A simple microfluidic device for live-imaging of the vertical section of epithelial cells

S, Araki; Nakano, Masayoshi; Tsugane, Mamiko; Sunaga, Fumiko; Hattori, Mitsuru; Nakano, Masahiro; Nagai, Takeharu; Suzuki, Hiroaki

doi:10.1039/c9an02165e

Cited by 11 publications

(7 citation statements)

References 40 publications

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“…Analyzing single-cell data will enable us to identify whether the difference is because of all the cells behaving differently, or a population of cells showing higher or lower responses. Future studies should also expand the panel of cellular readouts to provide subcellular information using microfluidic single-cell assays. , For instance, it is possible that PM is particularly toxic to mitochondrial function in cells with particular levels of intrinsic ROS or a particular profile of gene expression. Our single-cell assay would allow for visualizing mitochondrial localization, facilitating assessments of mitochondrial health.…”

Section: Resultsmentioning

confidence: 99%

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

Liu

Whitley

et al. 2020

Environ. Sci. Technol.

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Health risks associated with exposure to ambient particulate matter (PM) are a major concern around the world. Adverse PM health effects have been proposed to be linked to oxidative stress through the generation of reactive oxygen species (ROS). In vitro cellular assays can provide insights into components or characteristics of PM that best account for its toxicity at a cellular level. However, most current assays report cell population averages and are mostly time endpoint measurements and thus provide no temporal information. This poses limitations on our understanding of PM health effects. In this study, we developed a microfluidic assay that can measure cellular ROS responses at the singlecell level and evaluate temporal dynamic behavior of single cells. We first established a protocol that enables culturing cells in our microfluidic platform and that can provide reproducible ROS readouts. We further examined the heterogeneous ROS responses of cell populations and tracked the dynamics of individual cellular responses upon exposure to different concentrations of PM extracts. Our results show that in an alveolar macrophage cell line, cellular ROS responses are highly heterogeneous. ROS responses from different cells can vary over an order of magnitude, and large coefficients of variation at each timepoint measurement indicate a high variability. The dynamic behavior of single-cell responses is strongly dependent on PM concentrations. Our work serves as a proof-of-principle demonstration of the capability of our microfluidic technology to study time-resolved single-cell responses upon PM exposure. We envision applying this high-resolution, high-content assay to investigate a wide array of single-cell responses (beyond ROS) upon exposure to different types of PM in the future.

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Section: Resultsmentioning

confidence: 99%

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

Liu

Whitley

et al. 2020

Environ. Sci. Technol.

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“…In summary, the proposed microfluidic technology can serve as a new platform approach, which has the potential to advance studies at the cellular level [95]. Fluorescence live-cell imaging is an indispensable tool for studying the structure and dynamics of cells and their internal/external building blocks [96].…”

Section: Discussionmentioning

confidence: 99%

Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

Capuzzo¹,

Vigo²

2021

Preprint

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One of the most surprising differences observed when comparing cell cultures in 2D and 3D is morphological dissimilarity and their evolution over time. Cells grown in a monolayer tend to flatten in the lower part of the plate adhering to and spreading in the horizontal plane without expanding in the vertical dimension. The result is that cells grown in 2D have a forced apex-basal polarity. 3D cultures support co-cultivation and crosstalking between multiple cell types, which regulate development and formation in the in vivo counterpart. 3D models culture, with or without a scaffold matrix, can exhibit more in vivo-like morphology and physiology. 3D cultures recapitulate relevant physiological cellular processes, transforming into unique platforms for drug screening. To support and guarantee the functional maintenance of a 3D structure, one must consider the structures and dynamics of regulatory networks, increasingly studied with liveimaging microscopy. However, commercially available technologies that can be used for current laboratory needs are limited, although there is a need to facilitate the acquisition of cellular kinetics with a high spatial and temporal resolution, to elevate visual performance and consequently that of experimentation. The CELLviewer is a newly conceived and developed multi-technology instrumentation, combining and synchronizing the work of different scientific disciplines. This 2 work aims to test the system with two models: the first model is a single Jurkat cell while the second is an MCF-7 spheroid. After having grown both models, the two models used are loaded into the microfluidic cartridge for each experiment and recorded in time-lapse for a total of 4 hours. After adaptive autofocus, when sliding inside the cartridge chamber, the samples used are tracked under the action of the optics and the 3D rotation was experimentally successfully obtained. A cell viability assessment was then used using the MitoGreen dye, a fluorescence marker selectively permeable to live cells. The ImageJ software was used to: calculate the model diameter, create fluorescence intensity graphs along a straight line passing through the cell, visualize the spatial fluorescence intensity distribution in 3D.

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“…In conclusion, the proposed microfluidic technology can be used as a new platform solution to advance studies at the cellular level [95]. Fluorescence live-cell imaging is a vital method for researching the structure and dynamics of cells, as well as the internal and external components that make them up [96].…”

Section: Discussionmentioning

confidence: 99%

Microfluidic Live Imaging Technology to Perform Research Activities in 3D Models

Martino¹

2021

AABSc

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Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-akind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 1 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, Image J software was used.

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A simple microfluidic device for live-imaging of the vertical section of epithelial cells

Abstract: Capability of simple microfluidic devices having vertical sidewalls for live-cell fluorescence imaging was investigated.

Cited by 11 publications

References 40 publications

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

Microfluidic Live Imaging Technology to Perform Research Activities in 3D Models

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