Westbrook M. Weaver

Label-free cell separation and sorting in microfluidic systems

AbstractCell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. FigureA wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties

Intrinsic particle-induced lateral transport in microchannels

In microfluidic systems at low Reynolds number, the flow field around a particle is assumed to maintain fore-aft symmetry, with fluid diverted by the presence of a particle, returning to its original streamline downstream. This current model considers particles as passive components of the system. However, we demonstrate that at finite Reynolds number, when inertia is taken into consideration, particles are not passive elements in the flow but significantly disturb and modify it. In response to the flow field, particles translate downstream while rotating. The combined effect of the flow of fluid around particles, particle rotation, channel confinement (i.e., particle dimensions approaching those of the channel), and finite fluid inertia creates a net recirculating flow perpendicular to the primary flow direction within straight channels that resembles the well-known Dean flow in curved channels. Significantly, the particle generating this flow remains laterally fixed as it translates downstream and only the fluid is laterally transferred. Therefore, as the particles remain inertially focused, operations can be performed around the particles in a way that is compatible with downstream assays such as flow cytometry. We apply this particle-induced transfer to perform fluid switching and mixing around rigid microparticles as well as deformable cells. This transport phenomenon, requiring only a simple channel geometry with no external forces to operate, offers a practical approach for fluid transfer at high flow rates with a wide range of applications, including sample preparation, flow reaction, and heat transfer.

Advances in High-Throughput Single-Cell Microtechnologies

Micro-scale biological tools that have allowed probing of individual cells--from the genetic, to proteomic, to phenotypic level--have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics.

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.

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments.

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.

Fluid Flow Induces Biofilm Formation in Staphylococcus epidermidis Polysaccharide Intracellular Adhesin-positive Clinical Isolates

ABSTRACT Staphylococcus epidermidis is a common cause of catheter-related bloodstream infections, resulting in significant morbidity and mortality and increased hospital costs. The ability to form biofilms plays a crucial role in pathogenesis; however, not all clinical isolates form biofilms under normal in vitro conditions. Strains containing the ica operon can display significant phenotypic variation with respect to polysaccharide intracellular adhesin (PIA)-based biofilm formation, including the induction of biofilms upon environmental stress. Using a parallel microfluidic approach to investigate flow as an environmental signal for S. epidermidis biofilm formation, we demonstrate that fluid shear alone induces PIA-positive biofilms of certain clinical isolates and influences biofilm structure. These findings suggest an important role of the catheter microenvironment, particularly fluid flow, in the establishment of S. epidermidis infections by PIA-dependent biofilm formation.

Research highlights: microfluidic point-of-care diagnostics

In this issue we highlight point-of-care (POC) diagnostic technologies to analyze cells, proteins, and small molecules from blood and other body fluids.

Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel

Abstract:Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases.

Well-plate mechanical confinement platform for studies of mechanical mutagenesis

Limited space for cell division, perhaps similar to the compressed microenvironment of a growing tumor, has been shown to induce phenotypic and karyotypic changes to a cell during mitosis. To expand understanding of this missegregation of chromosomes in aberrant multi-daughter or asymmetric cell divisions, we present a simple technique for subjecting mammalian cells to adjustable levels of confinement which allows subsequent interrogation of intracellular molecular components using high resolution confocal imaging. PDMS micropatterned confinement structures of subcellular height with neighboring taller media reservoir channels were secured on top of confluent cells with a custom compression well-plate system. The system improved ease of use over previous devices since confined cells could be initially grown on glass coverslips in a 12-well plate, and subsequently be imaged by high resolution confocal imaging, or during compression by live cell imaging. Live cell imaging showed a significant increase in abnormal divisions of confined cells across three different cell lines (HeLa, A375, and A549). Immunofluoresecence stains revealed a significant increase in cell diameter and chromosome area of confined cells, but no significant increase in centrosome-centromere distance upon division when compared to unconfined cells. The developed system could open up studies more broadly on confinement effects on mitotic processes, and increase the throughput of such studies.

Abstract A197: Size-selective isolation of viable and pure CTCs for molecular analysis using vortex technology.

Circulating tumor cells (CTCs) are cancer cells shed from a primary tumor, which enter the bloodstream and have the potential to metastasize. The detection and isolation of CTCs in bodily fluids can aid in cancer prognosis, characterizing genetic mutations for targeted therapies, and studying the biological mechanisms of metastasis. CTCs are very rare; there may only be tens of CTCs among millions of white blood cells and billions of red blood cells in one milliliter of blood. Current approaches for CTC isolation each have unique advantages and disadvantages: these techniques are often limited by speed, variable gene expression for immunocapture, long sample preparation steps, cost, and ability to deliver viable cells at high purity. Here, we use of arrays of microscale laminar fluid vortices to quickly isolate CTCs from large volumes of blood at high purity and without labels. Under high flow rates, large cells (> 15 μm) become stably trapped in laminar microvortices that form in simple rectangular reservoirs, while smaller red and white blood cells pass through. The large cancer cells are maintained in the microvortices, allowing for solution exchange, followed by release on lowering flow rate. The device is able to purify, enrich, and release a small volume (∼300 μL) of concentrated, viable cancer cells from blood at high throughput, concentration, and purity. The vortex chip successfully separated a variety of cancer cell lines from blood (∼20% capture efficiency), including those from melanoma, ovarian, breast, lung, and prostate cancer cell lines, with ∼10,000 fold enrichment. Captured cells remained viable and could be cultured off-chip. Finally, CTCs were successfully extracted and enumerated from the blood of patients with breast (N=4, 25-51 CTCs per 7.5 mL) and lung (N=8, 23-317 CTCs per 7.5 mL) cancers, and their sizes were measured. Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). Preliminary comparisons with FDA-approved CellSearch system highlighted improved results for CTC enumeration from breast and lung samples. Cells isolated from the vortex chip can also be utilized for downstream molecular analysis. We successfully utilized a qPCR-based approach to detect specific KRAS point mutations from lung cancer cells spiked into pleural fluids and then isolated using the vortex chip and demonstrated improved detection sensitivity. The high purity of our approach should allow for improved molecular sequencing results, and additional molecular analyses of CTCs from patient blood samples are underway, utilizing next generation transcriptome sequencing technologies. This vortex approach offers significant advantages over existing technologies, especially in terms of processing time, low cost, simplicity, cell integrity and purity. The ability to obtain viable CTCs provides flexible opportunities for the clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A197. Citation Format: Elodie Sollier, James Che, Derek E. Go, Westbrook M. Weaver, Nicolas Kummer, Matthew Rettig, Jonathan Goldman, Nicholas Nickols, Dino Di Carlo, Rajan P. Kulkarni. Size-selective isolation of viable and pure CTCs for molecular analysis using vortex technology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A197.