Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection.

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices.

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each thermoplastic of interest. Solvent bonding is a simple and versatile method that can be used to fabricate devices from a variety of plastics. An appropriate solvent is added between two device layers to be bonded, and heat and pressure are applied to the device to facilitate the bonding. By using an appropriate combination of solvent, plastic, heat, and pressure, the device can be sealed with a high quality bond, characterized as having high bond coverage, bond strength, optical clarity, durability over time, and low deformation or damage to microfeature geometry. We describe the procedure for bonding devices made from two popular thermoplastics, poly(methyl-methacrylate) (PMMA), and cyclo-olefin polymer (COP), as well as a variety of methods to characterize the quality of the resulting bonds, and strategies to troubleshoot low quality bonds. These methods can be used to develop new solvent bonding protocols for other plastic-solvent systems.

Liquid phase solvent bonding of plastic microfluidic devices assisted by retention grooves.

We report a novel method for achieving consistent liquid phase solvent bonding of plastic microfluidic devices via the use of retention grooves at the bonding interface. The grooves are patterned during the regular microfabrication process, and can be placed at the periphery of a device, or surrounding microfluidic features with open ports, where they effectively mitigate solvent evaporation, and thus substantially reduce poor bond coverage. This method is broadly applicable to a variety of plastics and solvents, and produces devices with high bond quality (i.e., coverage, strength, and microfeature fidelity) that are suitable for studies in physics, chemistry, and cell biology at the microscale.

Micromilling: a method for ultra-rapid prototyping of plastic microfluidic devices.

This tutorial review offers protocols, tips, insight, and considerations for practitioners interested in using micromilling to create microfluidic devices. The objective is to provide a potential user with information to guide them on whether micromilling would fill a specific need within their overall fabrication strategy. Comparisons are made between micromilling and other common fabrication methods for plastics in terms of technical capabilities and cost. The main discussion focuses on "how-to" aspects of micromilling, to enable a user to select proper equipment and tools, and obtain usable microfluidic parts with minimal start-up time and effort. The supplementary information provides more extensive discussion on CNC mill setup, alignment, and programming. We aim to reach an audience with minimal prior experience in milling, but with strong interests in fabrication of microfluidic devices.

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

BACKGROUND: Changes in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. METHODS: Decellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. RESULTS: Our findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Electrical control of protein conformation.

Conducting polymer devices that enable precise control of fibronectin conformation over macroscopic areas are reported. Single conformations as well as conformation gradients are achieved by applying an appropriate potential. These surfaces remain biologically relevant and support cell culture; hence, they may serve as a model to understand and control cell-surface interactions, with applications in basic research, medical diagnostics, and tissue engineering.

Electrical control of cell density gradients on a conducting polymer surface.

We describe a conducting polymer device that can induce electrically controlled density gradients of normal and cancerous cell lines, and hence can be used as a tool for the study of cell-cell interactions.