A microfluidic device composed of variable numbers of multiconstriction channels is reported in this paper to differentiate a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast cell line, MCF-10A. Differences between their mechanical properties were assessed by comparing the effect of single or multiple relaxations on their velocity profiles which is a novel measure of their deformation ability. Videos of the cells were recorded via a microscope using a smartphone, and imported to a tracking software to gain the position information on the cells. Our results indicated that a multiconstriction channel design with five deformation (50 μm in length, 10 μm in width, and 8 μm in height) separated by four relaxation (50 μm in length, 40 μm in width, and 30 μm in height) regions was superior to a single deformation design in differentiating MDA-MB-231 and MCF-10A cells. Velocity profile criteria can achieve a differentiation accuracy around 95% for both MDA-MB-231 and MCF-10A cells.

A multifunctional microfluidic platform combining on-demand aqueous-phase droplet generation, multi-droplet storage, and controlled merging of droplets selected from a storage library in a single integrated microfluidic device is described. A unique aspect of the technology is a microfluidic trap design comprising a droplet trap chamber and lateral bypass channels integrated with a microvalve that supports the capture and merger of multiple droplets over a wide range of individual droplet sizes. A storage unit comprising an array of microfluidic traps operates in a first-in first-out manner, allowing droplets stored within the library to be analyzed before sequentially delivering selected droplets to a downstream merging zone, while shunting other droplets to waste. Performance of the microfluidic trap is investigated for variations in bypass/chamber hydrodynamic resistance ratio, micro-chamber geometry, trapped droplet volume, and overall flow rate. The integrated microfluidic platform is then utilized to demonstrate the operational steps necessary for cell-based assays requiring the isolation of defined cell populations with single cell resolution, including encapsulation of individual cells within an aqueous-phase droplet carrier, screening or incubation of the immobilized cell-encapsulated droplets, and generation of controlled combinations of individual cells through the sequential droplet merging process. Beyond its utility for cell analysis, the presented platform represents a versatile approach to robust droplet generation, storage, and merging for use in a wide range of droplet-based microfluidics applications.

The mechanical response of a living cell is notoriously complicated. The complex, heterogeneous characteristics of cellular structure introduce difficulties that simple linear models of viscoelasticity cannot overcome, particularly at deep indentation depths. Herein, a nano-scale stress-relaxation analysis performed with an atomic force microscope reveals that isolated human breast cells do not exhibit simple exponential relaxation capable of being modeled by the standard linear solid (SLS) model. Therefore, this work proposes the application of the fractional Zener (FZ) model of viscoelasticity to extract mechanical parameters from the entire relaxation response, improving upon existing physical techniques to probe isolated cells. The FZ model introduces a new parameter that describes the fractional time-derivative dependence of the response. The results show an exceptional increase in conformance to the experimental data compared to that predicted by the SLS model, and the order of the fractional derivative (α) is remarkably homogeneous across the populations, with a median value of 0.48 ± 0.06 for the malignant population and 0.51 ± 0.07 for the benign. The cells' responses exhibit power-law behavior and complexity not associated with simple relaxation (SLS, α = 1) that supports the application of a fractional model. The distributions of some of the FZ parameters also preserve the distinction between the malignant and benign sample populations seen from the linear model and previous results while including the contribution of fast-relaxation behavior. The resulting viscosity, measured by a composite relaxation time, exhibits considerably less dispersion due to residual error than the distribution generated by the linear model and therefore serves as a more powerful marker for cell differentiation.

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures.

We herein report, for the first time, the mechanical properties of ovarian cancer stem-like/tumor initiating cells (CSC/TICs). The represented model is a spontaneously transformed murine ovarian surface epithelial (MOSE) cell line that mimics the progression of ovarian cancer from early/non-tumorigenic to late/highly aggressive cancer stages. Elastic Modulus measurements via Atomic Force Microscopy (AFM) illustrate that the enriched CSC/TICs population (0.32±0.12kPa) are 46%, 61%, and 72% softer (p<0.0001) than their aggressive late stage, intermediate, and non-malignant early stage cancer cells, respectively. Exposure to sphingosine, an anti-cancer agent, induced an increase in the elastic moduli of CSC/TICs by more than 46% (0.47±0.14kPa, p<0.0001). Altogether, our data demonstrate that the elastic modulus profile of CSC/TICs is unique and responsive to anti-cancer treatment strategies that impact the cytoskeleton architecture of cells. These findings increase the chance for obtaining distinctive cell biomechanical profiles with the intent of providing a means for effective cancer detection and treatment control.

