Single cells in confined volumes: microchambers and microdroplets.

Microfluidic devices capable of manipulating and guiding small fluid volumes open new methodical approaches in the fields of biology, pharmacy, and medicine. They have already proven their extraordinary value for cell analysis. The emergence of microfluidic platforms has paved the way to novel analytical strategies for the positioning, treatment and observation of living cells, for the creation of chemically defined liquid environments, and for tailoring biomechanical or physical conditions in small volumes. In this article, we particularly focus on two complementary approaches: (i) the isolation of cells in small chambers defined by microchannels and integrated valves and (ii) the encapsulation of cells in microdroplets. We review the advantages and limitations of both approaches and discuss their potential for single-cell analysis and related fields. Our intention is also to give a recommendation on which platform is most appropriate for a new question, i.e., a guideline to choose the most suitable platform.

[1]  Benjamin J Hindson,et al.  On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets. , 2007, Analytical chemistry.

[2]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[3]  Eric Brouzes,et al.  Rapid and continuous magnetic separation in droplet microfluidic devices. , 2015, Lab on a chip.

[4]  Liang Qiao,et al.  On-chip spyhole mass spectrometry for droplet-based microfluidics. , 2014, Angewandte Chemie.

[5]  Timothy K Lee,et al.  Single-cell NF-κB dynamics reveal digital activation and analogue information processing , 2010, Nature.

[6]  Luis F. Olguin,et al.  Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays. , 2009, Analytical chemistry.

[7]  P. Dittrich,et al.  On-chip enzyme quantification of single Escherichia coli bacteria by immunoassay-based analysis. , 2014, Analytical chemistry.

[8]  Helen Song,et al.  A microfluidic system for controlling reaction networks in time. , 2003, Angewandte Chemie.

[9]  Arum Han,et al.  A high-throughput microfluidic single-cell screening platform capable of selective cell extraction. , 2015, Lab on a chip.

[10]  D. Beebe,et al.  Biological implications of polydimethylsiloxane-based microfluidic cell culture. , 2009, Lab on a chip.

[11]  Luke P. Lee,et al.  Dynamic single cell culture array. , 2006, Lab on a chip.

[12]  D. Weitz,et al.  Dropspots: a picoliter array in a microfluidic device. , 2009, Lab on a chip.

[13]  Andrew B. Feldman,et al.  A high-throughput drop microfluidic system for virus culture and analysis. , 2015, Journal of virological methods.

[14]  J. Baret Surfactants in droplet-based microfluidics. , 2012, Lab on a chip.

[15]  G. Whitesides,et al.  Patterning proteins and cells using soft lithography. , 1999, Biomaterials.

[16]  H. Stone,et al.  Formation of dispersions using “flow focusing” in microchannels , 2003 .

[17]  S. Quake,et al.  Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth , 2007, Proceedings of the National Academy of Sciences.

[18]  D. Pang,et al.  A High Throughput Micro-Chamber Array Device for Single Cell Clonal Cultivation and Tumor Heterogeneity Analysis , 2015, Scientific Reports.

[19]  G. Stephanopoulos,et al.  Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption , 2014, Nature Biotechnology.

[20]  Bo Huang,et al.  Counting Low-Copy Number Proteins in a Single Cell , 2007, Science.

[21]  D. Ingber,et al.  Reconstituting Organ-Level Lung Functions on a Chip , 2010, Science.

[22]  Huaying Chen,et al.  High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices. , 2015, Lab on a chip.

[23]  J. Viovy,et al.  Programmable magnetic tweezers and droplet microfluidic device for high-throughput nanoliter multi-step assays. , 2012, Angewandte Chemie.

[24]  Raymond H. W. Lam,et al.  Building a better cell trap: Applying Lagrangian modeling to the design of microfluidic devices for cell biology , 2008 .

[25]  Petra S. Dittrich,et al.  Droplet microfluidics with magnetic beads: a new tool to investigate drug–protein interactions , 2011, Analytical and bioanalytical chemistry.

