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3 pages, 162 KiB

Open AccessEditorial

Editorial for the Special Issue on Microfluidics for Cells and Other Organisms

by Danny van Noort

Micromachines 2019, 10(8), 520; https://doi.org/10.3390/mi10080520 - 5 Aug 2019

Viewed by 2217

Abstract

It is my great pleasure to present to you this first volume of 13 papers on the subject of Microfluidics for Cells and other Organisms [...] Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

Research

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21 pages, 3746 KiB

Open AccessFeature PaperArticle

Self-Learning Microfluidic Platform for Single-Cell Imaging and Classification in Flow

by Iordania Constantinou, Michael Jendrusch, Théo Aspert, Frederik Görlitz, André Schulze, Gilles Charvin and Michael Knop

Micromachines 2019, 10(5), 311; https://doi.org/10.3390/mi10050311 - 9 May 2019

Cited by 20 | Viewed by 5361

Abstract

Single-cell analysis commonly requires the confinement of cell suspensions in an analysis chamber or the precise positioning of single cells in small channels. Hydrodynamic flow focusing has been broadly utilized to achieve stream confinement in microchannels for such applications. As imaging flow cytometry [...] Read more.

Single-cell analysis commonly requires the confinement of cell suspensions in an analysis chamber or the precise positioning of single cells in small channels. Hydrodynamic flow focusing has been broadly utilized to achieve stream confinement in microchannels for such applications. As imaging flow cytometry gains popularity, the need for imaging-compatible microfluidic devices that allow for precise confinement of single cells in small volumes becomes increasingly important. At the same time, high-throughput single-cell imaging of cell populations produces vast amounts of complex data, which gives rise to the need for versatile algorithms for image analysis. In this work, we present a microfluidics-based platform for single-cell imaging in-flow and subsequent image analysis using variational autoencoders for unsupervised characterization of cellular mixtures. We use simple and robust Y-shaped microfluidic devices and demonstrate precise 3D particle confinement towards the microscope slide for high-resolution imaging. To demonstrate applicability, we use these devices to confine heterogeneous mixtures of yeast species, brightfield-image them in-flow and demonstrate fully unsupervised, as well as few-shot classification of single-cell images with 88% accuracy. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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11 pages, 2575 KiB

Open AccessArticle

An Automated and Miniaturized Rotating-Disk Device for Rapid Nucleic Acid Extraction

by Rui Tong, Lijuan Zhang, Chuandeng Hu, Xuee Chen, Qi Song, Kai Lou, Xin Tang, Yongsheng Chen, Xiuqing Gong, Yibo Gao and Weijia Wen

Micromachines 2019, 10(3), 204; https://doi.org/10.3390/mi10030204 - 22 Mar 2019

Cited by 9 | Viewed by 5210

Abstract

The result of molecular diagnostic and detection greatly dependent on the quality and integrity of the isolated nucleic acid. In this work, we developed an automated miniaturized nucleic acid extraction device based on magnetic beads method, consisting of four components including a sample [...] Read more.

The result of molecular diagnostic and detection greatly dependent on the quality and integrity of the isolated nucleic acid. In this work, we developed an automated miniaturized nucleic acid extraction device based on magnetic beads method, consisting of four components including a sample processing disc and its associated rotary power output mechanism, a pipetting module, a magnet module and an external central controller to enable a customizable and automated robust nucleic acid sample preparation. The extracted nucleic acid using 293T cells were verified using real-time polymerase chain reaction (PCR) and the data implies a comparable efficiency to a manual process, with the advantages of performing a flexible, time-saving (~10 min), and simple nucleic acid sample preparation. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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11 pages, 2838 KiB

Open AccessArticle

A Bubble-Free Microfluidic Device for Easy-to-Operate Immobilization, Culturing and Monitoring of Zebrafish Embryos

by Zhen Zhu, Yangye Geng, Zhangyi Yuan, Siqi Ren, Meijing Liu, Zhaozheng Meng and Dejing Pan

Micromachines 2019, 10(3), 168; https://doi.org/10.3390/mi10030168 - 28 Feb 2019

Cited by 18 | Viewed by 4876

Abstract

The development of miniaturized devices for studying zebrafish embryos has been limited due to complicated fabrication and operation processes. Here, we reported on a microfluidic device that enabled the capture and culture of zebrafish embryos and real-time monitoring of dynamic embryonic development. The [...] Read more.

