Advertisement

Analysis of Biomechanical Properties of Hematopoietic Stem and Progenitor Cells Using Real-Time Fluorescence and Deformability Cytometry

  • Angela JacobiEmail author
  • Philipp Rosendahl
  • Martin Kräter
  • Marta Urbanska
  • Maik Herbig
  • Jochen Guck
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2017)

Abstract

Stem cell mechanics, determined predominantly by the cell’s cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner—a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s—similar to conventional flow cytometers—which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.

Key words

Mechanical phenotyping Hematopoietic stem and progenitor cells Cell mechanics Microfluidics Flow cytometry 

Notes

Acknowledgments

The authors would like to thank Prof. Martin Bornhäuser from the University Hospital Dresden for providing the patients material, Zellmechanik Dresden for providing materials for graphics, and the Microstructure Facility at the Center for Molecular and Cellular Bioengineering (CMCB) at Technische Universität Dresden (in part funded by the State of Saxony and the European Regional Development Fund) and Alejandro Riviera Prieto for help with the production of RT-DC chips. This work was financially supported by the Alexander von Humboldt-Stiftung (Alexander von Humboldt Professorship to J.G.) and the DKMS Mechthild Harf Research Grant (DKMS-SLS-MHG-2016-02 to A.J.).

References

  1. 1.
    Dicke KA, van Noord MJ, Maat B et al (1973) Identification of cells in primate bone marrow resembling the hemopoietic stem cell in the mouse. Blood 42:195–208PubMedGoogle Scholar
  2. 2.
    Abramson S (1977) The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J Exp Med 145:1567–1579.  https://doi.org/10.1084/jem.145.6.1567CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Visser JW, Bauman JG, Mulder AH et al (1984) Isolation of murine pluripotent hemopoietic stem cells. J Exp Med 159:1576–1590CrossRefGoogle Scholar
  4. 4.
    Bhatia M, Wang JC, Kapp U et al (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 94:5320–5325.  https://doi.org/10.1073/pnas.94.10.5320CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bhatia M, Bonnet D, Murdoch B et al (1998) A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 4:1038–1045.  https://doi.org/10.1038/2023CrossRefPubMedGoogle Scholar
  6. 6.
    Burt RK, Loh Y, Pearce W et al (2008) Clinical applications of blood-derived and marrow-derived stem cells for nonmalignant diseases. JAMA 299:925–936.  https://doi.org/10.1001/jama.299.8.925CrossRefPubMedGoogle Scholar
  7. 7.
    Cutler C, Antin JH (2001) Peripheral blood stem cells for allogeneic transplantation: a review. Stem Cells 19:108–117.  https://doi.org/10.1634/stemcells.19-2-108CrossRefPubMedGoogle Scholar
  8. 8.
    Arndt K, Grinenko T, Mende N et al (2013) CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells. Proc Natl Acad Sci U S A 110:5582–5587. 10.1073/pnas.1215438110/-/DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.1215438110CrossRefGoogle Scholar
  9. 9.
    Sharma S, Gurudutta GU, Satija NK et al (2016) Stem cell c-KIT and HOXB4 genes: critical roles and mechanisms in self-renewal, proliferation, and differentiation. Stem Cells Dev 778:755–778.  https://doi.org/10.1089/scd.2006.15.755CrossRefGoogle Scholar
  10. 10.
    Paschke S, Weidner AF, Paust T et al (2013) Technical advance: inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments. J Leukoc Biol 94:1091–1096.  https://doi.org/10.1189/jlb.1012510CrossRefPubMedGoogle Scholar
  11. 11.
