Microfluidic image cytometry for quantitative single-cell profiling of human pluripotent stem cells in chemically defined conditions

Kamei, Ken‐ichiro; Ohashi, Masao; Gschweng, Eric H.; Ho, Quinn; Suh, Jane; Tang, Jinghua; Yu, Zeta Tak For; Clark, Amander T.; Pyle, April D.; Teitell, Michael A.; Lee, Ki‐Bum; Witte, Owen N.; Tseng, Hsian‐Rong

doi:10.1039/b922884e

Cited by 45 publications

(39 citation statements)

References 31 publications

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“…Since a high level of ESR1 gene expression was shown in MCF7 cells and ESR1 was not expressed in HeLa cells in NASBA experiments, we used the same cells for ESR1 immunostaining. ESR1 protein is known to be localized in the nucleus of MCF7 cells 4 and absent in HeLa cells. 42 Additionally, we used other cell lines, the ovarian cancer cell line 59M and non-adherent plasma cells U-266 (MM) to investigate the protein expression.…”

Section: Resultsmentioning

confidence: 99%

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Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

et al. 2011

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Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (~100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.

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Section: Resultsmentioning

confidence: 99%

“…Microscale operations hold the promise of integrating complete protocols onto a single chip ( i.e. , lab-on-a-chip) 1–4 and the potential of significantly enhancing assay reproducibility and quantitation 5 relative to macroscale techniques.…”

Section: Introductionmentioning

confidence: 99%

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

et al. 2011

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“…These features have been exploited in the design of various types of cell culture devices for analyzing the growth of single bacteria [36], yeast [37] and various types of mammalian cells [38,39,40,41], including mouse ESCs that are normally passaged as single cells [42]. However, the use of microfluidic devices to analyze hESC growth has been largely restricted to devices containing small numbers of relatively large chambers (>1 mm 2 in area) [43,44,45,46,47,48,49,50,51], with limited reports of single hESC colony cultures in <650 nL volumes [45,46,47].…”

Section: Clonal Analysis Of Individual Human Embryonic Stem Cell Diffmentioning

confidence: 99%

Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures

Sikorski

Caron

VanInsberghe

et al. 2015

Biotechnology Journal

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Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated.

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“…As illustrated in Figure 1a, this approach utilized two microfluidic systems including a digital droplet generator (DDG; Supporting Information, Section 1.2–1.5, video available in Supporting Information) [20,22] and a microfluidic cell culture array [31,32] . To enable the capability of on-chip synthesis of MFSNPs using the DDG, we developed a modular assembly system based on the supramolecular synthetic strategy [15,26–30] (Figure 1b).…”

mentioning

confidence: 99%

A High‐Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors

Liu

Choi

et al. 2015

Angew Chem Int Ed

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Simultaneous delivery of multiple genes and proteins (e.g., transcription factors, TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications.

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Microfluidic image cytometry for quantitative single-cell profiling of human pluripotent stem cells in chemically defined conditions

Cited by 45 publications

References 31 publications

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures

A High‐Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors

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