[26]  Stephen R Quake,et al.  Microfluidic single-cell mRNA isolation and analysis. , 2006, Analytical chemistry.

[27]  U. Sauer,et al.  Real-time metabolome profiling of the metabolic switch between starvation and growth , 2015, Nature Methods.

[28]  M. Figge,et al.  Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. , 2013, Lab on a chip.

[29]  A. Griffiths,et al.  Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms. , 2014, Chemistry and Biology.

[30]  Sara Rigante,et al.  Large-scale arrays of picolitre chambers for single-cell analysis of large cell populations. , 2010, Lab on a chip.

[31]  Petra Schwille,et al.  A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices , 2005, Chembiochem : a European journal of chemical biology.

[32]  Rong Fan,et al.  Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells , 2011, Proceedings of the National Academy of Sciences.

[33]  J. Michael Ramsey,et al.  Microchip flow cytometry using electrokinetic focusing. , 1999, Analytical chemistry.

[34]  Q. Fang,et al.  Analytical detection techniques for droplet microfluidics--a review. , 2013, Analytica chimica acta.

[35]  Alexandra Ros,et al.  Single cell manipulation, analytics, and label‐free protein detection in microfluidic devices for systems nanobiology , 2005, Electrophoresis.

[36]  G M Whitesides,et al.  Biological surface engineering: a simple system for cell pattern formation. , 1999, Biomaterials.

[37]  Numrin Thaitrong,et al.  Integrated microfluidic bioprocessor for single-cell gene expression analysis , 2008, Proceedings of the National Academy of Sciences.

[38]  Chang Lu,et al.  Droplet sorting based on the number of encapsulated particles using a solenoid valve. , 2013, Lab on a chip.

[39]  Andrew D Griffiths,et al.  Selective droplet coalescence using microfluidic systems. , 2012, Lab on a chip.

[40]  Christoph A. Merten,et al.  Efficient cell pairing in droplets using dual-color sorting. , 2015, Lab on a chip.

[41]  Luis F. Olguin,et al.  Simultaneous measurement of reactions in microdroplets filled by concentration gradients. , 2009, Lab on a chip.

[42]  L. Mazutis,et al.  Dynamics of molecular transport by surfactants in emulsions , 2012 .

[43]  R. Ismagilov,et al.  Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics. , 2008, Lab on a chip.

[44]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[45]  Varun Trivedi,et al.  A modular approach for the generation, storage, mixing, and detection of droplet libraries for high throughput screening. , 2010, Lab on a chip.

[46]  Ying Zhu,et al.  Multifunctional picoliter droplet manipulation platform and its application in single cell analysis. , 2011, Analytical chemistry.

[47]  Manuel Théry,et al.  The extracellular matrix guides the orientation of the cell division axis , 2005, Nature Cell Biology.

[48]  Mehmet Toner,et al.  Controlled encapsulation of single-cells into monodisperse picolitre drops. , 2008, Lab on a chip.

[49]  Mart Min,et al.  Contactless sensing of the conductivity of aqueous droplets in segmented flow , 2011 .

[50]  Yanwei Jia,et al.  Control and measurement of the phase behavior of aqueous solutions using microfluidics. , 2007, Journal of the American Chemical Society.

[51]  A. Abate,et al.  Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops. , 2013, Analytical chemistry.

[52]  Thomas Henkel,et al.  Generation of larger numbers of separated microbial populations by cultivation in segmented-flow microdevices. , 2003, Lab on a chip.

[53]  N. Neff,et al.  Quantitative assessment of single-cell RNA-sequencing methods , 2013, Nature Methods.

[54]  D. Chiu,et al.  Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets. , 2005, Analytical chemistry.

[55]  Jianhong Xu,et al.  Correlations of droplet formation in T-junction microfluidic devices: from squeezing to dripping , 2008 .

[56]  Stephen R Quake,et al.  A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination. , 2006, Journal of the American Chemical Society.

[57]  Kenji Yasuda,et al.  On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells. , 2003, Biochemical and biophysical research communications.

