Protocol | Published:

Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering

Nature Protocols volume 6, pages 17261735 (2011) | Download Citation

Abstract

This protocol describes an effective method for the production of spherical microtissues (microspheres), which can be used for a variety of tissue-engineering purposes. The obtained microtissues are well suited for the study of osteogenesis in vitro when multipotent stem cells are used. The dimensions of the microspheres can easily be adjusted according to the cell numbers applied in an individual experiment. Thus, microspheres allow for the precise administration of defined cell numbers at well-defined sites. Here we describe a detailed workflow for the production of microspheres using unrestricted somatic stem cells from human umbilical cord blood and adapted protocols for the use of these microspheres in histological analysis. RNA extraction methods for mineralized microtissues are specifically modified for optimum yields. The duration of running the complete protocol without preparatory cell culture but including 2 weeks of microsphere incubation, histological staining and RNA isolation is about 3 weeks.

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Acknowledgements

We thank M. Hölbling for technical assistance. We also thank the group of G. Kögler (José Carreras Cord Blood Bank) for providing USSCs. F.L. was supported by a Deutsche Forschungsgemeinschaft (DFG) grant (HA 3228/2-1).

Author information

Author notes

    • Fabian Langenbach
    •  & Karin Berr

    These authors contributed equally to this work.

Affiliations

  1. Department of Oral and Maxillofacial Surgery, Heinrich Heine University Medical Center, Düsseldorf, Germany.

    • Fabian Langenbach
    • , Karin Berr
    • , Christian Naujoks
    • , Andrea Hassel
    • , Michael Hentschel
    • , Rita Depprich
    • , Norbert R Kubler
    •  & Jörg Handschel
  2. MKG Münster, Münster, Germany.

    • Ulrich Meyer
  3. Institute for Materials Science, Technische Universität Dresden, Dresden, Germany.

    • Hans-Peter Wiesmann
  4. Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Düsseldorf, Germany.

    • Gesine Kögler

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Contributions

F.L. and A.H. performed the majority of the experiments and wrote the manuscript, K.B. supervised the project and wrote the manuscript. U.M. and H.-P.W. established the method. J.H. and C.N. supervised the project and adapted the protocol for USSCs and ESCs. M.H. performed the microscopy, R.D. supported data analysis. N.R.K. administered the project and G.K. provided the cells.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Christian Naujoks.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Microsphere–ICBM construct was incubated for 3 days in DAG medium. Hemalum/eosin stain. The microsphere is visible as a densely packed cell mass with outgrowing cells connecting with the collagen matrix (see arrow) (bar: 250 µm).

  2. 2.

    Supplementary Fig. 2

    Autologous chondrocytes previously harvested from minipigs were multiplicated in culture and assembled to microspheres. Cartilage defects were surgically set into the femoropatellar joints of minipigs. A) The defects were then treated by implanting the microspheres. B) Defects without cell transplantation served as a control. After 60 days animals were sacrificed and subsequent histological sections were stained with safraninO/Fast-Green and counter stained with Weigert's ferric hematoxylin for the presence of hyaline-specific, acidic glycosaminoglycans. In the defects supplemented with microspheres a cartilage-specific layer of acidic glycosaminoglycans was secreted from the cells (bars: 1 mm).

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DOI

https://doi.org/10.1038/nprot.2011.394

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