Uncovering the Contribution of Microchannel Deformation to Impedance-Based Flow Rate Measurements.

Changes in electrical impedance have previously been used to measure fluid flow rate in microfluidic channels. Ionic redistribution within the electrical double layer by fluid flow has been considered to be the primary mechanism underlying such impedance based microflow sensors. Here we describe a previously unappreciated contribution of microchannel deformation to such measurements. We found that flow-induced microchannel deformation contributes significantly to the change in electrical impedance of solutions, in particular to those solutions producing an electrical double layer in the order of a few tens of nanometers (i.e., containing relatively high ionic strength). Since the flow velocity at the measurement surface is near zero, due to the laminar nature of the flow, the contribution of the double layer under the conditions mentioned above should be negligible. In contrast, an increase in the fluid flow rate results in an increase in the microchannel cross-sectional area (because of higher local pressure), therefore, producing a decrease in solution resistance between the two electrodes. Our results suggest that microflow sensors based on the concept of elastic deformation could be designed for in situ monitoring and fine control of fluid flow in flexible microfluidics. Finally, we show that purposefully engineering a larger deformability of the microchannel, by changing the geometry and the Young's modulus of the microchannel, enhances the sensitivity of this flow rate measurement.

Cell response to matrix mechanics: focus on collagen.

Many model systems and measurement tools have been engineered for observing and quantifying the effect of mechanics on cellular response. These have contributed greatly to our current knowledge of the molecular events by which mechanical cues affect cell biology. Cell responses to the mechanical properties of type 1 collagen gels are discussed, followed by a description of a model system of very thin, mechanically tunable collagen films that evoke similar responses from cells as do gel systems, but have additional advantages. Cell responses to thin films of collagen suggest that at least some of the mechanical cues that cells can respond to in their environment occur at the sub-micron scale. Mechanical properties of thin films of collagen can be tuned without altering integrin engagement, and in some cases without altering topology, making them useful in addressing questions regarding the roles of specific integrins in transducing or mitigating responses to mechanical cues. The temporal response of cells to differences in ECM may provide insight into mechanisms of signal transduction.

Using surface plasmon resonance imaging to probe dynamic interactions between cells and extracellular matrix.

Spatially resolved details of the interactions of cells with a fibronectin modified surface were examined using surface plasmon resonance imaging (SPRI). SPRI is a label-free technique that is based on the spatial measurement of interfacial refractive index. SPRI is sensitive to short range interactions between cells and their substratum. The high contrast in SPR signal between cell edges and substratum facilitates identification of cell edges and segmentation of cell areas. With this novel technique, we demonstrate visualization of cell-substratum interactions, and how cell-substratum interactions change over time as cells spread, migrate, and undergo membrane ruffling.

Nanomechanical properties of thin films of type I collagen fibrils.

The mechanical cues that adherent cells derive from the extracellular matrix (ECM) can effect dramatic changes in cell migration, proliferation, differentiation, and apoptosis. Model ECMs composed of collagen fibrils formed from purified collagen are an important experimental system to study cell responses to mechanical properties of the ECM. Using a self-assembled model system of a film composed of 100-200 nm diameter collagen fibrils overlaying a bed of smaller fibrils, we have previously demonstrated changes in cellular response to systematically controlled changes in mechanical properties of the collagen. In this study, we describe an experimental and modeling approach to calculate the elastic modulus of individual collagen fibrils, and thereby the effective stiffness of the entire collagen thin film matrix, from atomic force microscopy force spectroscopy data. These results demonstrate an approach to the analysis of fundamental properties of thin, heterogeneous, and organic films and add further insights into the mechanical and topographical properties of collagen fibrils that are relevant to cell responses to the ECM.

Lens-Free Imaging as a Sensor for Dynamic Cell Viability Detection Using the Neutral Red Uptake Assay.

Neutral red is a low-cost supravital stain for determining cell viability. The standard protocol relies on a destructive extraction process to release the accumulated dye for endpoint spectrophotometric quantification. We report a non-destructive, live-cell quantification of neutral red uptake using a compact lens-free system. Two light sources indentify the cell perimeter and quantify neutral red uptake. The quantification occurs during staining, thus eliminating the destructive extraction process and reducing assay time. Our system enables live quantification for continuous high-throughput screening of cell viability within confined spaces such as incubators.

Surface plasmon resonance imaging of cells and surface-associated fibronectin.

BACKGROUND: A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells. RESULTS: Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h. CONCLUSION: SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.

Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Engineering amount of cell-cell contact demonstrates biphasic proliferative regulation through RhoA and the actin cytoskeleton.

Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation.

The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype.

Cells receive signals from the extracellular matrix through receptor-dependent interactions, but they are also influenced by the mechanical properties of the matrix. Although bulk properties of substrates have been shown to affect cell behavior, we show here that nanoscale properties of collagen fibrils also play a significant role in determining cell phenotype. Type I collagen fibrils assembled into thin films provide excellent viewing of cells interacting with individual fibrils. Cells can be observed to extensively manipulate the fibrils, and this behavior seems to result in an incompletely spread stellate morphology and a nonproliferative phenotype that is typical of these cells in collagen gels. We show here that thin films of collagen fibrils can be dehydrated, and when seeded on these dehydrated fibrils, smooth muscle cells spread and proliferate extensively. The dehydrated collagen fibrils appear to be similar to the fully hydrated collagen fibrils in topology and in presentation of beta(1) integrin ligation sites, but they are mechanically stiffer. This decrease in compliance of dehydrated fibrils is seen by a failure of cell movement of dehydrated fibrils compared to their ability to rearrange fully hydrated fibrils and from direct measurements by nanoindentation and quantitative atomic force measurements. We suggest that increase in the nanoscale rigidity of collagen fibrils can cause these cells to assume a proliferative phenotype.