[58]  Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay. , 2014, The Analyst.

[59]  Xin Li,et al.  Sensitive, high throughput detection of proteins in individual, surfactant-stabilized picoliter droplets using nanoelectrospray ionization mass spectrometry. , 2013, Analytical chemistry.

[60]  Rong Fan,et al.  A Clinical Microchip for Evaluation of Single Immune Cells Reveals High Functional Heterogeneity in Phenotypically Similar T Cells Nih Public Access Author Manuscript Design Rationale and Detection Limit of the Scbc Online Methods Microchip Fabrication On-chip Secretion Profiling Supplementary Mater , 2022 .

[61]  Burak Dura,et al.  Profiling lymphocyte interactions at the single-cell level by microfluidic cell pairing , 2015, Nature Communications.

[62]  Godfrey L. Smith,et al.  Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform. , 2006, Lab on a chip.

[63]  Jean-Louis Viovy,et al.  Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells , 2008, Proceedings of the National Academy of Sciences.

[64]  Wolfgang Wiechert,et al.  Microfluidic growth chambers with optical tweezers for full spatial single-cell control and analysis of evolving microbes. , 2013, Journal of microbiological methods.

[65]  A. Griffiths,et al.  Reliable microfluidic on-chip incubation of droplets in delay-lines. , 2009, Lab on a chip.

[66]  Dan Bratton,et al.  Static microdroplet arrays: a microfluidic device for droplet trapping, incubation and release for enzymatic and cell-based assays. , 2009, Lab on a chip.

[67]  Klaus Eyer,et al.  A microchamber array for single cell isolation and analysis of intracellular biomolecules. , 2012, Lab on a chip.

[68]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[69]  Minh Do-Quang,et al.  Droplet dynamics in a bifurcating channel , 2010 .

[70]  P. Dittrich,et al.  Confined synthesis and integration of functional materials in sub-nanoliter volumes. , 2013, ACS nano.

[71]  Allon M. Klein,et al.  Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells , 2015, Cell.

[72]  David J Beebe,et al.  From the cellular perspective: exploring differences in the cellular baseline in macroscale and microfluidic cultures. , 2009, Integrative biology : quantitative biosciences from nano to macro.

[73]  Wilhelm T S Huck,et al.  Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics. , 2013, Lab on a chip.

[74]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[75]  G. Fuhr,et al.  Three-dimensional electric field traps for manipulation of cells--calculation and experimental verification. , 1993, Biochimica et biophysica acta.

[76]  A. Folch,et al.  Large-scale single-cell trapping and imaging using microwell arrays. , 2005, Analytical chemistry.

[77]  Michele Zagnoni,et al.  On-chip electrocoalescence of microdroplets as a function of voltage, frequency and droplet size. , 2009, Lab on a chip.

[78]  G. Whitesides,et al.  Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). , 1998, Analytical chemistry.

[79]  G. Whitesides,et al.  Using an elastomeric phase mask for sub-100 nm photolithography in the optical near field , 1997 .

[80]  A. Griffiths,et al.  Microfluidic Approaches for the Study of Emulsions: Transport of Solutes , 2013 .

[81]  Klaus Eyer,et al.  A new mechanobiological era: microfluidic pathways to apply and sense forces at the cellular level. , 2012, Current opinion in chemical biology.

[82]  Niels W. Hanson,et al.  A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities , 2012, Proceedings of the National Academy of Sciences.

[83]  S. Quake,et al.  A microfabricated fluorescence-activated cell sorter , 1999, Nature Biotechnology.

[84]  S. Anna,et al.  Microfluidic methods for generating continuous droplet streams , 2007 .

[85]  Ismail Emre Araci,et al.  Microfluidic very large scale integration (mVLSI) with integrated micromechanical valves. , 2012, Lab on a chip.

[86]  Ryan A. Kellogg,et al.  High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics , 2014, Nature Protocols.

[87]  K. Jefimovs,et al.  High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis. , 2014, Analytical chemistry.

