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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Langenbach, F. et al. Improvement of the cell-loading efficiency of biomaterials by inoculation with stem cell-based microspheres, in osteogenesis. J. Biomater. Appl., published online, doi:10.1177/0885328210377675 (6 September 2010).
Handschel, J. et al. Comparison of ectopic bone formation of embryonic stem cells and cord blood stem cells in vivo. Tissue Eng. Part A 16, 2475–2483 (2010).
Langenbach, F. et al. Osteogenic differentiation influences stem cell migration out of scaffold-free microspheres. