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

  • Josef JarosEmail author
  • Michal Petrov
  • Marketa Tesarova
  • Ales Hampl
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

3D visualization Image reconstruction Image segmentation Morphology Organoid SBF-SEM Scanning electron microscopy Serial block-face Spheroid Stem cell Ultrastructure 

Notes

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.

Supplementary material

Supplementary Video:

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

References

  1. 1.
    Caliari SR, Burdick JA (2016) A practical guide to hydrogels for cell culture. Nat Methods 13:405–414CrossRefPubMedGoogle Scholar
  2. 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 12:207–218CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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 31:23–31CrossRefPubMedGoogle Scholar
  4. 4.
    Rimann M, Graf-Hausner U (2012) Synthetic 3D multicellular systems for drug development. Curr Opin Biotechnol 23:803–809CrossRefPubMedGoogle Scholar
  5. 5.
    Bock DD, Lee W-CA, Kerlin AM et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mishchenko Y (2009) Automation of 3D reconstruction of neural tissue from large volume of conventional serial section transmission electron micrographs. J Neurosci Methods 176:276–289CrossRefPubMedGoogle Scholar
  7. 7.
    Takemura S, Bharioke A, Lu Z et al (2013) A visual motion detection circuit suggested by drosophila connectomics. Nature 500:175–181CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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 6:845–858CrossRefPubMedGoogle Scholar
  10. 10.
    Kremer A, Lippens S, Bartunkova S et al (2015) Developing 3D SEM in a broad biological context. J Microsc 259:80–96CrossRefPubMedPubMedCentralGoogle Scholar
  11. 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 2:246–254CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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.2Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Josef Jaros
    • 1
    • 2
    Email author
  • Michal Petrov
    • 3
  • Marketa Tesarova
    • 4
  • Ales Hampl
    • 1
    • 2
  1. 1.Faculty of Medicine, Department of Histology and EmbryologyMasaryk UniversityBrnoCzech Republic
  2. 2.Cell and Tissue Regeneration, International Clinical Research CenterSt. Anne’s University Hospital BrnoBrnoCzech Republic
  3. 3.TESCAN Brno, s.r.o.BrnoCzech Republic
  4. 4.CEITEC BUT, Brno University of TechnologyBrnoCzech Republic

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