, Volume 68, Issue 6, pp 2479–2490 | Cite as

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

  • Chiara Bellotti
  • Serena Duchi
  • Alessandro Bevilacqua
  • Enrico Lucarelli
  • Filippo PiccininiEmail author
Original Article


Three-dimensional (3D) spheroids of mesenchymal stromal cells (MSC) have been demonstrated to improve a wide range of MSC features, such as multilineage potential, secretion of therapeutic factors, and resistance against hypoxic condition. Accordingly, they represent a promising tool in regenerative medicine for several biological and clinical applications. Many approaches have been proposed to generate MSC spheroids. They usually require specific generation systems, such as rotatory bioreactors or low-attachment plates, and each approach has its own disadvantages. Furthermore, an over-time analysis of morphological homogeneity and architectural stability of the spheroids generated is rarely provided. In this work we adapted the “pellet culture” method to obtain homogenous spheroids of MSC and maintain them in vitro for long term studies. We analysed their outer and inner structure over a 2-month period to provide morphological and architectural information regarding the spheroids generated. Quantitative and qualitative data were obtained using brightfield and confocal microscope imaging coupled to a computational analysis to estimate volume, sphericity, and jagging degree. In addition, histological evaluation was performed to more thoroughly assess the cellular composition and the internal architecture of the 3D spheroids. The results provided show that MSC spheroids generated with the proposed approach are homogeneous and stable, from both morphological and architectural points of view, for a period of at least 15 days, approximately between day 15 and day 30 after their generation. Accordingly, the approach proposed serves as a rapid, cost-effective, and efficient method to generate and maintain MSC spheroids using common entry-level laboratory equipment only.


Mesenchymal stromal/stem cells 3D multicellular spheroids Pellet culture method Morphological analysis Microscopy Computational analysis 



The authors would like to thank Dr. Davide Donati and his staff of the Third Orthopedics and Traumatology Clinic (IOR, Bologna), for providing the cells used in this work and Ms. Charlotte Story (University of North Carolina at Chapel Hill, USA) for editorial assistance and English revision of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Chiara Bellotti
    • 1
    • 2
  • Serena Duchi
    • 1
    • 2
  • Alessandro Bevilacqua
    • 3
    • 4
  • Enrico Lucarelli
    • 1
  • Filippo Piccinini
    • 3
    Email author
  1. 1.Osteoarticular Regeneration LaboratoryRizzoli Orthopedic InstituteBolognaItaly
  2. 2.Department of Biomedical and Neuromotor Sciences (DIBINEM)University of BolognaBolognaItaly
  3. 3.Advanced Research Center on Electronic Systems for Information and Communication Technologies “E. De Castro” (ARCES)University of BolognaBolognaItaly
  4. 4.Department of Computer Science and Engineering (DISI)University of BolognaBolognaItaly

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