Visco-Node-Pore Sensing: A Microfluidic Rheology Platform to Characterize Viscoelastic Properties of Epithelial Cells.

Viscoelastic properties of cells provide valuable information regarding biological or clinically relevant cellular characteristics. Here, we introduce a new, electronic-based, microfluidic platform-visco-node-pore sensing (visco-NPS)-which quantifies cellular viscoelastic properties under periodic deformation. We measure the storage (G) and loss (G″) moduli (i.e., elasticity and viscosity, respectively) of cells. By applying a wide range of deformation frequencies, our platform quantifies the frequency dependence of viscoelastic properties. G and G″ measurements show that the viscoelastic properties of malignant breast epithelial cells (MCF-7) are distinctly different from those of non-malignant breast epithelial cells (MCF-10A). With its sensitivity, visco-NPS can dissect the individual contributions of different cytoskeletal components to whole-cell mechanical properties. Moreover, visco-NPS can quantify the mechanical transitions of cells as they traverse the cell cycle or are initiated into an epithelial-mesenchymal transition. Visco-NPS identifies viscoelastic characteristics of cell populations, providing a biophysical understanding of cellular behavior and a potential for clinical applications

Cell viscoelasticity is linked to fluctuations in cell biomass distributions.

The viscoelastic properties of mammalian cells can vary with biological state, such as during the epithelial-to-mesenchymal (EMT) transition in cancer, and therefore may serve as a useful physical biomarker. To characterize stiffness, conventional techniques use cell contact or invasive probes and as a result are low throughput, labor intensive, and limited by probe placement. Here, we show that measurements of biomass fluctuations in cells using quantitative phase imaging (QPI) provides a probe-free, contact-free method for quantifying changes in cell viscoelasticity. In particular, QPI measurements reveal a characteristic underdamped response of changes in cell biomass distributions versus time. The effective stiffness and viscosity values extracted from these oscillations in cell biomass distributions correlate with effective cell stiffness and viscosity measured by atomic force microscopy (AFM). This result is consistent for multiple cell lines with varying degrees of cytoskeleton disruption and during the EMT. Overall, our study demonstrates that QPI can reproducibly quantify cell viscoelasticity

Mechanical fluidity of fully suspended biological cells

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity---hysteresivity normalized to the extremes of an elastic solid or a viscous liquid---can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance vs. time, complex modulus vs. frequency, and phase lag vs. frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences around a time scale of 1 s. We find that fluidity estimates are consistent in the time and the frequency domains under a structural damping (power-law or fractional derivative)model, but not under an equivalent-complexity lumpedcomponent (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical crosslinking, we find that adenosine triphosphate (ATP) depletion in the cell does not measurably alter the parameter, and thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature---now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion

The effects of rapid stretch injury on rat neocortical cultures

Several key biological mechanisms of traumatic injury to axons have been elucidated using in vitro stretch injury models. These models, however, are based on the experimentation of single cultures keeping productivity slow. Indeed, low yield has hindered important and well founded investigations requiring high throughput methods such as proteomic analyses. To meet this need, a multi-well high throughput injury device is engineered to accelerate and accommodate the next generation of traumatic brain injury research. This modular system stretch-injures neuronal cultures in either a 24-well culture plate format or six individual wells simultaneously. Custom software control allows the user to accurately program the pressure pulse parameters to achieve the desired substrate deformation and injury parameters. Classically, in vitro research in TBI has shown increases in [Ca2+]i levels following injury. The Ca2+ sensitive fluorescent dye, Fluo-4AM, is used to observe the effects of strain rate on the changes in [Ca2+]i levels following injury. Neuronal cultures are injured at three strain levels: 20%, 40% and 60% strain. At each of these strain levels, two strain rates are applied; 30s-1 (slow) and 70s-1 (rapid). At each strain level, the data show that neurons injured at 70s-1 experience larger maximum F/F0 and longer sustained Ca2+ fluorescence than neurons injured at 30s-1. It is also shown that at high strain rates TTx no longer blocks increases in [Ca2+]i levels after injury. Traumatic injury to the brain is known to cause dysfunction in surviving neurons. The effects of simulated traumatic injury of rat neocortical neurons cultures are investigated. These neurons are subjected to a stretch injury of 60% strain over 20 ms using a custom in vitro injury device. Spontaneous and stimulated electrical properties are measured 20-60 minutes after stretch using current and voltage clamp techniques. The same measurements are performed in non-stretched neurons. All neurons display TTX-inhibitable action potentials when basal membrane potential was set at -60 mV, and many display bursting behavior in response to depolarizing current injection. No differences in resting membrane potential (-40 ± 1 mV [n=20]) or input resistance (1.0 ± 0.1 GΩ [n=20]) are observed in injured and non-injured neurons. Interestingly, stretch injury reduces the frequency of spontaneous action potentials (33 ± 6 min-1 [ n=13] and 11 ± 3 min-1 [n=16] in non-injured and injured neurons, respectively) and decreases spontaneous bursting activity by almost 90%. ADP50 and action potential amplitude are unchanged. However, A D P90 is significantly prolonged in injured neurons and characterized by a less pronounced action potential after-hyperpolarization. These data are consistent with an alteration in kinetics of K+ currents in injured neurons. Since spontaneous action potentials are blocked by 20 µM bicuculline and 3 mM kyneuri nic acid, the frequency of subthreshold depolarizations is measured to estimate overall neuronal network activity. The frequency of spontaneous subthreshold depolarizations is not significantly different in injured and non-injured neurons. These data show that spontaneous electrical signaling is acutely altered and suggest that action potential initiation is reduced by in vitro stretch in neuronal cell cultures