The development of miniaturized devices for studying zebrafish embryos has been limited due to complicated fabrication and operation processes. Here, we reported on a microfluidic device that enabled the capture and culture of zebrafish embryos and real-time monitoring of dynamic embryonic development. The device was simply fabricated by bonding two layers of polydimethylsiloxane (PDMS) structures replicated from three-dimensional (3D) printed reusable molds onto a flat glass substrate. Embryos were easily loaded into the device with a pipette, docked in traps by gravity, and then retained in traps with hydrodynamic forces for long-term culturing. A degassing chamber bonded on top was used to remove air bubbles from the embryo-culturing channel and traps so that any embryo movement caused by air bubbles was eliminated during live imaging. Computational fluid dynamics simulations suggested this embryo-trapping and -retention regime to exert low shear stress on the immobilized embryos. Monitoring of the zebrafish embryogenesis over 20 h during the early stages successfully verified the performance of the microfluidic device for culturing the immobilized zebrafish embryos. Therefore, this rapid-prototyping, low-cost and easy-to-operate microfluidic device offers a promising platform for the long-term culturing of immobilized zebrafish embryos under continuous medium perfusion and the high-quality screening of the developmental dynamics. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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12 pages, 5474 KiB

Open AccessArticle

On-Chip Cell Incubator for Simultaneous Observation of Culture with and without Periodic Hydrostatic Pressure

by Mitsuhiro Horade, Chia-Hung Dylan Tsai and Makoto Kaneko

Micromachines 2019, 10(2), 133; https://doi.org/10.3390/mi10020133 - 17 Feb 2019

Cited by 7 | Viewed by 4380

Abstract

This paper proposes a microfluidic device which can perform simultaneous observation on cell growth with and without applying periodic hydrostatic pressure (Yokoyama et al. Sci. Rep. 2017, 7, 427). The device is called on-chip cell incubator. It is known that culture [...] Read more.

This paper proposes a microfluidic device which can perform simultaneous observation on cell growth with and without applying periodic hydrostatic pressure (Yokoyama et al. Sci. Rep. 2017, 7, 427). The device is called on-chip cell incubator. It is known that culture with periodic hydrostatic pressure benefits the elasticity of a cultured cell sheet based on the results in previous studies, but how the cells respond to such a stimulus during the culture is not yet clear. In this work, we focused on cell behavior under periodic hydrostatic pressure from the moment of cell seeding. The key advantage of the proposed device is that we can compare the results with and without periodic hydrostatic pressure while all other conditions were kept the same. According to the results, we found that cell sizes under periodic hydrostatic pressure increase faster than those under atmospheric pressure, and furthermore, a frequency-dependent fluctuation of cell size was found using Fourier analysis. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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15 pages, 6759 KiB

Open AccessArticle

A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells

by Gwo-Chin Ma, Wen-Hsiang Lin, Chung-Er Huang, Ting-Yu Chang, Jia-Yun Liu, Ya-Jun Yang, Mei-Hui Lee, Wan-Ju Wu, Yun-Shiang Chang and Ming Chen

Micromachines 2019, 10(2), 132; https://doi.org/10.3390/mi10020132 - 17 Feb 2019

Cited by 19 | Viewed by 5641

Abstract

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell Reveal^TM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform [...] Read more.

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell Reveal^TM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform is recently optimized to enhance the capture efficiency and system automation. In this study, spiking tests of SK-BR-3 breast cancer cells were used for the evaluation of capture efficiency. Then, peripheral bloods from 14 pregnant women whose fetuses have evidenced non-maternal genomic markers (e.g., de novo pathogenic copy number changes) were tested for the capture of circulating fetal nucleated red blood cells (fnRBCs). Captured cells were subjected to fluorescent in situ hybridization (FISH) on chip or recovered by an automated cell picker for molecular genetic analyses. The capture rate for the spiking tests is estimated as 88.1%. For the prenatal study, 2–71 fnRBCs were successfully captured from 2 mL of maternal blood in all pregnant women. The captured fnRBCs were verified to be from fetal origin. Our results demonstrated that the Cell Reveal^TM system has a high capture efficiency and can be used for fnRBC capture that is feasible for the genetic diagnosis of fetuses without invasive procedures. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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12 pages, 2698 KiB

