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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (20 October 2018) | Viewed by 63881

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Special Issue Editors

Dr. Tuhin Subhra Santra

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Guest Editor

Department of Engineering Design, Indian Institute of Technology Madras, Chennai, India
Interests: MEMS; Bio-NEMS; single- cell technology; biomedical micro/nano devices; micro/nanofluidics; nanomedicine
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Fan-Gang Tseng

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Guest Editor

Distinguished Professor, Department of Engineering and System Science, National Tsing Hua University (NTHU), Affiliated Research Fellow, Academia Sinica, Hsinchu, Taiwan
Interests: organ on a chip; microfluidic systems; biosensors; CTCs/CTM diagnosis; single cell analysis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The cell is the most fundamental building block in our body and it is presently a great challenge in cell biology to understand cell-to-cell responses, and how an individual cell delivers its information to others. The average ensemble measurement of cells cannot provide information regarding the detailed behaviors of any individual cell; however, each cell in heterogeneous populations has its own unique behavior and it has different responses, even after treatment with the same reagents or drugs as other cells. On the other hand, single cell sequencing (SCS) is able to empirically infer driver mutations and to map sequential mutation events during cancer development. The integration of single-cell genomics and transcriptomics is able to provide functional consequences of mutations and the copy number variations of cells. Thus, single cell technology (SCT) has the power to understand fundamental cell biology in embryonic development, detailed knowledge of cell lineage trees in higher organisms, to dissect tumor heterogeneity and disease, etc. Recently, the development of micro/nanofluidic technologies have enabled the characterization of a single cell in the micro/nanoscale environments with a massively parallel platform, which can distinguish cell-to-cell heterogeneity via tracking responses over time. The devices are, not only useful for cell manipulation, isolation, separation, and analysis, but are also able to control cellular parameters at the single cell level.

This Special Issue will invite the latest research articles and reviews dealing with SCT, and the role of SCT in omics. Applications and future prospects, with their advantages and limitations, are also welcome.

Dr. Tuhin Subhra Santra
Prof. Dr. Fan-Gang Tseng
Guest Editors

Manuscript Submission Information

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Keywords

Single cells interaction, perturbation, cultivation, cellular heterogeneity
single cell in system biology
single cell cytometry
micro/nano fluidic devices for single cell analysis
single cell manipulation, separation, detection
single cell genomics, proteomics, transcriptomics, metabolomics and fluxomics
single cell diagnostics and imaging

Related Special Issue

Single Cell Analysis in Biotechnology and Systems Biology in International Journal of Molecular Sciences (10 articles)

Published Papers (10 papers)

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Research

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11 pages, 3169 KiB

Open AccessArticle

Combining TIR and FRET in Molecular Test Systems

by Herbert Schneckenburger, Petra Weber, Michael Wagner, Sandra Enderle, Bernd Kalthof, Linn Schneider, Claudia Herzog, Julian Weghuber and Peter Lanzerstorfer

Int. J. Mol. Sci. 2019, 20(3), 648; https://doi.org/10.3390/ijms20030648 - 02 Feb 2019

Cited by 6 | Viewed by 3748

Abstract

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples. Full article

(This article belongs to the Special Issue Single Cell Technology)

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13 pages, 3469 KiB

Open AccessArticle

Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

by Kiki C. Andree, Fikri Abali, Lisa Oomens, Fiona R. Passanha, Joska J. Broekmaat, Jaco Kraan, Pauline A.J. Mendelaar, Stefan Sleijfer and Leon W.M.M. Terstappen

Int. J. Mol. Sci. 2019, 20(3), 477; https://doi.org/10.3390/ijms20030477 - 23 Jan 2019

Cited by 10 | Viewed by 4140

Abstract

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, α-EpCAM+, α-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0–5% as compared with CTC counts obtained with the CellSearch^® system. Viability of the detected CTC ranged from 0–36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses. Full article

(This article belongs to the Special Issue Single Cell Technology)

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11 pages, 3784 KiB

Open AccessCommunication

Noradrenaline Sensitivity Is Severely Impaired in Immortalized Adipose-Derived Mesenchymal Stem Cell Line

by Pyotr A. Tyurin-Kuzmin, Vadim I. Chechekhin, Anastasiya M. Ivanova, Daniyar T. Dyikanov, Veronika Y. Sysoeva, Natalia I. Kalinina and Vsevolod A. Tkachuk

Int. J. Mol. Sci. 2018, 19(12), 3712; https://doi.org/10.3390/ijms19123712 - 22 Nov 2018

Cited by 7 | Viewed by 3452

Abstract

Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called “heterologous sensitization”, and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca²⁺-mobilization in single cells, we found that these cells did not demonstrate the sensitization seen in primary adMSCs. Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology. Full article

(This article belongs to the Special Issue Single Cell Technology)

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11 pages, 1730 KiB

Open AccessArticle

Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons

by Daniel Andersson, David Svec, Cathrine Pedersen, Jørn Remi Henriksen and Anders Ståhlberg

Int. J. Mol. Sci. 2018, 19(10), 3185; https://doi.org/10.3390/ijms19103185 - 16 Oct 2018

Cited by 5 | Viewed by 5792

Abstract

Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification. Full article

(This article belongs to the Special Issue Single Cell Technology)

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10 pages, 6853 KiB

Open AccessCommunication

Localization Microscopy of Actin Cytoskeleton in Human Platelets

by Sandra Mayr, Fabian Hauser, Anja Peterbauer, Andreas Tauscher, Christoph Naderer, Markus Axmann, Birgit Plochberger and Jaroslaw Jacak

Int. J. Mol. Sci. 2018, 19(4), 1150; https://doi.org/10.3390/ijms19041150 - 11 Apr 2018

Cited by 16 | Viewed by 6468

Abstract

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm. Full article

(This article belongs to the Special Issue Single Cell Technology)

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2568 KiB

Open AccessArticle

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAM^high and EpCAM^low/negativeCTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer

by Rita Lampignano, Liwen Yang, Martin H. D. Neumann, André Franken, Tanja Fehm, Dieter Niederacher and Hans Neubauer

Int. J. Mol. Sci. 2017, 18(9), 1885; https://doi.org/10.3390/ijms18091885 - 31 Aug 2017

Cited by 37 | Viewed by 6442

Abstract

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAM^high and EpCAM^low/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch^® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients′ blood samples both EpCAM^high and EpCAM^low/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAM^low/negative cells vs. 28% of EpCAM^high cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAM^high and EpCAM^low/negative CTCs. Our workflow is suitable for single CTC analysis, permitting—for the first time—assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAM^high and EpCAM^low/negative CTCs. Full article

(This article belongs to the Special Issue Single Cell Technology)

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1826 KiB

Open AccessArticle

The Instrumentation of a Microfluidic Analyzer Enabling the Characterization of the Specific Membrane Capacitance, Cytoplasm Conductivity, and Instantaneous Young’s Modulus of Single Cells

by Ke Wang, Yang Zhao, Deyong Chen, Chengjun Huang, Beiyuan Fan, Rong Long, Chia-Hsun Hsieh, Junbo Wang, Min-Hsien Wu and Jian Chen

Int. J. Mol. Sci. 2017, 18(6), 1158; https://doi.org/10.3390/ijms18061158 - 19 Jun 2017

Cited by 4 | Viewed by 4201

Abstract

This paper presents the instrumentation of a microfluidic analyzer enabling the characterization of single-cell biophysical properties, which includes seven key components: a microfluidic module, a pressure module, an imaging module, an impedance module, two LabVIEW platforms for instrument operation and raw data processing, respectively, and a Python code for data translation. Under the control of the LabVIEW platform for instrument operation, the pressure module flushes single cells into the microfluidic module with raw biophysical parameters sampled by the imaging and impedance modules and processed by the LabVIEW platform for raw data processing, which were further translated into intrinsic cellular biophysical parameters using the code developed in Python. Based on this system, specific membrane capacitance, cytoplasm conductivity, and instantaneous Young’s modulus of three cell types were quantified as 2.76 ± 0.57 μF/cm², 1.00 ± 0.14 S/m, and 3.79 ± 1.11 kPa for A549 cells (n_cell = 202); 1.88 ± 0.31 μF/cm², 1.05 ± 0.16 S/m, and 3.74 ± 0.75 kPa for 95D cells (n_cell = 257); 2.11 ± 0.38 μF/cm², 0.87 ± 0.11 S/m, and 5.39 ± 0.89 kPa for H460 cells (n_cell = 246). As a semi-automatic instrument with a throughput of roughly 1 cell per second, this prototype instrument can be potentially used for the characterization of cellular biophysical properties. Full article

(This article belongs to the Special Issue Single Cell Technology)

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Review

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47 pages, 12682 KiB

Open AccessReview

Current Trends of Microfluidic Single-Cell Technologies

by Pallavi Shinde, Loganathan Mohan, Amogh Kumar, Koyel Dey, Anjali Maddi, Alexander N. Patananan, Fan-Gang Tseng, Hwan-You Chang, Moeto Nagai and Tuhin Subhra Santra

Int. J. Mol. Sci. 2018, 19(10), 3143; https://doi.org/10.3390/ijms19103143 - 12 Oct 2018

Cited by 61 | Viewed by 8476

Abstract

The investigation of human disease mechanisms is difficult due to the heterogeneity in gene expression and the physiological state of cells in a given population. In comparison to bulk cell measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. In this review, we describe the recent advances in single-cell technologies and their applications in single-cell manipulation, diagnosis, and therapeutics development. Full article

(This article belongs to the Special Issue Single Cell Technology)

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20 pages, 513 KiB

Open AccessReview

Platforms for Single-Cell Collection and Analysis

by Lukas Valihrach, Peter Androvic and Mikael Kubista

Int. J. Mol. Sci. 2018, 19(3), 807; https://doi.org/10.3390/ijms19030807 - 11 Mar 2018

Cited by 132 | Viewed by 14359

Abstract

Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments. Full article

(This article belongs to the Special Issue Single Cell Technology)

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2359 KiB

Open AccessReview

Recent Advances in Experimental Whole Genome Haplotyping Methods

by Mengting Huang, Jing Tu and Zuhong Lu

Int. J. Mol. Sci. 2017, 18(9), 1944; https://doi.org/10.3390/ijms18091944 - 11 Sep 2017

Cited by 12 | Viewed by 5670

Abstract

Haplotype plays a vital role in diverse fields; however, the sequencing technologies cannot resolve haplotype directly. Pioneers demonstrated several approaches to resolve haplotype in the early years, which was extensively reviewed. Since then, numerous methods have been developed recently that have significantly improved phasing performance. Here, we review experimental methods that have emerged mainly over the past five years, and categorize them into five classes according to their maximum scale of contiguity: (i) encapsulation, (ii) 3D structure capture and construction, (iii) compartmentalization, (iv) fluorography, (v) long-read sequencing. Several subsections of certain methods are attached to each class as instances. We also discuss the relative advantages and disadvantages of different classes and make comparisons among representative methods of each class. Full article

(This article belongs to the Special Issue Single Cell Technology)

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