    Lautenschlager F, Paschke S, Schinkinger S et al (2009) The regulatory role of cell mechanics for migration of differentiating myeloid cells. Proc Natl Acad Sci U S A 106:15696–15701.  https://doi.org/10.1073/pnas.0811261106CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ekpenyong AE, Whyte G, Chalut K et al (2012) Viscoelastic properties of differentiating blood cells are fate- and function-dependent. PLoS One 7(9):e45237.  https://doi.org/10.1371/journal.pone.0045237CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    González-Cruz RD, Fonseca VC, Darling EM (2012) Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells. Proc Natl Acad Sci U S A 109:E1523–E1529.  https://doi.org/10.1073/pnas.1120349109CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Maloney JM, Nikova D, Lautenschläger F et al (2010) Mesenchymal stem cell mechanics from the attached to the suspended state. Biophys J 99:2479–2487.  https://doi.org/10.1016/j.bpj.2010.08.052CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Radmacher M (2007) Studying the mechanics of cellular processes by atomic force microscopy. Methods Cell Biol 83:347–372.  https://doi.org/10.1016/S0091-679X(07)83015-9CrossRefPubMedGoogle Scholar
  16. 16.
    Hochmuth RM (2000) Micropipette aspiration of living cells. J Biomech 33:15–22.  https://doi.org/10.1016/S0021-9290(99)00175-XCrossRefPubMedGoogle Scholar
  17. 17.
    Guck J, Ananthakrishnan R, Mahmood H et al (2001) The optical stretcher: a novel laser tool to micromanipulate cells. Biophys J 81:767–784.  https://doi.org/10.1016/S0006-3495(01)75740-2CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lincoln B, Wottawah F, Schinkinger S et al (2007) High-throughput rheological measurements with an optical stretcher. Methods Cell Biol 83:397–423.  https://doi.org/10.1016/S0091-679X(07)83017-2CrossRefPubMedGoogle Scholar
  19. 19.
    Mietke A, Otto O, Girardo S et al (2015) Extracting cell stiffness from real-time deformability cytometry: theory and experiment. Biophys J 109:2023–2036.  https://doi.org/10.1016/j.bpj.2015.09.006CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Otto O, Rosendahl P, Golfier S et al (2015) Real-time deformability cytometry as a label-free indicator of cell function. Conf Proc IEEE Eng Med Biol Soc 2015:1861–1864PubMedGoogle Scholar
  21. 21.
    Mokbel M, Mokbel D, Mietke A et al (2017) Numerical simulation of real-time deformability cytometry to extract cell mechanical properties. ACS Biomater Sci Eng 3:2962–2973.  https://doi.org/10.1021/acsbiomaterials.6b00558CrossRefGoogle Scholar
  22. 22.
    Rosendahl P, Plak K, Jacobi A et al (2018) Real-time fluorescence and deformability cytometry. Nat Methods 15(5):355–358.  https://doi.org/10.1038/nmeth.4639CrossRefPubMedGoogle Scholar
  23. 23.
    Herbig M, Kräter M, Plak K et al (2018) Real-time deformability cytometry: label-free functional characterization of cells. In: Hawley TS, Hawley RG (eds) Flow cytometry protocols. Springer New York, New York, NY, pp 347–369CrossRefGoogle Scholar
  24. 24.
    Toepfner N, Herold C, Otto O et al (2018) Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood. elife 7:1–22.  https://doi.org/10.7554/eLife.29213CrossRefGoogle Scholar
  25. 25.
    Elson EL (1988) Cellular mechanics as an indicator of cytoskeletal structure and function. Annu Rev Biophys Biophys Chem 17:397–430.  https://doi.org/10.1146/annurev.bb.17.060188.002145CrossRefPubMedGoogle Scholar
  26. 26.
    Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463:485–492.  https://doi.org/10.1038/nature08908CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Guzniczak E, Mohammad Zadeh M, Dempsey F et al (2017) High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing. Sci Rep 7:1–11.  https://doi.org/10.1038/s41598-017-14958-wCrossRefGoogle Scholar
  28. 28.
    Zhu G, Trung Nguyen N (2010) Particle sorting in microfluidic systems. Micro Nanosyst 2:202–216.  https://doi.org/10.2174/1876402911002030202CrossRefGoogle Scholar
  29. 29.
    Nawaz AA, Chen Y, Nama N et al (2015) Acoustofluidic fluorescence activated cell sorter. Anal Chem 87:12051–12058.  https://doi.org/10.1021/acs.analchem.5b02398CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Angela Jacobi
    • 1
    Email author
  • Philipp Rosendahl
    • 1
  • Martin Kräter
    • 1
  • Marta Urbanska
    • 1
  • Maik Herbig
    • 1
  • Jochen Guck
    • 1
  1. 1.Biotechnology Center, Center for Molecular and Cellular BioengineeringTechnische Universität DresdenDresdenGermany

Personalised recommendations