Open AccessArticle

A Microfluidic Micropipette Aspiration Device to Study Single-Cell Mechanics Inspired by the Principle of Wheatstone Bridge

by Yong-Jiang Li, Yu-Nong Yang, Hai-Jun Zhang, Chun-Dong Xue, De-Pei Zeng, Tun Cao and Kai-Rong Qin

Micromachines 2019, 10(2), 131; https://doi.org/10.3390/mi10020131 - 16 Feb 2019

Cited by 24 | Viewed by 5791

Abstract

The biomechanical properties of single cells show great potential for early disease diagnosis and effective treatments. In this study, a microfluidic device was developed for quantifying the mechanical properties of a single cell. Micropipette aspiration was integrated into a microfluidic device that mimics [...] Read more.

The biomechanical properties of single cells show great potential for early disease diagnosis and effective treatments. In this study, a microfluidic device was developed for quantifying the mechanical properties of a single cell. Micropipette aspiration was integrated into a microfluidic device that mimics a classical Wheatstone bridge circuit. This technique allows us not only to effectively alter the flow direction for single-cell trapping, but also to precisely control the pressure exerted on the aspirated cells, analogous to the feature of the Wheatstone bridge that can precisely control bridge voltage and current. By combining the micropipette aspiration technique into the microfluidic device, we can effectively trap the microparticles and Hela cells as well as measure the deformability of cells. The Young’s modulus of Hela cells was evaluated to be 387 ± 77 Pa, which is consistent with previous micropipette aspiration studies. The simplicity, precision, and usability of our device show good potential for biomechanical trials in clinical diagnosis and cell biology research. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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11 pages, 1745 KiB

Open AccessCommunication

Bacterial Concentration Detection using a PCB-based Contactless Conductivity Sensor

by Xiao-Yan Zhang, Zhe-Yu Li, Yu Zhang, Xiao-Qian Zang, Kosei Ueno, Hiroaki Misawa and Kai Sun

Micromachines 2019, 10(1), 55; https://doi.org/10.3390/mi10010055 - 14 Jan 2019

Cited by 11 | Viewed by 5487

Abstract

Capacitively coupled contactless conductivity detection (C⁴D) is an improved approach to avoid the problems of labor-intensive, time-consuming and insufficient accuracy of plate count as well as the high-cost apparatus of flow cytometry (FCM) in bacterial counting. This article describes a novel [...] Read more.

Capacitively coupled contactless conductivity detection (C⁴D) is an improved approach to avoid the problems of labor-intensive, time-consuming and insufficient accuracy of plate count as well as the high-cost apparatus of flow cytometry (FCM) in bacterial counting. This article describes a novel electrode-integrated printed-circuit-board (PCB)-based C⁴D device, which supports the simple and safe exchange of capillaries and improves the sensitivity and repeatability of the contactless detection. Furthermore, no syringe pump is needed in the detection, it reduces the system size, and, more importantly, avoids the effect on the bacteria due to high pressure. The recovered bacteria after C⁴D detection at excitation of 25 Vpp and 60–120 kHz were analyzed by flow cytometry, and a survival rate higher than 96% was given. It was verified that C⁴D detection did not influence the bacterial viability. Moreover, bacteria concentrations from 10⁶ cells/mL to 10⁸ cells/mL were measured in a linear range, and relative standard deviation (RSD) is below 0.2%. In addition, the effects on bacteria and C⁴D from background solutions were discussed. In contrast to common methods used in most laboratories, this method may provide a simple solution to in situ detection of bacterial cultures. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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13 pages, 4098 KiB

Open AccessArticle

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells

by Maho Kaminaga, Tadashi Ishida, Tetsuya Kadonosono, Shinae Kizaka-Kondoh and Toru Omata

Micromachines 2019, 10(1), 41; https://doi.org/10.3390/mi10010041 - 9 Jan 2019

Cited by 4 | Viewed by 3864

Abstract

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on [...] Read more.

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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11 pages, 8380 KiB

Open AccessArticle

In Situ Analysis of Interactions between Fibroblast and Tumor Cells for Drug Assays with Microfluidic Non-Contact Co-Culture

by Hongmei Chen, Wenting Liu, Bin Wang and Zhifeng Zhang

Micromachines 2018, 9(12), 665; https://doi.org/10.3390/mi9120665 - 17 Dec 2018

Cited by 4 | Viewed by 3580

Abstract

Fibroblasts have significant involvement in cancer progression and are an important therapeutic target for cancer. Here, we present a microfluidic non-contact co-culture device to analyze interactions between tumor cells and fibroblasts. Further, we investigate myofibroblast behaviors induced by lung tumor cells as responses [...] Read more.

Fibroblasts have significant involvement in cancer progression and are an important therapeutic target for cancer. Here, we present a microfluidic non-contact co-culture device to analyze interactions between tumor cells and fibroblasts. Further, we investigate myofibroblast behaviors induced by lung tumor cells as responses to gallic acid and baicalein. Human lung fibroblast (HLF) and lung cancer cell line (A549) cells were introduced into neighboring, separated regions by well-controlled laminar flows. The phenotypic behavior and secretion activity of the tumor cells indicate that fibroblasts could become activated through paracrine signaling to create a supportive microenvironment for cancer cells when HLF is co-cultured with A549. Furthermore, both gallic acid (GA) and baicalein (BAE) could inhibit the activation of fibroblasts. In situ analysis of various cell communications via the paracrine pathway could be realizable in this contactless co-culture single device. This device facilitates a better understanding of interactions between heterotypic cells, thus exploring the mechanism of cancer, and performs anti-invasion drug assays in a relatively complex microenvironment. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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12 pages, 2901 KiB

Open AccessArticle

Separation and Characterization of Prostate Cancer Cell Subtype according to Their Motility Using a Multi-Layer CiGiP Culture

by Lin-Xiang Wang, Ying Zhou, Jing-Jing Fu, Zhisong Lu and Ling Yu

Micromachines 2018, 9(12), 660; https://doi.org/10.3390/mi9120660 - 14 Dec 2018

Cited by 10 | Viewed by 3041

Abstract

Cancer cell metastasis has been recognized as one hallmark of malignant tumor progression; thus, measuring the motility of cells, especially tumor cell migration, is important for evaluating the therapeutic effects of anti-tumor drugs. Here, we used a paper-based cell migration platform to separate [...] Read more.

Cancer cell metastasis has been recognized as one hallmark of malignant tumor progression; thus, measuring the motility of cells, especially tumor cell migration, is important for evaluating the therapeutic effects of anti-tumor drugs. Here, we used a paper-based cell migration platform to separate and isolate cells according to their distinct motility. A multi-layer cells-in-gels-in-paper (CiGiP) stack was assembled. Only a small portion of DU 145 prostate cancer cells seeded in the middle layer could successfully migrate into the top and bottom layers of the stack, showing heterogeneous motility. The cells with distinct migration were isolated for further analysis. Quantitative PCR assay results demonstrated that cells with higher migration potential had increased expression of the ALDH1A1, SRY (sex-determining region Y)-box 2, NANOG, and octamer-binding transcription 4. Increased doxorubicin tolerance was also observed in cells that migrated through the CiGiP layers. In summary, the separation and characterization of prostate cancer cell subtype can be achieved by using the multi-layer CiGiP cell migration platform. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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9 pages, 2785 KiB

Open AccessArticle

Microfluidic Analyzer Enabling Quantitative Measurements of Specific Intracellular Proteins at the Single-Cell Level

by Lixing Liu, Beiyuan Fan, Diancan Wang, Xiufeng Li, Yeqing Song, Ting Zhang, Deyong Chen, Yixiang Wang, Junbo Wang and Jian Chen

Micromachines 2018, 9(11), 588; https://doi.org/10.3390/mi9110588 - 12 Nov 2018

Cited by 2 | Viewed by 3405

Abstract

This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic [...] Read more.

This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic module with fluorescent intensities quantified by a fluorescent module and translated into the numbers of specific intracellular proteins at the single-cell level using a MATLAB platform. Detection ranges and resolutions of the analyzer were characterized as 896.78–6.78 × 10⁵ and 334.60 nM for Alexa 488, 314.60–2.11 × 10⁵ and 153.98 nM for FITC, and 77.03–5.24 × 10⁴ and 37.17 nM for FITC-labelled anti-beta-actin antibodies. As a demonstration, the numbers of single-cell beta-actins of two paired oral tumor cell types and two oral patient samples were quantified as: 1.12 ± 0.77 × 10⁶/cell (salivary adenoid cystic carcinoma parental cell line (SACC-83), n_cell = 13,689) vs. 0.90 ± 0.58 × 10⁵/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), n_cell = 15,341); 0.89 ± 0.69 × 10⁶/cell (oral carcinoma cell line (CAL 27), n_cell = 7357) vs. 0.93 ± 0.69 × 10⁶/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), n_cell = 6276); and 0.86 ± 0.52 × 10⁶/cell (patient I) vs. 0.85 ± 0.58 × 10⁶/cell (patient II). These results (1) validated the developed analyzer with a throughput of 10 cells/s and a processing capability of ~10,000 cells for each cell type, and (2) revealed that as an internal control in cell analysis, the expressions of beta-actins remained stable in oral tumors with different malignant levels. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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Review

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31 pages, 4310 KiB

Open AccessReview

Microfluidic Single-Cell Manipulation and Analysis: Methods and Applications

by Tao Luo, Lei Fan, Rong Zhu and Dong Sun

Micromachines 2019, 10(2), 104; https://doi.org/10.3390/mi10020104 - 1 Feb 2019

Cited by 152 | Viewed by 16885

Abstract

In a forest of a hundred thousand trees, no two leaves are alike. Similarly, no two cells in a genetically identical group are the same. This heterogeneity at the single-cell level has been recognized to be vital for the correct interpretation of diagnostic [...] Read more.

In a forest of a hundred thousand trees, no two leaves are alike. Similarly, no two cells in a genetically identical group are the same. This heterogeneity at the single-cell level has been recognized to be vital for the correct interpretation of diagnostic and therapeutic results of diseases, but has been masked for a long time by studying average responses from a population. To comprehensively understand cell heterogeneity, diverse manipulation and comprehensive analysis of cells at the single-cell level are demanded. However, using traditional biological tools, such as petri-dishes and well-plates, is technically challengeable for manipulating and analyzing single-cells with small size and low concentration of target biomolecules. With the development of microfluidics, which is a technology of manipulating and controlling fluids in the range of micro- to pico-liters in networks of channels with dimensions from tens to hundreds of microns, single-cell study has been blooming for almost two decades. Comparing to conventional petri-dish or well-plate experiments, microfluidic single-cell analysis offers advantages of higher throughput, smaller sample volume, automatic sample processing, and lower contamination risk, etc., which made microfluidics an ideal technology for conducting statically meaningful single-cell research. In this review, we will summarize the advances of microfluidics for single-cell manipulation and analysis from the aspects of methods and applications. First, various methods, such as hydrodynamic and electrical approaches, for microfluidic single-cell manipulation will be summarized. Second, single-cell analysis ranging from cellular to genetic level by using microfluidic technology is summarized. Last, we will also discuss the advantages and disadvantages of various microfluidic methods for single-cell manipulation, and then outlook the trend of microfluidic single-cell analysis. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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18 pages, 4022 KiB

Open AccessCommentary

Integrating Microfabrication into Biological Investigations: the Benefits of Interdisciplinarity

by Gianluca Grenci, Cristina Bertocchi and Andrea Ravasio

Micromachines 2019, 10(4), 252; https://doi.org/10.3390/mi10040252 - 16 Apr 2019

Cited by 13 | Viewed by 4758

Abstract

The advent of micro and nanotechnologies, such as microfabrication, have impacted scientific research and contributed to meaningful real-world applications, to a degree seen during historic technological revolutions. Some key areas benefitting from the invention and advancement of microfabrication platforms are those of biological [...] Read more.

The advent of micro and nanotechnologies, such as microfabrication, have impacted scientific research and contributed to meaningful real-world applications, to a degree seen during historic technological revolutions. Some key areas benefitting from the invention and advancement of microfabrication platforms are those of biological and biomedical sciences. Modern therapeutic approaches, involving point-of-care, precision or personalized medicine, are transitioning from the experimental phase to becoming the standard of care. At the same time, biological research benefits from the contribution of microfluidics at every level from single cell to tissue engineering and organoids studies. The aim of this commentary is to describe, through proven examples, the interdisciplinary process used to develop novel biological technologies and to emphasize the role of technical knowledge in empowering researchers who are specialized in a niche area to look beyond and innovate. Full article

(This article belongs to the Special Issue Microfluidics for Cells and Other Organisms)

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