Microfluidic Single-Cell Manipulation and Analysis: Methods and Applications

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.

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

[2]  Hywel Morgan,et al.  Negative DEP traps for single cell immobilisation. , 2009, Lab on a chip.

[3]  Lloyd M Smith,et al.  Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. , 2010, Analytical chemistry.

[4]  Andrew J deMello,et al.  Micro- and nanofluidic systems for high-throughput biological screening. , 2009, Drug discovery today.

[5]  Vincent J Sieben,et al.  An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization. , 2008, Lab on a chip.

[6]  Guangyu Liu,et al.  Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer. , 2016, Small.

[7]  R. Tompkins,et al.  Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes. , 2005, Lab on a chip.

[8]  Michael Wagner,et al.  Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. , 2007, Environmental microbiology.

[9]  Shoji Takeuchi,et al.  A trap-and-release integrated microfluidic system for dynamic microarray applications , 2007, Proceedings of the National Academy of Sciences.

[10]  James R Heath,et al.  Microfluidics-based single-cell functional proteomics for fundamental and applied biomedical applications. , 2014, Annual review of analytical chemistry.

[11]  Zechen Chong,et al.  Single-cell isolation by a modular single-cell pipette for RNA-sequencing. , 2016, Lab on a chip.

[12]  Pak Kin Wong,et al.  Electrokinetic focusing and separation of mammalian cells in conductive biological fluids. , 2012, The Analyst.

[13]  Gabriel P López,et al.  Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. , 2015, Lab on a chip.

[14]  Amy E Herr,et al.  Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response. , 2009, Analytical chemistry.

[15]  B. Yan,et al.  Oscillatory flow beneath a free surface , 1998 .

[16]  Maria Dimaki,et al.  FISHprep: A Novel Integrated Device for Metaphase FISH Sample Preparation , 2011, Micromachines.

[17]  Paul C. Blainey,et al.  A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages , 2016, Nature Communications.

[18]  David Klenerman,et al.  Electrochemical nanoprobes for single-cell analysis. , 2014, ACS nano.

[19]  Hongshen Ma,et al.  Microfluidic micropipette aspiration for measuring the deformability of single cells. , 2012, Lab on a chip.

[20]  Hywel Morgan,et al.  Single-cell microfluidic impedance cytometry: a review , 2010 .

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

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

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

[24]  Jorge S Reis-Filho,et al.  Genetic heterogeneity and cancer drug resistance. , 2012, The Lancet. Oncology.

[25]  William H. Grover,et al.  Measuring single cell mass, volume, and density with dual suspended microchannel resonators. , 2013, Lab on a chip.

[26]  Mingming Wu,et al.  Interstitial flows promote amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model. , 2015, Integrative biology : quantitative biosciences from nano to macro.

[27]  Xing Cheng,et al.  A planar dielectrophoresis-based chip for high-throughput cell pairing. , 2017, Lab on a chip.

[28]  Elinore M Mercer,et al.  Microfluidic sorting of mammalian cells by optical force switching , 2005, Nature Biotechnology.

[29]  Nikolai Dechev,et al.  A novel permalloy based magnetic single cell micro array. , 2009, Lab on a chip.

[30]  Dino Di Carlo,et al.  Hydrodynamic stretching of single cells for large population mechanical phenotyping , 2012, Proceedings of the National Academy of Sciences.

[31]  Dino Di Carlo,et al.  Microtechnology for Cell Manipulation and Sorting. , 2017, Anticancer research.

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

[33]  Pingan Zhu,et al.  Passive and active droplet generation with microfluidics: a review. , 2016, Lab on a chip.

[34]  David A. Weitz,et al.  Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices , 2017, Nature Reviews Genetics.

[35]  David J. Beebe,et al.  Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates , 2017, SLAS technology.

[36]  Tomoyuki Yasukawa,et al.  Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device. , 2008, Analytical chemistry.

[37]  N. Riley.  Acoustic Streaming , 1998 .

[38]  D. Di Carlo.  Inertial microfluidics. , 2009, Lab on a chip.

[39]  Paul C H Li,et al.  Microfluidic Devices for Circulating Tumor Cells Isolation and Subsequent Analysis. , 2016, Current pharmaceutical biotechnology.

[40]  Yong Wang,et al.  Direct measurement of cell protrusion force utilizing a robot-aided cell manipulation system with optical tweezers for cell migration control , 2014, Int. J. Robotics Res..

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

[42]  Doryaneh Ahmadpour,et al.  Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications , 2013, Micromachines.

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

[44]  David J. Collins,et al.  Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves , 2015, Nature Communications.

[45]  Ashwin A. Seshia,et al.  Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device , 2016, Biosensors & bioelectronics.

[46]  Hongyuan Jiang,et al.  Large-Scale Single Particle and Cell Trapping based on Rotating Electric Field Induced-Charge Electroosmosis. , 2016, Analytical chemistry.

[47]  Carmen Bartic,et al.  Local electrical stimulation of single adherent cells using three-dimensional electrode arrays with small interelectrode distances , 2009, 2009 Annual International Conference of the IEEE Engineering in Medicine and Biology Society.