[88]  Albert Folch,et al.  Microfluidic devices with tunable microtopographies , 2005 .

[89]  A. Abate,et al.  High-throughput injection with microfluidics using picoinjectors , 2010, Proceedings of the National Academy of Sciences.

[90]  D. Moras,et al.  Quantitative cell-based reporter gene assays using droplet-based microfluidics. , 2010, Chemistry & biology.

[91]  F. Hollfelder,et al.  A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets. , 2012, Lab on a chip.

[92]  C. Kim,et al.  An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS. , 2006, Lab on a chip.

[93]  D. Weitz,et al.  Electric control of droplets in microfluidic devices. , 2006, Angewandte Chemie.

[94]  R. Jaenisch,et al.  Microfluidic Control of Cell Pairing and Fusion , 2009, Nature Methods.

[95]  K. Jefimovs,et al.  High-density micro-arrays for mass spectrometry. , 2010, Lab on a chip.

[96]  Euisik Yoon,et al.  Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal. , 2014, Lab on a chip.

[97]  Nitish V Thakor,et al.  Single cell kinase signaling assay using pinched flow coupled droplet microfluidics. , 2014, Biomicrofluidics.

[98]  Todd P. Lagus,et al.  High-throughput co-encapsulation of self-ordered cell trains: cell pair interactions in microdroplets , 2013 .

[99]  Dan S. Tawfik,et al.  High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. , 2005, Chemistry & biology.

[100]  Aaron R Wheeler,et al.  Electrowetting-based microfluidics for analysis of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry. , 2004, Analytical chemistry.

[101]  J. Nielsen,et al.  High-throughput screening for industrial enzyme production hosts by droplet microfluidics. , 2014, Lab on a chip.

[102]  Martin Fischlechner,et al.  One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution , 2014, Analytical chemistry.

[103]  N. Friedman,et al.  Stochastic protein expression in individual cells at the single molecule level , 2006, Nature.

[104]  A. Abate,et al.  On-chip background noise reduction for cell-based assays in droplets. , 2011, Lab on a chip.

[105]  I. Shmulevich,et al.  Dynamic analysis of MAPK signaling using a high-throughput microfluidic single-cell imaging platform , 2009, Proceedings of the National Academy of Sciences.

[106]  K. Griswold,et al.  A high‐throughput screen for antibiotic drug discovery , 2014, Biotechnology and bioengineering.

[107]  G. Whitesides,et al.  Fabrication of microfluidic systems in poly(dimethylsiloxane) , 2000, Electrophoresis.

[108]  Daniel T Chiu,et al.  Microfluidic and optical systems for the on-demand generation and manipulation of single femtoliter-volume aqueous droplets. , 2006, Analytical chemistry.

[109]  D. Kent,et al.  High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays , 2011, Nature Methods.

[110]  Christoph A. Merten,et al.  Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms. , 2008, Chemistry & biology.

[111]  A. Griffiths,et al.  New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications. , 2011, Analytical Chemistry.

[112]  P S Dittrich,et al.  Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells. , 2013, Analytical chemistry.

[113]  Richard Novak,et al.  High-performance single cell genetic analysis using microfluidic emulsion generator arrays. , 2010, Analytical chemistry.

[114]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[115]  Haakan N Joensson,et al.  Droplet size based separation by deterministic lateral displacement-separating droplets by cell--induced shrinking. , 2011, Lab on a chip.

[116]  Russell H. Cole,et al.  Compact and modular multicolour fluorescence detector for droplet microfluidics. , 2015, Lab on a chip.

[117]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[118]  Samuel Aparicio,et al.  High-throughput microfluidic single-cell RT-qPCR , 2011, Proceedings of the National Academy of Sciences.

[119]  Aaron M. Streets,et al.  Microfluidic single-cell whole-transcriptome sequencing , 2014, Proceedings of the National Academy of Sciences.

[120]  Stephen R. Quake,et al.  Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm , 2012, Cell.

[121]  Helen Song,et al.  Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants. , 2005, Analytical chemistry.

[122]  Chang Lu,et al.  Chemical transfection of cells in picoliter aqueous droplets in fluorocarbon oil. , 2011, Analytical chemistry.

[123]  Mehmet Toner,et al.  Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device. , 2004, Analytical chemistry.

[124]  Jessica Melin,et al.  Microfluidic large-scale integration: the evolution of design rules for biological automation. , 2007, Annual review of biophysics and biomolecular structure.

[125]  Helen Song,et al.  Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels. , 2003, Applied physics letters.

[126]  J. M. Watson,et al.  The behaviour of water in poly(dimethylsiloxane) , 1996 .

[127]  Adam Sciambi,et al.  Accurate microfluidic sorting of droplets at 30 kHz. , 2015, Lab on a chip.

[128]  Daniel Bratton,et al.  The electrochemical detection of droplets in microfluidic devices. , 2008, Lab on a chip.

[129]  Colin J Ingham,et al.  The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms , 2007, Proceedings of the National Academy of Sciences.

[130]  N. Munce,et al.  Microfabricated system for parallel single-cell capillary electrophoresis. , 2004, Analytical chemistry.

[131]  Anupam Singhal,et al.  Megapixel digital PCR , 2011, Nature Methods.

[132]  David K. Wood,et al.  Single cell trapping and DNA damage analysis using microwell arrays , 2010, Proceedings of the National Academy of Sciences.

[133]  S. Quake,et al.  Dynamic pattern formation in a vesicle-generating microfluidic device. , 2001, Physical review letters.

[134]  David A Weitz,et al.  DNA sequence analysis with droplet-based microfluidics. , 2013, Lab on a chip.

[135]  D. Weitz,et al.  Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter. , 2014, Lab on a chip.

[136]  Jean-Pierre Delville,et al.  An optical toolbox for total control of droplet microfluidics. , 2007, Lab on a chip.

[137]  Microfluidic platform for electrophysiological studies on Xenopus laevis oocytes under varying gravity levels. , 2011, Lab on a chip.

[138]  Chaitanya P. Pabbati,et al.  Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis. , 2006, Analytical chemistry.

[139]  A. Abate,et al.  Identification and genetic analysis of cancer cells with PCR-activated cell sorting , 2014, Nucleic acids research.

[140]  Konstantins Jefimovs,et al.  Mass spectrometry-based metabolomics of single yeast cells , 2013, Proceedings of the National Academy of Sciences.

[141]  Effects of ultrasmall orifices on the electrogeneration of femtoliter-volume aqueous droplets. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[142]  R. Zare,et al.  Chemical cytometry on a picoliter-scale integrated microfluidic chip. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[143]  P. E. Verboket,et al.  A New Microfluidics-Based Droplet Dispenser for ICPMS , 2014, Analytical chemistry.

[144]  A. Franco-Obregón,et al.  Transient receptor potential vanilloid 2‐mediated shear‐stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[145]  A. deMello,et al.  The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. , 2015, Lab on a chip.

[146]  Luke P. Lee,et al.  Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays. , 2006, Analytical chemistry.

[147]  F. Hollfelder,et al.  Interfacing microwells with nanoliter compartments: a sampler generating high-resolution concentration gradients for quantitative biochemical analyses in droplets. , 2015, Analytical chemistry.

[148]  Hiroyuki Moriguchi,et al.  An agar-microchamber cell-cultivation system: flexible change of microchamber shapes during cultivation by photo-thermal etching. , 2002, Lab on a chip.

[149]  Wei-Hua Huang,et al.  Transport, location, and quantal release monitoring of single cells on a microfluidic device. , 2004, Analytical chemistry.

[150]  Hongsheng Song,et al.  A droplet-based microfluidic electrochemical sensor using platinum-black microelectrode and its application in high sensitive glucose sensing. , 2014, Biosensors & bioelectronics.

[151]  Nancy Allbritton,et al.  Micromolded arrays for separation of adherent cells. , 2010, Lab on a chip.