Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets

Um, Eujin; Rha, Eugene; Choi, Su-Lim; Lee, Dae-Hee; Park, Je‐Kyun

doi:10.1039/c2lc21266h

Cited by 33 publications

(25 citation statements)

References 20 publications

(22 reference statements)

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“…To achieve this, cells must be isolated in compartments that can sustain cell viability and growth while permitting conventional optical analysis over many hours to days. Dropletbased microfluidics, which enables single-cell encapsulation in nano-and subnanoliter droplets by surrounding microscopic aqueous medium with an immiscible carrier fluid (4)(5)(6)(7)(8), recently gained interest with the appearance of digital PCR (9)(10)(11). Much of the work thus far has been directed toward improving droplet manipulation capabilities (12)(13)(14)(15)(16).…”

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confidence: 99%

“…However, upon their adjustment to prolonged mammalian assays, they require multilayer fabrication, either involving high-pressure cell loading or not allowing for different substrates to be used (26,27). Several single-cell platforms that encapsulate cells in stationary droplet arrays are available (5,(28)(29)(30)(31)(32)(33)(34)(35); however, the ability to culture adherent cells for long periods remains difficult (36).…”

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confidence: 99%

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Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Shemesh

Ben-Arye

Avesar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.single cell | nanoliter array | diagnostics C ommon single-cell analysis methods, such as flow cytometry and mass cytometry (1), offer high throughput and accurate single-cell marker quantification, yet they lack the ability to monitor large numbers of single cells continuously and simultaneously in performance-based assays (2, 3). Conventional microscopy may be used for these assays; however, in the case of single cells, they cannot analyze extracellular events, such as secretion. To achieve this, cells must be isolated in compartments that can sustain cell viability and growth while permitting conventional optical analysis over many hours to days. Dropletbased microfluidics, which enables single-cell encapsulation in nano-and subnanoliter droplets by surrounding microscopic aqueous medium with an immiscible carrier fluid (4-8), recently gained interest with the appearance of digital PCR (9-11). Much of the work thus far has been directed toward improving droplet manipulation capabilities (12-16). With these methods, droplets are mobile, and thus cytometry is performed under flow conditions (17), making continuous monitoring of single cells difficult. Continuous monitoring may be achieved by using stationary indexed droplets, but many current droplet immobilization techniques are limited by pressure coupling between droplet generation and capture events, as well as the requirement to adjust droplet volume to nanowell size (6,18,19). The vast majority of methods used to generate water-in-oil droplets begin by priming a continuous oil phase in a microfluidic channel followed by an injection of a dispersed (aqueous) medium (2...

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confidence: 99%

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confidence: 99%

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Shemesh

Ben-Arye

Avesar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…7.3c). Due to miniaturization, the device provides high-throughput screening of the droplets (Um et al ., 2012), but multistep reactions using these devices are still a big challenge.…”

Section: Synthesis Of Drug Librariesmentioning

confidence: 99%

“…(c) Schematic of a microdroplet manipulator, including functions for (i) droplet generation, (ii) transfer of droplets to a microwell array, (iii) migration of droplets into the wells, (iv) trapping of second droplets and (v) oil change to induce droplet merging (Um et al ., 2012).…”

Section: Synthesis Of Drug Librariesmentioning

confidence: 99%

Microfluidic devices for drug discovery and analysis

Kochhar

Chan

Ong

et al. 2013

Microfluidic Devices for Biomedical Applications

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“…Zeng et al (2010) randomly seeded E. coli cells into droplets containing primer-adhered microspheres and real-time PCR reagents. Um et al (2012) encapsulated single E. coli cells in a pico-liter volume droplet. They employed a secondary breakup of droplets, thereby reducing stochastic loading of the cells and increasing the single-cell occupancy from 30% to around 50%.…”

Section: Dilution-to-extinctionmentioning

confidence: 99%

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

Shi

Gao

Wang

et al. 2014

Critical Reviews in Biotechnology

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Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.

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Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets

Cited by 33 publications

References 20 publications

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Microfluidic devices for drug discovery and analysis

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

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