Advertisement

Introduction: Why Analyze Single Cells?

  • Dino Di Carlo
  • Henry Tat Kwong Tse
  • Daniel R. Gossett
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 853)

Abstract

Powerful methods in molecular biology are abundant; however, in many fields including hematology, stem cell biology, tissue engineering, and cancer biology, data from tools and assays that analyze the average signals from many cells may not yield the desired result because the cells of interest may be in the minority—their behavior masked by the majority—or because the dynamics of the populations of interest are offset in time. Accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. In this chapter, we discuss the rationale for performing analyses on individual cells in more depth, cover the fields of study in which single-cell behavior is yielding new insights into biological and clinical questions, and speculate on how single-cell analysis will be critical in the future.

Key words

Single-cell analysis Microfluidics Biomicrofluidics Cellular heterogeneity 

References

  1. 1.
    Elowitz MB, Levine AJ, Siggia ED & Swain PS (2002) Stochastic Gene Expression in a Single Cell. Science 297, 1183–1186.PubMedCrossRefGoogle Scholar
  2. 2.
    Miller MJ, Safrina O, Parker I & Cahalan MD (2004) Imaging the Single Cell Dynamics of CD4+ T Cell Activation by Dendritic Cells in Lymph Nodes. The Journal of Experimental Medicine 200, 847–856.PubMedCrossRefGoogle Scholar
  3. 3.
    Fiering S, Northrop JP, Nolan GP, Mattila PS, Crabtree GR & Herzenberg LA (1990) Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes & Development 4, 1823–1834.CrossRefGoogle Scholar
  4. 4.
    Levsky JM, Shenoy SM, Pezo RC & Singer RH (2002) Single-Cell Gene Expression Profiling. Science 297, 836–840.PubMedCrossRefGoogle Scholar
  5. 5.
    Dexter DL, Kowalski HM, Blazar BA, Fligiel Z, Vogel R & Heppner GH (1978) Heterogeneity of Tumor Cells from a Single Mouse Mammary Tumor. Cancer Research 38, 3174–3181.PubMedGoogle Scholar
  6. 6.
    Dexter DL, Spremulli EN, Fligiel Z, Barbosa JA, Vogel R, VanVoorhees A & Calabresi P (1981) Heterogeneity of cancer cells from a single human colon carcinoma. Am. J. Med 71, 949–956.PubMedCrossRefGoogle Scholar
  7. 7.
    Vermeulen L, Sprick MR, Kemper K, Stassi G & Medema JP (2008) Cancer stem cells - old concepts, new insights. Cell Death Differ 15, 947–958.PubMedCrossRefGoogle Scholar
  8. 8.
    Navin N, Krasnitz A, Rodgers L, Cook K, Meth J, Kendall J, Riggs M, Eberling Y, Troge J, Grubor V, Levy D, Lundin P, Månér S, Zetterberg A, Hicks J & Wigler M (2010) Inferring tumor progression from genomic heterogeneity. Genome Research 20, 68–80.PubMedCrossRefGoogle Scholar
  9. 9.
    Orth JD, Tang Y, Shi J, Loy CT, Amendt C, Wilm C, Zenke FT & Mitchison TJ (2008) Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate. Molecular Cancer Therapeutics 7, 3480–3489.PubMedCrossRefGoogle Scholar
  10. 10.
    Cohen AA, Geva-Zatorsky N, Eden E, Frenkel-Morgenstern M, Issaeva I, Sigal A, Milo R, Cohen-Saidon C, Liron Y, Kam Z, Cohen L, Danon T, Perzov N & Alon U (2008) Dynamic Proteomics of Individual Cancer Cells in Response to a Drug. Science 322, 1511–1516.PubMedCrossRefGoogle Scholar
  11. 11.
