Skip to main content
SpringerLink
Log in

Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications

  • Protocol
  • First Online:
Microchip Diagnostics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1547))

Abstract

Cell populations are heterogeneous: they can comprise different cell types or even cells at different stages of the cell cycle and/or of biological processes. Furthermore, molecular processes taking place in cells are stochastic in nature. Therefore, cellular analysis must be brought down to the single cell level to get useful insight into biological processes, and to access essential molecular information that would be lost when using a cell population analysis approach. Furthermore, to fully characterize a cell population, ideally, information both at the single cell level and on the whole cell population is required, which calls for analyzing each individual cell in a population in a parallel manner. This single cell level analysis approach is particularly important for diagnostic applications to unravel molecular perturbations at the onset of a disease, to identify biomarkers, and for personalized medicine, not only because of the heterogeneity of the cell sample, but also due to the availability of a reduced amount of cells, or even unique cells. This chapter presents a versatile platform meant for the parallel analysis of individual cells, with a particular focus on diagnostic applications and the analysis of cancer cells. We first describe one essential step of this parallel single cell analysis protocol, which is the trapping of individual cells in dedicated structures. Following this, we report different steps of a whole analytical process, including on-chip cell staining and imaging, cell membrane permeabilization and/or lysis using either chemical or physical means, and retrieval of the cell molecular content in dedicated channels for further analysis. This series of experiments illustrates the versatility of the herein-presented platform and its suitability for various analysis schemes and different analytical purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 119.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Haselgrubler T, Haider M, Ji BZ et al (2014) High-throughput, multiparameter analysis of single cells. Anal Bioanal Chem 406(14):3279–3296

    Article  Google Scholar 

  2. Young EWK (2013) Cells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment. Integr Biol-Uk 5(9):1096–1109

    Article  CAS  Google Scholar 

  3. Tian TH, Olson S, Whitacre JM, Harding A (2011) The origins of cancer robustness and evolvability. Integr Biol-Uk 3(1):17–30

    Article  CAS  Google Scholar 

  4. Ligthart ST, Bidard FC, Decraene C et al (2013) Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non-metastatic breast cancer. Ann Oncol 24(5):1231–1238

    Article  CAS  Google Scholar 

  5. van den Brink FT, Gool E, Frimat JP et al (2011) Parallel single-cell analysis microfluidic platform. Electrophoresis 32(22):3094–3100

    Article  Google Scholar 

  6. Harper JC, SenGupta SB (2012) Preimplantation genetic diagnosis: state of the ART 2011. Obstet Gynecol Surv 67(6):347–348

    Article  Google Scholar 

  7. Steele CD, Wapner RJ, Smith JB et al (1996) Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review. Clin Obstet Gynecol 39(4):801–813

    Article  CAS  Google Scholar 

  8. Whitesides GM (2006) The origins and the future of microfluidics. Nature 442(7101):368–373

    Article  CAS  Google Scholar 

  9. Le Gac S, van den Berg A (2010) Single cells as experimentation units in lab-on-a-chip devices. Trends Biotechnol 28(2):55–62

    Article  CAS  Google Scholar 

  10. Sims CE, Allbritton NL (2007) Analysis of single mammalian cells on-chip. Lab Chip 7(4):423–440

    Article  CAS  Google Scholar 

  11. Liberale C, Cojoc G, Bragheri F et al (2013) Integrated microfluidic device for single-cell trapping and spectroscopy. Sci Rep 3:1258. doi:10.1038/srep01258

    CAS  Google Scholar 

  12. Hochstetter A, Stellamanns E, Deshpande S et al (2015) Microfluidics-based single cell analysis reveals drug-dependent motility changes in trypanosomes. Lab Chip 15(8):1961–1968

    Article  CAS  Google Scholar 

  13. Valero A, Post JN, van Nieuwkasteele JW et al (2008) Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device. Lab Chip 8(1):62–67

    Article  CAS  Google Scholar 

  14. Adamo A, Jensen KF (2008) Microfluidic based single cell microinjection. Lab Chip 8(8):1258–1261

    Article  CAS  Google Scholar 

  15. Takayama S, McDonald JC, Ostuni E et al (1999) Patterning cells and their environments using multiple laminar fluid flows in capillary networks. P Natl Acad Sci USA 96(10):5545–5548

    Article  CAS  Google Scholar 

  16. Eriksson E, Enger J, Nordlander B et al (2007) A microfluidic system in combination with optical tweezers for analyzing rapid and reversible cytological alterations in single cells upon environmental changes. Lab Chip 7(1):71–76

    Article  CAS  Google Scholar 

  17. Kobel S, Valero A, Latt J et al (2010) Optimization of microfluidic single cell trapping for long-term on-chip culture. Lab Chip 10(7):857–863

    Article  CAS  Google Scholar 

  18. Le Gac S, Van den Berg A (2010) Cell captureand lysis on a chip. In: Bontoux N, Dauphinot L, Potier M-C (eds) Unravelling single cell genomics. vol RSC nanosciences & nanotechnology, RSC Publishing, pp 150–184.