[48]  Fumihito Arai,et al.  High-precision motion of magnetic microrobot with ultrasonic levitation for 3-D rotation of single oocyte , 2016, Int. J. Robotics Res..

[49]  William H. Grover,et al.  Using buoyant mass to measure the growth of single cells , 2010, Nature Methods.

[50]  H. Morgan,et al.  Pumping of liquids with ac voltages applied to asymmetric pairs of microelectrodes. , 2003, Physical review. E, Statistical, nonlinear, and soft matter physics.

[51]  Alois Würger,et al.  Thermal non-equilibrium transport in colloids , 2010 .

[52]  A. Bhagat,et al.  Inertial microfluidics for continuous particle separation in spiral microchannels. , 2009, Lab on a chip.

[53]  Dong Sun,et al.  A simplified sheathless cell separation approach using combined gravitational-sedimentation-based prefocusing and dielectrophoretic separation. , 2018, Lab on a chip.

[54]  Xiaolin Wang,et al.  Automated parallel cell isolation and deposition using microwell array and optical tweezers , 2012, 2012 IEEE International Conference on Robotics and Automation.

[55]  Zhao-Lun Fang,et al.  Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip. , 2004, Lab on a chip.

[56]  Dong Sun,et al.  Transportation of biological cells with robot-tweezer manipulation system , 2011, 2011 IEEE International Conference on Robotics and Automation.

[57]  A. Valero,et al.  Optimization of microfluidic single cell trapping for long-term on-chip culture. , 2010, Lab on a chip.

[58]  Monpichar Srisa-Art,et al.  Microdroplets: a sea of applications? , 2008, Lab on a chip.

[59]  Scott R. Manalis,et al.  Measuring the mass, density, and size of particles and cells using a suspended microchannel resonator , 2007 .

[60]  Amy E Herr,et al.  Single-Cell Western Blotting. , 2015, Methods in molecular biology.

[61]  Wenqi Hu,et al.  Micro-assembly using optically controlled bubble microrobots , 2011 .

[62]  Qiushui Chen,et al.  Biochemical analysis on microfluidic chips , 2016 .

[63]  Donald Wlodkowic,et al.  Microfluidic single-cell array cytometry for the analysis of tumor apoptosis. , 2009, Analytical chemistry.

[64]  Cheng Sun,et al.  Isolating single cells in a neurosphere assay using inertial microfluidics. , 2015, Lab on a chip.

[65]  Siva A. Vanapalli,et al.  Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters , 2017, Scientific Reports.

[66]  Feng Jin,et al.  Recent advances in single cell manipulation and biochemical analysis on microfluidics. , 2019, The Analyst.

[67]  Dong Sun,et al.  Microfluidic single-cell array platform enabling week-scale clonal expansion under chemical/electrical stimuli , 2017 .

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

[69]  R. Amann,et al.  Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques , 2008, Nature Reviews Microbiology.

[70]  M. Kirschner,et al.  Resonant microchannel volume and mass measurements show that suspended cells swell during mitosis , 2015, The Journal of cell biology.

[71]  Wei Liu,et al.  High-throughput analysis of yeast replicative aging using a microfluidic system , 2015, Proceedings of the National Academy of Sciences.

[72]  Roland Zengerle,et al.  Technologies for Single-Cell Isolation , 2015, International journal of molecular sciences.

[73]  Charles S Henry,et al.  Review: Microfluidic applications in metabolomics and metabolic profiling. , 2009, Analytica chimica acta.

[74]  Wei Gao,et al.  Nano/Microscale motors: biomedical opportunities and challenges. , 2012, ACS nano.

[75]  Yi-Hsing Hsiao,et al.  A microfluidic dual-well device for high-throughput single-cell capture and culture. , 2015, Lab on a chip.

[76]  W. Verboom,et al.  Optical sensing systems for microfluidic devices: a review. , 2007, Analytica chimica acta.

[77]  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 .

[78]  K. Woodhouse,et al.  Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories , 2008, Stem cells.

[79]  Allon M. Klein,et al.  Single-cell barcoding and sequencing using droplet microfluidics , 2016, Nature Protocols.

[80]  Barry R Lutz,et al.  Hydrodynamic tweezers: 1. Noncontact trapping of single cells using steady streaming microeddies. , 2006, Analytical chemistry.

[81]  Young-Ho Cho,et al.  A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process , 2005 .

[82]  Jan Wouters,et al.  Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip. , 2012, Lab on a chip.

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

[84]  Dinglong Hu,et al.  Deterministic sequential isolation of floating cancer cells under continuous flow. , 2016, Lab on a chip.

[85]  Rona S. Gertner,et al.  Single cell RNA Seq reveals dynamic paracrine control of cellular variation , 2014, Nature.

[86]  Dong Sun,et al.  Probing cell biophysical behavior based on actin cytoskeleton modeling and stretching manipulation with optical tweezers , 2013 .

[87]  Dong Sun,et al.  Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies. , 2011, Lab on a chip.

[88]  J. Sweedler,et al.  Profiling metabolites and peptides in single cells , 2011, Nature Methods.