    Zhong JF, Chen Y, Marcus JS, Scherer A, Quake SR, Taylor CR & Weiner LP (2008) A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab Chip 8, 68.PubMedCrossRefGoogle Scholar
  12. 12.
    Kim L, Vahey MD, Lee H & Voldman J (2006) Microfluidic arrays for logarithmically perfused embryonic stem cell culture. Lab Chip 6, 394–406.PubMedCrossRefGoogle Scholar
  13. 13.
    Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Perez Alea M, Richel DJ, Stassi G & Medema JP (2008) Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proceedings of the National Academy of Sciences 105, 13427–13432.CrossRefGoogle Scholar
  14. 14.
    Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES & Jeon NL (2005) Human neural stem cell growth and differentiation in a gradient-generating microfluidic device. Lab Chip 5, 401.PubMedCrossRefGoogle Scholar
  15. 15.
    Weaver WM, Dharmaraja S, Milisavljevic V & Di Carlo D (2011) The effects of shear stress on isolated receptor–ligand interactions of Staphylococcus epidermidis and human plasma fibrinogen using molecularly patterned microfluidics. Lab Chip 11, 883.PubMedCrossRefGoogle Scholar
  16. 16.
    Di Carlo D, Aghdam N & Lee LP (2006) Single-Cell Enzyme Concentrations, Kinetics, and Inhibition Analysis Using High-Density Hydrodynamic Cell Isolation Arrays. Analytical Chemistry 78, 4925–4930.PubMedCrossRefGoogle Scholar
  17. 17.
    Di Carlo, D & Lee LP (2006) Dynamic Single-Cell Analysis for Quantitative Biology. Analytical Chemistry 78, 7918–7925.PubMedCrossRefGoogle Scholar
  18. 18.
    Gossett DR, Weaver WM, Ahmed NS & Di Carlo D (2010) Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel. Ann Biomed Eng 39, 1328–1334.Google Scholar
  19. 19.
    Wright D, Rajalingam B, Selvarasah S, Dokmeci MR & Khademhosseini A (2007) Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils. Lab Chip 7, 1272–1279.PubMedCrossRefGoogle Scholar
  20. 20.
    Chung S, Sudo R, Mack PJ, Wan C, Vickerman V & Kamm RD (2009) Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab Chip 9, 269–275.PubMedCrossRefGoogle Scholar
  21. 21.
    Ochsner M, Dusseiller MR, Grandin HM, Luna-Morris S, Textor M, Vogel V & Smith ML (2007) Micro-well arrays for 3D shape control and high resolution analysis of single cells. Lab Chip 7, 1074.PubMedCrossRefGoogle Scholar
  22. 22.
    Hui EE & Bhatia SN (2007) Micromechanical control of cell–cell interactions. Proceedings of the National Academy of Sciences 104, 5722–5726.CrossRefGoogle Scholar
  23. 23.
    Toh Y, Ng S, Khong YM, Samper V & Yu H (2005) A configurable three-dimensional micro­environment in a microfluidic channel for primary hepatocyte culture. Assay Drug Dev Technol 3, 169–176.PubMedCrossRefGoogle Scholar
  24. 24.
    Zhang MY, Lee PJ, Hung PJ, Johnson T, Lee LP & Mofrad MRK (2007) Microfluidic environment for high density hepatocyte culture. Biomed Microdevices 10, 117–121.CrossRefGoogle Scholar
  25. 25.
    Chung S, Sudo R, Mack PJ, Wan C, Vickerman V & Kamm RD (2009) Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab Chip 9, 269.PubMedCrossRefGoogle Scholar
  26. 26.
    Rettig JR & Folch A (2005) Large-Scale Single-Cell Trapping And Imaging Using Microwell Arrays. Analytical Chemistry 77, 5628–5634.PubMedCrossRefGoogle Scholar
  27. 27.
