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12 pages, 8357 KiB

Open AccessFeature PaperEditor’s ChoiceCommunication

Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel

by Yifei Ye, Xiaofeng Luan, Lingqian Zhang, Wenjie Zhao, Jie Cheng, Mingxiao Li, Yang Zhao and Chengjun Huang

Micromachines 2020, 11(9), 856; https://doi.org/10.3390/mi11090856 - 16 Sep 2020

Cited by 11 | Viewed by 4358

Abstract

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a [...] Read more.

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a microfluidic system for precise and rapid single-cell electroporation and simultaneous impedance monitoring in a constriction microchannel. When single cells (A549) were continuously passing through the constriction microchannel, a localized high electric field was applied on the cell membrane, which resulted in highly efficient (up to 96.6%) electroporation. During a single cell entering the constriction channel, an abrupt impedance drop was noticed and demonstrated to be correlated with the occurrence of electroporation. Besides, while the cell was moving in the constriction channel, the stabilized impedance showed the capability to quantify the electroporation extent. The correspondence of the impedance variation and electroporation was validated by the intracellular delivery of the fluorescence indicator (propidium iodide). Based on the obtained results, this system is capable of precise control of electroporation and real-time, label-free impedance assessment, providing a potential tool for intracellular delivery and other biomedical applications. Full article

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

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15 pages, 3575 KiB

Open AccessArticle

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

by Michael Januszyk, Kellen Chen, Dominic Henn, Deshka S. Foster, Mimi R. Borrelli, Clark A. Bonham, Dharshan Sivaraj, Dhananjay Wagh, Michael T. Longaker, Derrick C. Wan and Geoffrey C. Gurtner

Micromachines 2020, 11(9), 815; https://doi.org/10.3390/mi11090815 - 28 Aug 2020

Cited by 37 | Viewed by 7192

Abstract

Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of [...] Read more.

Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations. Full article

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

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13 pages, 2027 KiB

Open AccessArticle

Quantitative Image-Based Cell Viability (QuantICV) Assay for Microfluidic 3D Tissue Culture Applications

by Louis Jun Ye Ong, Liang Zhu, Gabriel Jenn Sern Tan and Yi-Chin Toh

Micromachines 2020, 11(7), 669; https://doi.org/10.3390/mi11070669 - 9 Jul 2020

Cited by 11 | Viewed by 3817

Abstract

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose [...] Read more.

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available. Full article

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

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18 pages, 5029 KiB

Open AccessArticle

OOCHIP: Compartmentalized Microfluidic Perfusion System with Porous Barriers for Enhanced Cell–Cell Crosstalk in Organ-on-a-Chip

by Qasem Ramadan, Sajay Bhuvanendran Nair Gourikutty and Qingxin Zhang

Micromachines 2020, 11(6), 565; https://doi.org/10.3390/mi11060565 - 31 May 2020

Cited by 7 | Viewed by 4149

Abstract

Improved in vitro models of human organs for predicting drug efficacy, interactions, and disease modelling are crucially needed to minimize the use of animal models, which inevitably display significant differences from the human disease state and metabolism. Inside the body, cells are organized [...] Read more.

Improved in vitro models of human organs for predicting drug efficacy, interactions, and disease modelling are crucially needed to minimize the use of animal models, which inevitably display significant differences from the human disease state and metabolism. Inside the body, cells are organized either in direct contact or in close proximity to other cell types in a tightly controlled architecture that regulates tissue function. To emulate this cellular interface in vitro, an advanced cell culture system is required. In this paper, we describe a set of compartmentalized silicon-based microfluidic chips that enable co-culturing several types of cells in close proximity with enhanced cell–cell interaction. In vivo-like fluid flow into and/or from each compartment, as well as between adjacent compartments, is maintained by micro-engineered porous barriers. This porous structure provides a tool for mimicking the paracrine exchange between cells in the human body. As a demonstrating example, the microfluidic system was tested by culturing human adipose tissue that is infiltrated with immune cells to study the role if the interplay between the two cells in the context of type 2 diabetes. However, the system provides a platform technology for mimicking the structure and function of single- and multi-organ models, which could significantly narrow the gap between in vivo and in vitro conditions. Full article

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

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11 pages, 2345 KiB

Open AccessArticle

Integrated Microfluidic Preconcentration and Nucleic Amplification System for Detection of Influenza A Virus H1N1 in Saliva

by Yonghee Kim, Abdurhaman Teyib Abafogi, Buu Minh Tran, Jaewon Kim, Jinyeop Lee, Zhenzhong Chen, Pan Kee Bae, Kyoungsook Park, Yong-Beom Shin, Danny van Noort, Nae Yoon Lee and Sungsu Park

Micromachines 2020, 11(2), 203; https://doi.org/10.3390/mi11020203 - 16 Feb 2020

Cited by 27 | Viewed by 5964

Abstract

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (μFPNAS) which enables both preconcentration of influenza A virus H1N1 [...] Read more.

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (μFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the μFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device. Full article

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

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11 pages, 16931 KiB

Open AccessArticle

A Foldable Chip Array for the Continuous Investigation of Seed Germination and the Subsequent Root Development of Seedlings

by Zhao Xi Song, Hui Hui Chai, Feng Chen, Ling Yu and Can Fang

Micromachines 2019, 10(12), 884; https://doi.org/10.3390/mi10120884 - 17 Dec 2019

Cited by 3 | Viewed by 3652

Abstract

Seed germination and seedling root development are important indicators of plant development. This work designed and fabricated a foldable microfluidic chip array for conducting nondestructive and continuous evaluation of seed germination and subsequent seedling development in situ. Each plant chamber has two functional [...] Read more.

Seed germination and seedling root development are important indicators of plant development. This work designed and fabricated a foldable microfluidic chip array for conducting nondestructive and continuous evaluation of seed germination and subsequent seedling development in situ. Each plant chamber has two functional units: seed germination part and root-growth part. The root-growth parts are themselves connected to a single channel designed to provide a uniform culture medium for plant growth. The individual chips are connected into an array using elastic hinges that facilitate the folding and unfolding of the array to accommodate different viewing purposes. In the folded state, the seed germination chambers form a closely spaced array platform to facilitate the comparison of seed germination and plant development characteristics. Unfolding the array facilitates a clear examination of root development within the root-growth parts. The observation window of an individual chip facilitates either the direct examination of the developing seedling (e.g., stems and leaves) or the use of a microscope for examining microscale features (e.g., root tips and root hairs). The potential of the proposed foldable chip array as a new cultivation platform for botanic studies is demonstrated by examining the seed germination and seedling development of tobacco (Nicotiana tabacum) under different cultivation conditions. Full article

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

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13 pages, 2063 KiB

Open AccessArticle

Determination of the Membrane Transport Properties of Jurkat Cells with a Microfluidic Device

by Tianhang Yang, Ji Peng, Zhiquan Shu, Praveen K. Sekar, Songjing Li and Dayong Gao

Micromachines 2019, 10(12), 832; https://doi.org/10.3390/mi10120832 - 29 Nov 2019

Cited by 20 | Viewed by 3924

Abstract

The Jurkat cell is an immortalized line of human acute lymphocyte leukemia cells that is widely used in the study of adoptive cell therapy, a novel treatment of several advanced forms of cancer. The ability to transport water and solutes across the cell [...] Read more.

The Jurkat cell is an immortalized line of human acute lymphocyte leukemia cells that is widely used in the study of adoptive cell therapy, a novel treatment of several advanced forms of cancer. The ability to transport water and solutes across the cell membrane under different temperatures is an important factor for deciding the specific protocol for cryopreservation of the Jurkat cell. In this study we propose a comprehensive process for determination of membrane transport properties of Jurkat cell. using a novel microfluidic controlled single cell-trapping system. The osmotic behavior of an individual Jurkat cell to water and dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA), under constant temperature, was recorded under a microscope utilizing the modified microfluidic system. The images of the Jurkat cell under osmotic change were processed to obtain a relationship between cell volume change and time. The experimental results were fitted using a two-parameter transport numeric model to calculate the Jurkat cell membrane permeability to water and DMSO at room temperature (22 °C). This model and the calculated parameters can help scientists optimize the cryopreservation protocol for any cell type with optimal cryoprotective agents and cooling rate for future experiments. Full article

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

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Review

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27 pages, 8705 KiB

Open AccessReview

Nanotechnology-Assisted Isolation and Analysis of Circulating Tumor Cells on Microfluidic Devices

by Jie Cheng, Yang Liu, Yang Zhao, Lina Zhang, Lingqian Zhang, Haiyang Mao and Chengjun Huang

Micromachines 2020, 11(8), 774; https://doi.org/10.3390/mi11080774 - 14 Aug 2020

Cited by 33 | Viewed by 6142

Abstract

Circulating tumor cells (CTCs), a type of cancer cell that spreads from primary tumors into human peripheral blood and are considered as a new biomarker of cancer liquid biopsy. It provides the direction for understanding the biology of cancer metastasis and progression. Isolation [...] Read more.

Circulating tumor cells (CTCs), a type of cancer cell that spreads from primary tumors into human peripheral blood and are considered as a new biomarker of cancer liquid biopsy. It provides the direction for understanding the biology of cancer metastasis and progression. Isolation and analysis of CTCs offer the possibility for early cancer detection and dynamic prognosis monitoring. The extremely low quantity and high heterogeneity of CTCs are the major challenges for the application of CTCs in liquid biopsy. There have been significant research endeavors to develop efficient and reliable approaches to CTC isolation and analysis in the past few decades. With the advancement of microfabrication and nanomaterials, a variety of approaches have now emerged for CTC isolation and analysis on microfluidic platforms combined with nanotechnology. These new approaches show advantages in terms of cell capture efficiency, purity, detection sensitivity and specificity. This review focuses on recent progress in the field of nanotechnology-assisted microfluidics for CTC isolation and detection. Firstly, CTC isolation approaches using nanomaterial-based microfluidic devices are summarized and discussed. The different strategies for CTC release from the devices are specifically outlined. In addition, existing nanotechnology-assisted methods for CTC downstream analysis are summarized. Some perspectives are discussed on the challenges of current methods for CTC studies and promising research directions. Full article

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

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