[89]  S. Merajver,et al.  Single-cell Migration Chip for Chemotaxis-based Microfluidic Selection of Heterogeneous Cell Populations , 2015, Scientific Reports.

[90]  Yaoyao Zhao,et al.  Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis , 2016, Scientific Reports.

[91]  Andreas Hierlemann,et al.  Characterization of subcellular morphology of single yeast cells using high frequency microfluidic impedance cytometer. , 2014, Lab on a chip.

[92]  Ming C. Wu,et al.  Massively parallel manipulation of single cells and microparticles using optical images , 2005, Nature.

[93]  Benjamin B Yellen,et al.  Magnetic separation of acoustically focused cancer cells from blood for magnetographic templating and analysis. , 2016, Lab on a chip.

[94]  Ling-Sheng Jang,et al.  Electrical characteristics analysis of various cancer cells using a microfluidic device based on single-cell impedance measurement , 2012 .

[95]  J. Voldman,et al.  A scalable addressable positive-dielectrophoretic cell-sorting array. , 2005, Analytical chemistry.

[96]  Wilhelm T S Huck,et al.  A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single-Cell Metabolism in Droplet-Based Microfluidics. , 2016, Angewandte Chemie.

[97]  Noemi Rozlosnik,et al.  Cell-Based Biosensors: Electrical Sensing in Microfluidic Devices , 2012, Diagnostics.

[98]  Saeid Nahavandi,et al.  A self-sufficient micro-droplet generation system using highly porous elastomeric sponges: A versatile tool for conducting cellular assays , 2018, Sensors and Actuators B: Chemical.

[99]  Bifeng Liu,et al.  High-throughput single cell multidrug resistance analysis with multifunctional gradients-customizing microfluidic device , 2016 .

[100]  M. Bennett,et al.  Microfluidic devices for measuring gene network dynamics in single cells , 2009, Nature Reviews Genetics.

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

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

[103]  R Puers,et al.  Digital microfluidics for time-resolved cytotoxicity studies on single non-adherent yeast cells. , 2015, Lab on a chip.

[104]  Jin‐Ming Lin,et al.  Recent advances in microfluidics combined with mass spectrometry: technologies and applications. , 2013, Lab on a chip.

[105]  David A. Weitz,et al.  Single step emulsification for the generation of multi-component double emulsions , 2012 .

[106]  Wenqi Hu,et al.  An opto-thermocapillary cell micromanipulator. , 2013, Lab on a chip.

[107]  Henry K. Chu,et al.  Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device , 2015 .

[108]  Liang Huang,et al.  A fluidic circuit based, high-efficiency and large-scale single cell trap. , 2016, Lab on a chip.

[109]  Fumihito Arai,et al.  On-chip microfluid induced by oscillation of microrobot for noncontact cell transportation , 2017 .

[110]  Fumihito Arai,et al.  On-chip cell sorting by high-speed local-flow control using dual membrane pumps. , 2017, Lab on a chip.

[111]  Aaron R Wheeler,et al.  Electrochemistry, biosensors and microfluidics: a convergence of fields. , 2015, Chemical Society reviews.

[112]  Lianqing Liu,et al.  Programmable micrometer-sized motor array based on live cells. , 2017, Lab on a Chip.

[113]  Lani F. Wu,et al.  Cellular Heterogeneity: Do Differences Make a Difference? , 2010, Cell.

[114]  R. Beroukhim,et al.  Molecular definition of breast tumor heterogeneity. , 2007, Cancer cell.

[115]  Ran Wang,et al.  Development of a magnetic microrobot for carrying and delivering targeted cells , 2018, Science Robotics.

[116]  James M Piret,et al.  Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures. , 2015, Biotechnology journal.

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

[118]  Han Wei Hou,et al.  Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation. , 2011, Lab on a chip.

[119]  F. Arai,et al.  On-chip 3D rotation of oocyte based on a vibration-induced local whirling flow , 2015, Microsystems & Nanoengineering.

[120]  Jongyoon Han,et al.  Characterizing Deformability and Electrical Impedance of Cancer Cells in a Microfluidic Device. , 2018, Analytical chemistry.

[121]  A. Ashkin,et al.  Optical trapping and manipulation of single cells using infrared laser beams , 1987, Nature.

[122]  Xuefeng Yin,et al.  Single-cell analysis by electrochemical detection with a microfluidic device. , 2005, Journal of chromatography. A.

[123]  Kyujung Kim,et al.  Microfluidic assay-based optical measurement techniques for cell analysis: A review of recent progress. , 2016, Biosensors & bioelectronics.

[124]  Joonwon Kim,et al.  Integration of a microfluidic chip with a size-based cell bandpass filter for reliable isolation of single cells. , 2015, Lab on a chip.

[125]  Liang Huang,et al.  3D cell electrorotation and imaging for measuring multiple cellular biophysical properties. , 2018, Lab on a chip.

[126]  Fabian Rudolf,et al.  Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells , 2015, Scientific Reports.

[127]  Peng Zhang,et al.  Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms , 2017, Scientific Reports.