Live single cell functional phenotyping in droplet nano-liter reactors

Konry, Tania; Golberg, Alexander; Yarmush, Martin L.

doi:10.1038/srep03179

Cited by 83 publications

(76 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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).…”

mentioning

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.

View full text Add to dashboard Cite

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...

show abstract

mentioning

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.

View full text Add to dashboard Cite

show abstract

“…The first group includes platforms that first distribute cell populations randomly into microchannels and then isolate cell groups into nanoliter-volume microchambers by on-chip microvalves or clamp systems [20,21] (Figure 1b). The second group, referred to as 'droplet microfluidics' (Figure 1c), co-encapsulate cell pairs or clusters in picoliter droplets generated by emulsions of immiscible phases [22,23], and can exceed Poisson-limits by controlled ordering of cells before droplet formation [24][25][26]. Conceivably, electrowetting-based approaches could further enable manipulation and transport of droplets containing cells in open format in addition to closed format configuration [27].…”

Section: Defining Intercellular Interactionsmentioning

confidence: 98%

Spatially and temporally controlled immune cell interactions using microscale tools

Dura

Voldman

2015

Current Opinion in Immunology

View full text Add to dashboard Cite

Many critical immunological responses are mediated by cell-cell interactions. Despite their capabilities, traditional techniques that rely on snapshot analysis or ensemble measurements can only provide a fragmentary picture of the complexity of these interactions. Emerging classes of new and versatile microscale tools hold great potential for enabling detailed investigation of these interactions with precise control in space and time, multiplexed measurement capability and high-throughput single-cell analysis. These features allow new ways of examining immune cell interactions that are not possible with traditional methods, improve the extent of information extracted from experiments, and reveal new findings. Here, we review recent developments in microscale tools that are paving the way for comprehensive analyses of cell-cell interactions in the immune system.

show abstract

“…Since then, the droplet platform has been used extensively for single cell isolation and protein detection in bacterial infection [52,64], cancers [65,66], hybridoma screening [30,67], and immunology [68][69][70]. Shim et al developed a droplet microfluidic platform for compartmentalizing single bacteria in droplets and measuring secretory enzyme activity of individual clones in a time-dependent fashion [49].…”

Section: Cellular Encapsulation and Protein Detection In Dropletsmentioning

confidence: 98%

“…They performed subsequent studies with simultaneous detection of surface marker CD86 on dendritic cells and secreted cytokine, IL-6 upon dendritic cell-T cell immune synapse (IS) formation in droplets [69]. This system facilitated the dynamic and simultaneous detection of cell-surface markers and secreted cytokines, with concurrent monitoring of phenotypical changes upon IS formation while accounting for heterogeneity in the cell population.…”

Section: Cellular Encapsulation and Protein Detection In Dropletsmentioning

confidence: 99%

Droplet Microfluidics for Screening of Surface-Marker and Secretory Protein Expression

Sabhachandani

Sarkar

Konry

2016

Microfluidic Methods for Molecular Biology

Self Cite

View full text Add to dashboard Cite

Current techniques of biomarker detection employ population-based studies, which fail to provide information on cellular and molecular heterogeneity and variances in functional behavior, which is important for diseased state characterization. Additionally, low concentration of samples in biological fluids creates hurdles in detection using these methods. Thus, improvement in detection techniques of clinical biomarkers has become imperative to halt disease progression at the earliest possible stage. Droplet microfluidics has become quite popular as a diagnostic platform due to its ability to sensitively detect low volume of clinical samples in a rapid and high-throughput manner. These advantages make the droplets an ideal platform for low-abundance biomarker detection. This chapter focuses on various techniques for detection of surface and secreted protein biomarkers using droplet microfluidics platform.

show abstract

Live single cell functional phenotyping in droplet nano-liter reactors

Cited by 83 publications

References 27 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

Spatially and temporally controlled immune cell interactions using microscale tools

Droplet Microfluidics for Screening of Surface-Marker and Secretory Protein Expression

Contact Info

Product

Resources

About