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy.

BACKGROUND: In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells. RESULTS: Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC50 for myosin light chain phosphorylation using Y27632 was determined to be 2.1 +/- 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods. CONCLUSION: Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.

Fibronectin in Layer-by-Layer Assembled Films Switches Tumor Cells between 2D and 3D Morphology.

Tumor cells showing a 3D morphology and in coculture with endothelial cells are a valuable in vitro model for studying cell-cell interactions and for the development of pharmaceuticals. Here, we found that HepG2 cells, unlike endothelial cells, show differences in adhesion to fibronectin alone, or in combination with poly(allylamine hydrochloride). This response allowed us to engineer micropatterned heterotypic cultures of the two cell types using microfluidics to pattern cell adhesion. The resulting cocultures exhibit spatially encoded and physiologically relevant cell function. Further, we found that the protrusive, migratory and 3D morphological responses of HepG2 are synergistically modulated by the constituents of the hybrid extracellular matrix. Treating the hybrid material with the cross-linking enzyme transglutaminase inhibited 3D morphogenesis of tumor cells. Our results extend previous work on the role of fibronectin in layer-by-layer assembled films, and demonstrate that cell-specific differences in adhesion to fibronectin can be used to engineer tumor cell cocultures.

MIST: Accurate and Scalable Microscopy Image Stitching Tool with Stage Modeling and Error Minimization.

Automated microscopy can image specimens larger than the microscope's field of view (FOV) by stitching overlapping image tiles. It also enables time-lapse studies of entire cell cultures in multiple imaging modalities. We created MIST (Microscopy Image Stitching Tool) for rapid and accurate stitching of large 2D time-lapse mosaics. MIST estimates the mechanical stage model parameters (actuator backlash, and stage repeatability 'r') from computed pairwise translations and then minimizes stitching errors by optimizing the translations within a (4r)2 square area. MIST has a performance-oriented implementation utilizing multicore hybrid CPU/GPU computing resources, which can process terabytes of time-lapse multi-channel mosaics 15 to 100 times faster than existing tools. We created 15 reference datasets to quantify MIST's stitching accuracy. The datasets consist of three preparations of stem cell colonies seeded at low density and imaged with varying overlap (10 to 50%). The location and size of 1150 colonies are measured to quantify stitching accuracy. MIST generated stitched images with an average centroid distance error that is less than 2% of a FOV. The sources of these errors include mechanical uncertainties, specimen photobleaching, segmentation, and stitching inaccuracies. MIST produced higher stitching accuracy than three open-source tools. MIST is available in ImageJ at isg.nist.gov.

Lineage mapper: A versatile cell and particle tracker.

The ability to accurately track cells and particles from images is critical to many biomedical problems. To address this, we developed Lineage Mapper, an open-source tracker for time-lapse images of biological cells, colonies, and particles. Lineage Mapper tracks objects independently of the segmentation method, detects mitosis in confluence, separates cell clumps mistakenly segmented as a single cell, provides accuracy and scalability even on terabyte-sized datasets, and creates division and/or fusion lineages. Lineage Mapper has been tested and validated on multiple biological and simulated problems. The software is available in ImageJ and Matlab at isg.nist.gov.

Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths.

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.

Engineering cellular microenvironments to improve cell-based drug testing.

Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.

The effect of high vacuum on the mechanical properties and bioactivity of collagen fibril matrices.

The extracellular matrix (ECM) environment plays a critical role in organism development and disease. Surface sensitive microscopy techniques for studying the structural and chemical properties of ECMs are often performed in high vacuum (HV) environments. In this report, we examine the affect HV conditions have on the bioactivity and mechanical properties of type I collagen fibrillar matrices. We find that HV exposure has an unappreciable affect on the cell spreading response and mechanical properties of these collagen fibril matrices. Conversely, low vacuum environments cause fibrils to become mechanically rigid as indicated by force microscopy, resulting in greater cell spreading. Time-of-flight secondary ion mass spectrometry results show no noticeable spectral differences between HV-treated and dehydrated matrices. While previous reports have shown that HV can denature proteins in monolayers, these observations indicate that HV-exposure does not mechanically or biochemically alter collagen in its supramolecular configuration. These results may have implication for complex ECM matrices such as decellularized scaffolds.

Quantitative methods to characterize morphological properties of cell lines.

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

The treatment of collagen fibrils by tissue transglutaminase to promote vascular smooth muscle cell contractile signaling.

The enzyme tissue transglutaminase 2 (TG2) appears to play an important role in several physiological processes such as wound healing, the progression of cancer and of vascular disease. Additionally, TG2 has been proposed as a means of stabilizing collagen extracellular matrix (ECM) scaffolds for tissue engineering applications. In this report, we examined the effect of TG2 treatment on the mechanical properties of the ECM, and associated cell responses. Using a model ECM of fibrillar collagen, we quantitatively examined vascular smooth muscle cell (vSMC) response to untreated, or TG2 treated collagen. We show that cells respond to TG2 treated collagen with increased spreading, an increase in contractile response as indicated by elevated F-actin polymerization and myosin light chain phosphorylation, and increased proliferation, without apparent changes in integrin specificity or matrix topography. Comparative atomic force microscopy loading studies indicate that TG2 treated fibrils are 3 times more resistant to shearing force from an AFM tip than untreated fibrils. The data suggest that TG2 treatment of collagen increases matrix mechanical stiffness, which apparently alters the contractile and proliferative response of vSMC.