The existing approach to characterize cell biomechanical properties typically utilizes switch-like models of mechanotransduction in which cell responses are analyzed in response to a single nanomechanical indentation or a transient pulsed stress. Although this approach provides effective descriptors at population-level, at a single-cell-level, there are significant overlaps in the biomechanical descriptors of non-metastatic and metastatic cells which precludes the use of biomechanical markers for single cell metastatic phenotyping. This study presents a new promising marker for biosensing metastatic and non-metastatic cells at a single-cell-level using the effects of a dynamic microenvironment on the biomechanical properties of cells. Two non-metastatic and two metastatic epithelial breast cell lines are subjected to a pulsed stresses regimen exerted by atomic force microscopy. The force-time data obtained for the cells revealed that the non-metastatic cells increase their resistance against deformation and become more stiffened when subjected to a series of nanomechanical indentations. On the other hand, metastatic cells become slightly softened when their mechanical microenvironment is subjected to a similar dynamical changes. This distinct behavior of the non-metastatic and metastatic cells to the pulsed stresses paradigm provided a signature for single-cell-level metastatic phenotyping with a high confidence level of ∼95%.

This study describes the development of a microfluidic biosensor called the iterative mechanical characteristics (iMECH) analyzer which enables label-free biomechanical profiling of individual cells for distinction between metastatic and non-metastatic human mammary cell lines. Previous results have demonstrated that pulsed mechanical nanoindentation can modulate the biomechanics of cells resulting in distinctly different biomechanical responses in metastatic and non-metastatic cell lines. The iMECH analyzer aims to move this concept into a microfluidic, clinically more relevant platform. The iMECH analyzer directs a cyclic deformation regimen by pulling cells through a test channel comprised of narrow deformation channels and interspersed with wider relaxation regions which together simulate a dynamic microenvironment. The results of the iMECH analysis of human breast cell lines revealed that cyclic deformations produce a resistance in non-metastatic 184A1 and MCF10A cells as determined by a drop in their average velocity in the iterative deformation channels after each relaxation. In contrast, metastatic MDA-MB-231 and MDA-MB-468 cells exhibit a loss of resistance as measured by a velocity raise after each relaxation. These distinctive modulatory mechanical responses of normal-like non-metastatic and metastatic cancer breast cells to the pulsed indentations paradigm provide a unique bio-signature. The iMECH analyzer represents a diagnostic microchip advance for discriminating metastatic cancer at the single-cell level.

Cancer progression is associated with an increased deformability of cancer cells and reduced resistance to mechanical forces, enabling motility and invasion. This is important for metastases survival and outgrowth and as such could be a target for chemopreventive strategies. In this study, we determined the differential effects of exogenous sphingolipid metabolites on the elastic modulus of mouse ovarian surface epithelial cells as they transition to cancer. Treatment with ceramide or sphingosine-1-phosphate in non-toxic concentrations decreased the average elastic modulus by 21% (p ≤ 0.001) in transitional and 15% (p ≤ 0.02) in aggressive stages while exerting no appreciable effect on non-malignant cells. In contrast, sphingosine treatment on average increased the elastic modulus by 33% (p ≤ 0.0002) in aggressive cells while not affecting precursor cells. These results indicate that tumor-supporting sphingolipid metabolites act by making cells softer, while the anti-cancer metabolite sphingosine partially reverses the decreased elasticity associated with cancer progression. Thus, sphingosine may be a valid alternative to conventional chemotherapeutics in ovarian cancer prevention or treatment.