    Di Carlo D, Edd JF, Irimia D, Tompkins RG & Toner M (2008) Equilibrium separation and filtration of particles using differential inertial focusing. Anal. Chem 80, 2204–2211.PubMedCrossRefGoogle Scholar
  28. 28.
    Wang X, Yang J, Huang Y, Vykoukal J, Becker FF & Gascoyne PRC (2000) Cell Separation by Dielectrophoretic Field-flow-fractionation. Analytical Chemistry 72, 832–839.PubMedCrossRefGoogle Scholar
  29. 29.
    Evander M, Johansson L, Lilliehorn T, Piskur J, Lindvall M, Johansson S, Almqvist M, Laurell T & Nilsson J (2007) Noninvasive Acoustic Cell Trapping in a Microfluidic Perfusion System for Online Bioassays. Analytical Chemistry 79, 2984–2991.PubMedCrossRefGoogle Scholar
  30. 30.
    Choi J, Oh KW, Thomas JH, Heineman WR, Halsall HB, Nevin JH, Helmicki AJ, Henderson HT & Ahn CH (2002) An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities. Lab Chip 2, 27.PubMedCrossRefGoogle Scholar
  31. 31.
    Lu H, Koo LY, Wang WM, Lauffenburger DA, Griffith LG & Jensen KF (2004) Microfluidic Shear Devices for Quantitative Analysis of Cell Adhesion. Analytical Chemistry 76, 5257–5264.PubMedCrossRefGoogle Scholar
  32. 32.
    Khandurina J, McKnight TE, Jacobson SC, Waters LC, Foote RS & Ramsey JM (2000) Integrated System for Rapid PCR-Based DNA Analysis in Microfluidic Devices. Analytical Chemistry 72, 2995–3000.PubMedCrossRefGoogle Scholar
  33. 33.
    Sato K, Yamanaka M, Takahashi H, Tokeshi M, Kimura H & Kitamori T (2002) Microchip-based immunoassay system with branching multichannels for simultaneous determination of interferon-gamma. Electrophoresis 23, 734–739.PubMedCrossRefGoogle Scholar
  34. 34.
    Sato K, Tokeshi M, Odake T, Kimura H, Ooi T, Nakao M & Kitamori T (2000) Integration of an immunosorbent assay system: analysis of secretory human immunoglobulin A on polystyrene beads in a microchip. Anal. Chem 72, 1144–1147.PubMedCrossRefGoogle Scholar
  35. 35.
    Zare RN & Kim S (2010) Microfluidic platforms for single-cell analysis. Annu Rev Biomed Eng 12, 187–201.PubMedCrossRefGoogle Scholar
  36. 36.
    Jin A, Ozawa T, Tajiri K, Obata T, Kondo S, Kinoshita K, Kadowaki S, Takahashi K, Sugiyama T, Kishi H & Muraguchi A (2009) A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 15, 1088–1092.PubMedCrossRefGoogle Scholar
  37. 37.
    Yamamura S, Kishi H, Tokimitsu Y, Kondo S, Honda R, Rao SR, Omori M, Tamiya E & Muraguchi A (2005) Single-Cell Microarray for Analyzing Cellular Response. Analytical Chemistry 77, 8050–8056.PubMedCrossRefGoogle Scholar
  38. 38.
    Lindström S, Larsson R & Andersson Svahn H (2008) Towards high-throughput single cell/clone cultivation and analysis. Electrophoresis 29, 1219–1227.PubMedCrossRefGoogle Scholar
  39. 39.
    Lindström S, Hammond M, Brismar H, Andersson-Svahn H & Ahmadian A (2009) PCR amplification and genetic analysis in a microwell cell culturing chip. Lab Chip 9, 3465.PubMedCrossRefGoogle Scholar
  40. 40.
    Vanherberghen B, Manneberg O, Christakou A, Frisk T, Ohlin M, Hertz HM, Önfelt B & Wiklund M (2010) Ultrasound-controlled cell aggregation in a multi-well chip. Lab Chip 10, 2727.PubMedCrossRefGoogle Scholar
  41. 41.
    Shapiro HM (2003) Practical Flow Cytometry, 4th ed. Wiley-Liss, New York.CrossRefGoogle Scholar
  42. 42.
