Electroporation of Cells in Microfluidic Droplets

Zhan, Yihong; Wang, Jun; Bao, Ning; Lu, Chao

doi:10.1021/ac9001172

Cited by 130 publications

(125 citation statements)

References 27 publications

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“…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 (20)(21)(22). Using these approaches, droplets can be trapped by surface energy minimization.…”

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.

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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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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.

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“…In addition to compartmentalizing reactions, droplet-based microfluidics can also be used to encapsulate prokaryotic [14][15][16][17][18] and eukaryotic cells [19][20][21][22][23], and even the embryos of multicellular organisms [24,25], which opens up a new avenue for cell analysis. Recently, Brouzes et al [19] developed a droplet-based viability assay that permitted quantitative analysis of cell viability and growth within compartmentalized aqueous droplets.…”

Section: Introductionmentioning

confidence: 99%

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

Liu

Chen

et al. 2016

Talanta

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“…Recently, electroporation was integrated into microfluidic devices by using various types of mechanisms [13][14][15][16][17] . In comparison to conventional electroporation methods, microfluidic systems have some advantages, including the use of a lower applied electric field, lower sample and reagent consumption and reduced Joule heating 6 .…”

Section: Introductionmentioning

confidence: 99%

Continuous medium exchange and optically induced electroporation of cells in an integrated microfluidic system

et al. 2015

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Optically induced electroporation (OIE) is a promising microfluidic-based approach for the electroporation of cell membranes. However, previously proposed microfluidic cell-electroporation devices required tedious sample pre-treatment steps, specifically, periodic media exchange. To enable the use of this OIE process in a practical protocol, we developed a new design for a microfluidic device that can perform continuous OIE; i.e., it is capable of automatically replacing the culture medium with electroporation buffers. Integrating medium exchanges on-chip with OIE minimises critical issues such as cell loss and damage, both of which are common in traditional, centrifuge-based approaches. Most importantly, our new system is suitable for handling small or rare cell populations. Two medium exchange modules, including a micropost array railing structure and a deterministic lateral displacement structure, were first adopted and optimised for medium exchange and then integrated with the OIE module. The efficacy of these integrated microfluidic systems was demonstrated by transfecting an enhanced green fluorescent protein (EGFP) plasmid into human embryonic kidney 293T cells, with an efficiency of 8.3%. This result is the highest efficiency reported for any existing OIE-based microfluidic system. In addition, successful co-transfections of three distinct plasmids (EGFP, DsRed and ECFP) into cells were successfully achieved. Hence, we demonstrated that this system is capable of automatically performing multiple gene transfections into mammalian cells.

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Electroporation of Cells in Microfluidic Droplets

Cited by 130 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

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip

Continuous medium exchange and optically induced electroporation of cells in an integrated microfluidic system

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