Internally and externally driven flows of complex fluids: viscoelastic active matter, flows in porous media and contact line dynamics

We consider three varied soft matter topics from a continuum fluid mechanics perspective, namely: viscoelastic active matter, viscoelastic flows in porous media, and contact line dynamics. Active matter. For the purposes of this thesis, the term active matter describes a collection of active particles which absorb energy from their local environment or from an internal fuel tank and dissipate it to the surrounding fluid. We explore the stability and dynamics of active matter in a biological context in the presence of a polymeric background fluid. Using a novel coarse-grained model, we generalise earlier linear stability analyses (without polymer) and demonstrate that the bulk orientationally ordered phase remains intrinsically unstable to spontaneous flow instabilities. This instability remains even as one takes an ’elastomeric limit’ in which the polymer relaxation time τC → ∞. The 1D nonlinear dynamics in this limit are oscillatory on a timescale set by the rate of active forcing. Then, by considering the rheological response of our model under shear, we explore the mechanism behind the above generic flow instability, which we show exists not only for orientationally ordered phases but also for disordered states deep in the isotropic phase. Our linear stability analysis in 1D for sheared suspensions predicts that initially homogeneous states represented by negatively sloping regions of the constitutive curve are unstable to shear-banding flow instabilities. In some cases, the shear-bands themselves are unstable which leads to a secondary instability that produces rheochaotic flow states. Consistent with recent experiments on active cellular extracts (without applied shear) which show apparently chaotic flow states, we find that the dynamics of active matter are significantly more complex in 2D. Focusing on the turbulent phase that occurs when the activity ζ (or energy input) is large, we show that the characteristic lengthscale of structure in the fluid l∗ scales as l∗ ∝ 1/ √ζ. While this lengthscale decreases with ζ, it also increases with the polymer relaxation time. This can produce a novel ‘drag reduction’ effect in confined geometries where the system forms more coherent flow states, characterised by net material transport. In the elastomeric limit spontaneous flows may still occur, though these appear to be transient in nature. Examples of exotic states that arise when the polymer is strongly coupled to the active particles are also given. Flows in porous media. The second topic treats viscoelastic flows in porous media, which we approximate numerically using geometries consisting of periodic arrays of cylinders. Experimentally, the normalised drag χ (i.e., the ratio of the pressure drop to the flow rate) is observed to undergo a large increase as the Weissenberg number We (which describes the ratio of the polymer relaxation time to the characteristic velocity-gradient timescale) is increased. An analysis of steady flow in the Newtonian limit identifies regions dominated by shear and extension; these are mapped to the rheological behaviour of several popular models for polymer viscoelasticity in simple viscometric protocols, allowing us to study and influence the upturn in the drag. We also attempt to reproduce a recent study in the literature which reported fluctuations for cylinders confined to a channel at high We. At low numerical resolution, we observe fluctuations which increase in magnitude with the same scaling observed in that study. However, these disappear at very high resolutions, suggesting that numerical convergence was not properly obtained by the earlier authors. Contact line dynamics. We finish by investigating the dynamics of the contact line, i.e., the point at which a fluid-fluid interface meets a solid surface, under an externally applied shear flow. The contact line moves relative to the wall, apparently contradicting the conventional no-slip boundary conditions employed in continuum fluid dynamics. A mechanism where material is transported within a ‘slip region’ via diffusive processes resolves this paradox, though the question of how the size of this region (i.e., slip length ξ) scales with fluid properties such as the viscosity η and the width of the interface between phases l, remains disputed within the literature. We reconcile two apparently contradictory scalings, which are shown to describe different limits: (a) a diffuse interface limit where ξ/l is small and (b) a sharp interface limit for large ξ/l. We demonstrate that the physics of the latter (which more closely resembles real fluids in macroscopic experimental geometries) can be captured using simulations in the former regime (which are numerically more accessible)

WIDE-RANGE COMPRESSION FORCES TO INVESTIGATE SINGLE-CELL IN-FLOW MOTIONS, MECHANOBIOLOGICAL RESPONSES AND INTRACELLULAR DELIVERY

The aim of the PhD work is to create a new microfluidic approach to finely tune applied in-flow forces in order to explore controlled single-cell deformation. In fact, we propose a microfluidic device based on compression forces arising from a viscoelastic fluid solution that firstly align cells and then deform them. By simply changing the rheological properties and the imposed fluid-flow conditions, our approach represents an easy-to-use and versatile tool to collect a comprehensive mapping of single-cell properties, investigating both biophysical and biomechanical characteristics. In a wide-range of applied compression, we observe how different degrees of deformation lead to cell-specific deformation-dependent in-flow dynamics, which correlate the classical deformation parameters (e.g. cell aspect-ratio), with dynamic quantities (e.g. revolution time of rotation during in-flow motion). Thus, a precise in-flow label-free cell phenotyping is achieved allowing the distinction of different cell classes. The observation of different degrees of deformation corresponding to variable compression, lead us to interrogate the inner cell structures possibly involved into the mechanical responses. We demonstrate that re-organization phenomena of actin cortex and microtubules as well as of nuclear envelope and chromatin content, occur. Also in this case, cell-specific responses are collected, allowing us to distinguish healthy from pathological cells depending on the structural mechanical reaction. Furthermore, by playing with the high levels of compression, we show preliminary results about the possibility to induce a nanoparticle intracellular delivery process by escaping physiological endocytosis. In fact, cells result to be able to incorporate nanoparticles into the cytoplasm, without involving a vesicle formation for the entry. These outcome open up new interesting scenarios about the possibility to use the microfluidic device as a platform for cell phenotyping and intracellular delivery, properly engineered for both diagnostic and therapeutic purposes