Skip to main content
SpringerLink
Log in

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy

  • Protocol
  • First Online:
3D Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1612))

Abstract

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

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 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.00
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

References

  1. Caliari SR, Burdick JA (2016) A practical guide to hydrogels for cell culture. Nat Methods 13:405–414

    Article  CAS  PubMed  Google Scholar 

  2. Edmondson R, Broglie JJ, Adcock AF et al (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12:207–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pasquinelli G, Tazzari P, Ricci F et al (2007) Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta. Ultrastruct Pathol 31:23–31

    Article  PubMed  Google Scholar 

  4. Rimann M, Graf-Hausner U (2012) Synthetic 3D multicellular systems for drug development. Curr Opin Biotechnol 23:803–809

    Article  CAS  PubMed  Google Scholar 

  5. Bock DD, Lee W-CA, Kerlin AM et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mishchenko Y (2009) Automation of 3D reconstruction of neural tissue from large volume of conventional serial section transmission electron micrographs. J Neurosci Methods 176:276–289

    Article  PubMed  Google Scholar 

  7. Takemura S, Bharioke A, Lu Z et al (2013) A visual motion detection circuit suggested by drosophila connectomics. Nature 500:175–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bushby AJ, P’ng KMY, Young RD et al (2011) Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat Protoc 6:845–858

    Article  CAS  PubMed  Google Scholar 

  10. Kremer A, Lippens S, Bartunkova S et al (2015) Developing 3D SEM in a broad biological context. J Microsc 259:80–96

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kunova M, Matulka K, Eiselleova L et al (2013) Adaptation to robust monolayer expansion produces human pluripotent stem cells with improved viability. Stem Cells Transl Med 2:246–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ludwig T, Thomson JA (2007) Defined, feeder-independent medium for human embryonic stem cell culture. Curr Protoc Stem Cell Biol Chapter 1:Unit 1C.2

    Google Scholar 

Download references

Acknowledgment

This study was supported by: the project FP7 Regpot ICRC-ERA-HumanBridge no. 316345, the project no. LQ1605 from the National Program of Sustainability II (MEYS CR), Czech Science Foundation (GA16-02702S), the project HistoPARK from the Centre for Analysis and Modeling of Tissues and Organs (CZ.1.07/2.3.00/20.0185, European Social Fund in the Czech Republic), funds from the Faculty of Medicine of Masaryk University (MUNI/M/1050/2013), and the project CEITEC 2020 no. LQ1601 by the Ministry of Education, Youth and Sports of the Czech Republic.

Author information

Authors and Affiliations

  1. Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Kamenice 3/A1, 625 00, Brno, Czech Republic

    Josef Jaros Ph.D. & Ales Hampl

  2. Cell and Tissue Regeneration, International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91, Brno, Czech Republic

    Josef Jaros Ph.D. & Ales Hampl

  3. TESCAN Brno, s.r.o., Libusina Trida 1, 623 00, Brno, Czech Republic

    Michal Petrov

  4. CEITEC BUT, Brno University of Technology, Purkynova 123, 612 00, Brno, Czech Republic

    Marketa Tesarova

Authors
  1. Josef Jaros Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michal Petrov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marketa Tesarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ales Hampl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Josef Jaros Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Histology and Embryology, Masaryk University, Brno, Czech Republic

    Zuzana Koledova

1 Electronic Supplementary Material

Animation of 3D visualization of the stem cell spheroid (AVI 48109 kb)

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Jaros, J., Petrov, M., Tesarova, M., Hampl, A. (2017). Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy. In: Koledova, Z. (eds) 3D Cell Culture. Methods in Molecular Biology, vol 1612. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7021-6_30

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7021-6_30

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7019-3

  • Online ISBN: 978-1-4939-7021-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation