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      Technologies for Single-Cell Isolation

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          Abstract

          The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

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          The impact of next-generation sequencing technology on genetics.

          Elaine R. Mardis (2008)
          If one accepts that the fundamental pursuit of genetics is to determine the genotypes that explain phenotypes, the meteoric increase of DNA sequence information applied toward that pursuit has nowhere to go but up. The recent introduction of instruments capable of producing millions of DNA sequence reads in a single run is rapidly changing the landscape of genetics, providing the ability to answer questions with heretofore unimaginable speed. These technologies will provide an inexpensive, genome-wide sequence readout as an endpoint to applications ranging from chromatin immunoprecipitation, mutation mapping and polymorphism discovery to noncoding RNA discovery. Here I survey next-generation sequencing technologies and consider how they can provide a more complete picture of how the genome shapes the organism.
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            Droplet microfluidic technology for single-cell high-throughput screening.

            Eric Brouzes, Martina Medkova, Neal Savenelli … (2009)
            We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.
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              Label-free cell separation and sorting in microfluidic systems

              Daniel Gossett, Westbrook Weaver, Albert Mach … (2010)
              Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties
              Article 0 comments Cited 228 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Fan-Gang Tseng: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: MDPI
                ISSN (Electronic): 1422-0067
                Publication date (Electronic): 24 July 2015
                Publication date Collection: August 2015
                Volume: 16
                Issue: 8
                Pages: 16897-16919
                Affiliations
                [1 ]Laboratory for MEMS Applications, IMTEK–Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, Freiburg 79110, Germany; E-Mails: andre.gross@ 123456imtek.uni-freiburg.de (A.G.); jonas.schoendube@ 123456imtek.uni-freiburg.de (J.S.); stefan.zimmermann@ 123456imtek.uni-freiburg.de (S.Z.); maximilian.steeb@ 123456outlook.de (M.S.); Roland.Zengerle@ 123456imtek.uni-freiburg.de (R.Z.)
                [2 ]Cytena GmbH, Georges-Koehler-Allee 103, Freiburg 79110, Germany
                [3 ]Hahn-Schickard, Georges-Koehler-Allee 103, Freiburg 79110, Germany
                [4 ]BIOSS–Centre for Biological Signalling Studies, University of Freiburg, Freiburg 79110, Germany
                Author notes
                [†]

                These authors contributed equally to this work.

                [* ]Author to whom correspondence should be addressed; E-Mail: Peter.Koltay@ 123456imtek.uni-freiburg.de ; Tel.: +49-761-2037-3240; Fax: +49-761-2037-3299.
                Article
                Publisher ID: ijms-16-16897
                DOI: 10.3390/ijms160816897
                PMC ID: 4581176
                PubMed ID: 26213926
                SO-VID: 8262aa09-a22e-4df2-8f20-d11ef8dd04f9
                Copyright © © 2015 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 29 May 2015
                Date accepted : 14 July 2015
                Categories
                Subject: Review

                ScienceOpen disciplines: Molecular biology
                Keywords: single-cell analysis,single-cell handling,single-cell separation,single-cell technologies,flow cytometry,laser microdissection,limiting dilution,microfluidics
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: single-cell analysis, single-cell handling, single-cell separation, single-cell technologies, flow cytometry, laser microdissection, limiting dilution, microfluidics

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