High-throughput microfluidic single-cell RT-qPCR

White, Alexander D.; VanInsberghe, Michael; Petriv, Oleh I.; Hamidi, M.; Sikorski, Darek; Marra, Marco A.; Piret, James M.; Aparicio, Samuel; Hansen, Carl L.

doi:10.1073/pnas.1019446108

Cited by 409 publications

(384 citation statements)

References 32 publications

(37 reference statements)

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“…Such approaches, which allowed for trapping single cells by actuating integrated valve systems 31 or exploiting physical obstacles as traps 14 , have a drawback in terms of device fabrication. Recently a new approach was presented to successfully isolate single cells within small trapping elements 15,19 .…”

Section: Discussionmentioning

confidence: 99%

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

et al. 2015

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The widespreading of gene therapy as therapeutic tool relies on the development of DNAcarrying vehicles devoid of safety concerns. Opposite to viral vectors, non-viral gene carriers are promising in this perspective, although their low transfection efficiency leads to the necessity of further optimizations. Aiming at overcoming the limitation of traditional macroscale approaches, mainly consisting in time-consuming and simplified models, a microfluidic strategy was developed for transfection studies on single cells in a highthroughput and deterministic fashion. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. At this purpose, we conceived a roundshaped culture chamber integrated with a bottom trapping junction which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for the optimization of the single cell trapping efficacy. The optimized chamber layout was integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, (ii) a serial dilution generator provided with chaotic mixing properties, able to deliver to chambers both soluble factors and non-diffusive particles (i.e. polymer/DNA complexes, polyplexes) under spatio-temporally controlled chemical patterns. Devices were experimentally validated and allowed for trapping individual human glioblastoma-astrocytoma epithelial-like cells (U87-MG) with a trapping efficacy of about 40%. Cells were cultured within the device and underwent preliminary transfection experiments using 25kDa linear polyethylenimine (lPEI)-based polyplexes, confirming the potentiality of the proposed platform for the future high-throughput screening of gene delivery vectors and the optimization of transfection protocols.

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Section: Discussionmentioning

confidence: 99%

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

et al. 2015

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“…This field has been propelled by remarkable technological advances for efficiently isolating single cells 28, 29, and the reduction of the detection levels of protocols for profiling gene expression 30, 31, histone marks 25, 26, or chromatin accessibility 32, 33 in individual cells. These techniques allow researchers to overcome the limitations of population studies, in which averaging the measurements over a heterogeneous population of cells masks the variability at the epigenetics and transcriptional levels among cells within a culture or tissue 26, 34, 35.…”

Section: Single‐cell Profiling Is Key For Studying Pluripotent State mentioning

confidence: 99%

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

Angarica

Sol

2016

BioEssays

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Pluripotency can be considered a functional characteristic of pluripotent stem cells (PSCs) populations and their niches, rather than a property of individual cells. In this view, individual cells within the population independently adopt a variety of different expression states, maintained by different signaling, transcriptional, and epigenetics regulatory networks. In this review, we propose that generation of integrative network models from single cell data will be essential for getting a better understanding of the regulation of self‐renewal and differentiation. In particular, we suggest that the identification of network stability determinants in these integrative models will provide important insights into the mechanisms mediating the transduction of signals from the niche, and how these signals can trigger differentiation. In this regard, the differential use of these stability determinants in subpopulation‐specific regulatory networks would mediate differentiation into different cell fates. We suggest that this approach could offer a promising avenue for the development of novel strategies for increasing the efficiency and fidelity of differentiation, which could have a strong impact on regenerative medicine.

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“…Improved designs for fundamental components include normally closed valves (NCVs) [16 ,17], structured valves [18], cell traps [19], and capacitors and diodes ( Figure 1a) [20,21]; higher-level components that have been introduced include a chamber array [22], a high performance separation column [23 ], a gradient selector (Figure 1b) [23 ], a long term gradient generator [24 ], a 2D spatial gradient controller [25], an agar filled chamber [26] and a mutilaminate mixer [27].…”

Section: Component-level Developmentsmentioning

confidence: 99%

Recent developments in microfluidic large scale integration

Araci

Brisk

2014

Current Opinion in Biotechnology

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In 2002, Thorsen et al. integrated thousands of micromechanical valves on a single microfluidic chip and demonstrated that the control of the fluidic networks can be simplified through multiplexors [1]. This enabled realization of highly parallel and automated fluidic processes with substantial sample economy advantage. Moreover, the fabrication of these devices by multilayer soft lithography was easy and reliable hence contributed to the power of the technology; microfluidic large scale integration (mLSI). Since then, mLSI has found use in wide variety of applications in biology and chemistry. In the meantime, efforts to improve the technology have been ongoing. These efforts mostly focus on; novel materials, components, micromechanical valve actuation methods, and chip architectures for mLSI. In this review, these technological advances are discussed and, recent examples of the mLSI applications are summarized. Recent advancements in microfluidic technologies have created new opportunities in the fields of biology and chemistry through miniaturization and automation of common processes, including mixing, metering, trapping, reaction, filtering, and separation, among others. Microfluidic large scale integration (mLSI) enables the fabrication of microfluidic chips containing hundreds or more of these functions using well-established lithography techniques, similar in principle to electronic LSI in the semiconductor industry [1]. Small feature dimensions, abundant spatial parallelism, and control over fluid flow provide mLSI technology with advantages such as high throughput, precise metering, automation and low reagent consumption. The implications of mLSI technology can especially be seen in recent advances in single cell, genomic, and protein analysis.

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High-throughput microfluidic single-cell RT-qPCR

Cited by 409 publications

References 32 publications

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

Recent developments in microfluidic large scale integration

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