    Gossett DR, Weaver WM, Mach AJ, Hur SC, Tse HTK, Lee W, Amini H & Di Carlo D (2010) Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem 397, 3249–3267.PubMedCrossRefGoogle Scholar
  43. 43.
    Vahey MD & Voldman J (2009) High-Throughput Cell and Particle Characterization Using Isodielectric Separation. Analytical Chemistry 81, 2446–2455.PubMedCrossRefGoogle Scholar
  44. 44.
    Vahey MD & Voldman J (2008) An Equilibrium Method for Continuous-Flow Cell Sorting Using Dielectrophoresis. Analytical Chemistry 80, 3135–3143.PubMedCrossRefGoogle Scholar
  45. 45.
    Harvey TJ, Hughes C, Ward AD, Correia Faria E, Henderson A, Clarke NW, Brown MD, Snook RD & Gardner P (2009) Classification of fixed urological cells using Raman tweezers. J Biophotonics 2, 47–69.PubMedCrossRefGoogle Scholar
  46. 46.
    Snook RD, Harvey TJ, Correia Faria E & Gardner P (2009) Raman tweezers and their application to the study of singly trapped eukaryotic cells. Integr. Biol. 1, 43.CrossRefGoogle Scholar
  47. 47.
    Krylov SN, Arriaga E, Zhang Z, Chan NWC, Palcic MM & Dovichi NJ (2000) Single-cell analysis avoids sample processing bias. Journal of Chromatography B: Biomedical Sciences and Applications 741, 31–35.CrossRefGoogle Scholar
  48. 48.
    Brehm-Stecher BF & Johnson EA (2004) Single-Cell Microbiology: Tools, Technologies, and Applications. Microbiol. Mol. Biol. Rev. 68, 538–559.PubMedCrossRefGoogle Scholar
  49. 49.
    Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF & Liotta LA (2006) Laser-capture microdissection. Nat. Protocols 1, 586–603.CrossRefGoogle Scholar
  50. 50.
    Filliers M, De Spiegelaere W, Peelman L, Goossens K, Burvenich C, Vandaele L, Cornillie P & Van Soom A (2011) Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts. Analytical Biochemistry 408, 169–171.PubMedCrossRefGoogle Scholar
  51. 51.
    Brouzes E, Medkova M, Savenelli N, Marran D, Twardowski M, Hutchison JB, Rothberg JM, Link DR, Perrimon N & Samuels ML (2009) Droplet microfluidic technology for single-cell high-throughput screening. Proc. Natl. Acad. Sci. USA 106, 14195–14200.PubMedCrossRefGoogle Scholar
  52. 52.
    Zhang Y & Yu L (2008) Microinjection as a tool of mechanical delivery. Curr. Opin. Biotechnol 19, 506–510.PubMedCrossRefGoogle Scholar
  53. 53.
    Zhang Y & Yu L (2008) Single-cell microinjection technology in cell biology. Bioessays 30, 606–610.PubMedCrossRefGoogle Scholar
  54. 54.
    Valero A, Post JN, van Nieuwkasteele JW, ter Braak PM, Kruijer W & van den Berg A (2008) Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab Chip 8, 62.PubMedCrossRefGoogle Scholar
  55. 55.
    Larsson C, Grundberg I, Soderberg O & Nilsson M (2010) In situ detection and genotyping of individual mRNA molecules. Nat Meth 7, 395–397.CrossRefGoogle Scholar
  56. 56.
    Heidecker B & Hare JM (2007) The use of transcriptomic biomarkers for personalized medicine. Heart Fail Rev 12, 1–11.PubMedCrossRefGoogle Scholar
  57. 57.
    Liotta LA, Kohn EC & Petricoin EF (2001) Clinical Proteomics. JAMA: The Journal of the American Medical Association 286, 2211–2214.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Dino Di Carlo
    • 1
  • Henry Tat Kwong Tse
    • 1
  • Daniel R. Gossett
    • 1
  1. 1.Department of BioengineeringUniversity of CaliforniaLos AngelesUSA

Personalised recommendations