Ivan Pushkarsky

Automated single-cell motility analysis on a chip using lensfree microscopy

Quantitative cell motility studies are necessary for understanding biophysical processes, developing models for cell locomotion and for drug discovery. Such studies are typically performed by controlling environmental conditions around a lens-based microscope, requiring costly instruments while still remaining limited in field-of-view. Here we present a compact cell monitoring platform utilizing a wide-field (24 mm2) lensless holographic microscope that enables automated single-cell tracking of large populations that is compatible with a standard laboratory incubator. We used this platform to track NIH 3T3 cells on polyacrylamide gels over 20 hrs. We report that, over an order of magnitude of stiffness values, collagen IV surfaces lead to enhanced motility compared to fibronectin, in agreement with biological uses of these structural proteins. The increased throughput associated with lensfree on-chip imaging enables higher statistical significance in observed cell behavior and may facilitate rapid screening of drugs and genes that affect cell motility.

Metallization and biopatterning on ultra-flexible substrates via dextran sacrificial layers.

Micro-patterning tools adopted from the semiconductor industry have mostly been optimized to pattern features onto rigid silicon and glass substrates, however, recently the need to pattern on soft substrates has been identified in simulating cellular environments or developing flexible biosensors. We present a simple method of introducing a variety of patterned materials and structures into ultra-flexible polydimethylsiloxane (PDMS) layers (elastic moduli down to 3 kPa) utilizing water-soluble dextran sacrificial thin films. Dextran films provided a stable template for photolithography, metal deposition, particle adsorption, and protein stamping. These materials and structures (including dextran itself) were then readily transferrable to an elastomer surface following PDMS (10 to 70∶1 base to crosslinker ratios) curing over the patterned dextran layer and after sacrificial etch of the dextran in water. We demonstrate that this simple and straightforward approach can controllably manipulate surface wetting and protein adsorption characteristics of PDMS, covalently link protein patterns for stable cell patterning, generate composite structures of epoxy or particles for study of cell mechanical response, and stably integrate certain metals with use of vinyl molecular adhesives. This method is compatible over the complete moduli range of PDMS, and potentially generalizable over a host of additional micro- and nano-structures and materials.

Inhibition of PI3K promotes dilation of human small airways in a rho kinase‐dependent manner

Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.

Inhibition of Phosphoinositide 3‐Kinase (PI3K) promotes dilation of human small airways in a Rho kinase‐dependent manner

Asthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.

Research highlights: microfluidics and magnets

In this highlight we present a snapshot of recent work using magnetic forces and particles to perform lab on a chip operations. Magnetic micro- & nanoparticles have been widely used for separations in cell biology & clinical diagnostics and as solid phase supports for reactions and chemical assays. Microscale approaches to control and manipulate magnetic particles can enable new functionality; allowing parallel and complex automation of assays, manipulation of fluids themselves, and precise separations based on small differences in magnetic properties. Here we discuss recent work demonstrating advances in these three areas.

Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3‐kinase‐mediated activation in a RhoA‐dependent manner

PI3K‐dependent activation of Rho kinase (ROCK) is necessary for agonist‐induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist‐mediated PI3K/ROCK axis activation, however, remain unclear. Given that G12 family proteins activate ROCK pathways in other cell types, their role in M3 muscarinic acetylcholine receptor‐stimulated PI3K/ROCK activation and contraction was examined.

Research highlights: measuring and manipulating cell migration

Microfabricated systems and microfluidic tools are well-suited to interface with cells because of the matching length scales. In this issue, we highlight three recent papers in which unique tools were used to control or measure cell migration. Cell migration is a key biological process involved in normal physiology (e.g. in embryonic development, immune response, and wound healing) which can go awry in diseases such as cancer. We highlight work applying electric fields and surface patterning to direct the collective migration of epithelial cells using galvanotaxis, in which surprisingly larger patches of cells respond more uniformly to electric fields. Such a platform may yield insights into or be co-opted to control tissue formation. We also describe recent results on developing a simple system to measure the migration of neutrophils in response to chemoattractants and on using this system to discriminate between neutrophils from asthmatic and non-asthmatic patients. Finally, a micropillar system is highlighted in which the epithelial-to-mesenchymal transition is quantified at the single-cell level, and may aid in our understanding of the plastic process of progression to a malignant phenotype. Cell migration is a wonderfully complex process in which microscale systems can not only contribute to a better understanding, but might also improve its recording and manipulation for practical applications.

Elastomeric sensor surfaces for high-throughput single-cell force cytometry

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.A microtechnology involving force sensors embedded in elastomers for cell culture enables the high-throughput measurement of single-cell force generation from contractile cells in a scalable and highly parallelized manner.

Publisher Correction: Elastomeric sensor surfaces for high-throughput single-cell force cytometry

In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.

Obesity increases airway smooth muscle responses to contractile agonists.

The asthma-obesity syndrome represents a major public health concern that disproportionately contributes to asthma severity and induces insensitivity to therapy. To date, no study has shown an intrinsic difference between human airway smooth muscle (HASM) cells derived from nonobese subjects and those derived from obese subjects. The objective of this study was to address whether there is a greater response to agonist-induced calcium mobilization, phosphorylation of myosin light chain (MLC), and greater shortening in HASM cells derived from obese subjects. HASM cells derived from nonobese and obese subjects were age and sex matched. Phosphorylation of MLC was measured after having been stimulated by carbachol. Carbachol- or histamine-induced mobilization of calcium and cell shortening were assessed in HASM cells derived from nonobese and obese donors. Agonist-induced MLC phosphorylation, mobilization of calcium, and cell shortening were greater in obese compared with non-obese-derived HASM cells. The MLC response was comparable in HASM cells derived from obese nonasthma and nonobese fatal asthma subjects. HASM cells derived from obese female subjects were more responsive to carbachol than HASM cells derived from obese male subjects. Insulin pretreatment had little effect on these responses. Our results show an increase in agonist-induced calcium mobilization associated with an increase in MLC phosphorylation and an increase in ASM cell shortening in favor of agonist-induced hyperresponsiveness in HASM cells derived from obese subjects. Our studies suggest that obesity induces a retained phenotype of hyperresponsiveness in cultured human airway smooth muscle cells.