    Google Scholar 

  19. Rettig JR, Folch A (2005) Large-scale single-cell trapping and imaging using microwell arrays. Anal Chem 77(17):5628–5634

    Article  CAS  Google Scholar 

  20. Toriello NM, Douglas ES, Mathies RA (2005) Microfluidic device for electric field-driven single-cell capture and activation. Anal Chem 77(21):6935–6941

    Article  CAS  Google Scholar 

  21. Taff BM, Voldman J (2005) A scalable addressable positive-dielectrophoretic cell-sorting array. Anal Chem 77(24):7976–7983

    Article  CAS  Google Scholar 

  22. Clausell-Tormos J, Lieber D, Baret JC et al (2008) Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms. Chem Biol 15(5):427–437

    Article  CAS  Google Scholar 

  23. Thery M, Racine V, Pepin A et al (2005) The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol 7(10):947–U929

    Article  CAS  Google Scholar 

  24. Le Gac S, de Boer H, Wijnperlé D et al Parallel single cell analysis on an integrated microfluidic platform for cell trapping, lysis and analysis. In: MicroTAS, the 13th international conference on miniaturized systems for chemistry and life sciences, Jeju Island, Korea, 2009. Royal Society of Chemistry.

    Google Scholar 

  25. Walker G, Beebe DJ (2002) A passive pumping method for microfluidic devices. Lab Chip 2(3):131–134

    Article  CAS  Google Scholar 

  26. Rao CG, Chianese D, Doyle GV et al (2005) Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors. Int J Oncol 27(1):49–57

    CAS  Google Scholar 

  27. Olofsson J, Bridle H, Jesorka A et al (2009) Direct access and control of the intracellular solution environment in single cells. Anal Chem 81(5):1810–1818

    Article  CAS  Google Scholar 

  28. Neumann E, Schaeferridder M, Wang Y, Hofschneider PH (1982) Gene-transfer into Mouse Lyoma cells by electroporation in high electric-fields. Embo J 1(7):841–845

    CAS  Google Scholar 

  29. Di Carlo D, Aghdam N, Lee LP (2006) Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays. Anal Chem 78(14):4925–4930

    Article  CAS  Google Scholar 

  30. Yamada M, Kano K, Tsuda Y et al (2007) Microfluidic devices for size-dependent separation of liver cells. Biomed Microdevices 9(5):637–645

    Article  Google Scholar 

  31. Miyamoto K, Yamashita T, Tsukiyama T et al (2008) Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts. Cloning Stem Cells 10(4):535–542

    Article  CAS  Google Scholar 

  32. Gac SL, Zwaan E, van den Berg A, Ohl CD (2007) Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement. Lab Chip 7(12):1666–1672

    Article  Google Scholar 

  33. Quinto-Su PA, Lai HH, Yoon HH et al (2008) Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging. Lab Chip 8(3):408–414

    Article  CAS  Google Scholar 

  34. Salehi-Reyhani A, Gesellchen F, Mampallil D et al (2015) Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays. Anal Chem 87(4):2161–2169

    Article  CAS  Google Scholar 

  35. Di Carlo D, Jeong KH, Lee LP (2003) Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation. Lab Chip 3(4):287–291

    Article  CAS  Google Scholar 

  36. Sims CE, Allbritton NL (2003) Single-cell kinase assays: opening a window onto cell behavior. Curr Opin Biotech 14(1):23–28

    Article  CAS  Google Scholar 

  37. Perez-Toralla K, Mottet G, Guneri ET et al (2015) FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool. Lab Chip 15(3):811–822

    Article  CAS  Google Scholar 

  38. Scriven PN, Ogilvie CM (2010) FISH for pre-implantation genetic diagnosis. Method Mol Biol 659:269–282

    Article  CAS  Google Scholar 

  39. Dochow S, Krafft C, Neugebauer U et al (2011) Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments. Lab Chip 11(8):1484–1490

    Article  CAS  Google Scholar 

  40. El-Ali J, Gaudet S, Gunther A et al (2005) Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow. Anal Chem 77(11):3629–3636

    Article  CAS  Google Scholar 

  41. Marcus JS, Anderson WF, Quake SR (2006) Microfluidic single-cell mRNA isolation and analysis. Anal Chem 78(9):3084–3089

    Article  CAS  Google Scholar 

  42. Yang Y, Swennenhuis JF, Rho HS et al (2014) Parallel single cancer cell whole genome amplification using button-valve assisted mixing in nanoliter chambers. PloS One 9(9):e107958

    Article  Google Scholar 

  43. Lee WC, Rigante S, Pisano AP, Kuypers FA (2010) Large-scale arrays of picolitre chambers for single-cell analysis of large cell populations. Lab Chip 10(21):2952–2958

    Article  CAS  Google Scholar 

  44. Munce NR, Li J, Herman PR, Lilge L (2004) Microfabricated system for parallel single-cell capillary electrophoresis. Anal Chem 76(17):4983–4989

    Article  CAS  Google Scholar 

  45. Salehi-Reyhani A, Kaplinsky J, Burgin E et al (2011) A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection. Lab Chip 11(7):1256–1261

    Article  CAS  Google Scholar 

  46. Kovac JR, Voldman J (2007) Intuitive, image-based cell sorting using optofluidic cell sorting. Anal Chem 79(24):9321–9330

    Article  CAS  Google Scholar 

  47. Di Carlo D, Lee LP (2006) Dynamic single-cell analysis for quantitative biology. Anal Chem 78(23):7918–7925

    Article  CAS  Google Scholar 

  48. Gong Y, Ogunniyi AO, Love JC (2010) Massively parallel detection of gene expression in single cells using subnanolitre wells. Lab Chip 10(18):2334–2337

    Article  CAS  Google Scholar 

  49. Salehi-Reyhani A, Sharma S, Burgin E et al (2013) Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis. Lab Chip 13(11):2066–2074

    Article  CAS  Google Scholar 

  50. Eyer K, Kuhn P, Hanke C et al (2012) A microchamber array for single cell isolation and analysis of intracellular biomolecules. Lab Chip 12(4):765–772

    Article  CAS  Google Scholar 

  51. Brouzes E, Medkova M, Savenelli N et al (2009) Droplet microfluidic technology for single-cell high-throughput screening. P Natl Acad Sci USA 106(34):14195–14200

    Article  CAS  Google Scholar 

  52. Sarkar S, Cohen N, Sabhachandani P et al (2015) Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors. Lab Chip 15(23):4441–4450

    Article  CAS  Google Scholar 

  53. Phillips KS, Lai HH, Johnson E et al (2011) Continuous analysis of dye-loaded, single cells on a microfluidic chip. Lab Chip 11(7):1333–1341

    Article  CAS  Google Scholar 

Download references

Acknowledgment

The work presented in this chapter has been conducted in part by Floris van den Brink and Elmar Gool. Johan Bomer, Daniël Wijnperlé, and Hans de Boer are thanked for technical assistance (mold fabrication and development of the setup). Prof. Edwin Carlen is acknowledged for his supervision in the first year of the project. This work was supported by NanoNext NL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners as well as by the company OGT (Oxford Gene Technology).

Author information

Authors and Affiliations

  1. Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology and MIRA Institute for Biomedical Engineering and Technical Medicine, University of Twente, Postbus 217, 7500, AE, Enschede, The Netherlands

    Séverine Le Gac PhD

Authors
  1. Séverine Le Gac PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Séverine Le Gac PhD .

Editor information

Editors and Affiliations

  1. INSERM UMRS1147, CNRS SNC 5014, Université Paris Descartes, Equipe labellisée Ligue Nationale contre le cancer 2016, Paris, France

    Valérie Taly

  2. Macromolecules and Microsystems in Biology and Medicine, Institut Curie, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, PSL Research University, UMR 168, Paris, France

    Jean-Louis Viovy

  3. Macromolecules and Microsystems in Biology and Medicine, Institut Curie, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, PSL Research University, UMR 168, Paris, France

    Stéphanie Descroix

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Le Gac, S. (2017). Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications. In: Taly, V., Viovy, JL., Descroix, S. (eds) Microchip Diagnostics. Methods in Molecular Biology, vol 1547. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6734-6_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6734-6_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6732-2

  • Online ISBN: 978-1-4939-6734-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation