenvironments. Here we review recent applications of microfluidic technologies to the mechanics of living cells and synthetic cell-mimetic systems. We begin by discussing the use of microfluidic devices to dissect the mechanics of cellular mimics, such as capsules and vesicles. We then explore applications to circulating cells, including erythrocytes and other normal blood cells, and rare populations with potential disease diagnostic value, such as circulating tumor cells. We conclude by discussing how microfluidic devices have been used to investigate the mechanics, chemotaxis, and invasive migration of adherent cells. In these ways, microfluidic technologies represent an increasingly important toolbox for investigating cellular mechanics and motility at high throughput and in a format that lends itself to clinical translation.