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

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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Figure 1: Microspheres of different sizes.
Figure 2: Histological stains of microsphere sections after 3, 7 and 14 d of differentiation.
Figure 3: Fluorescent stain (OsteoImage) of microsphere sections after 3, 7 and 14 d of differentiation.

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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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Christian Naujoks.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

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). (PDF 8591 kb)

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). (PDF 1429 kb)

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Langenbach, F., Berr, K., Naujoks, C. et al. Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering. Nat Protoc 6, 1726–1735 (2011). https://doi.org/10.1038/nprot.2011.394

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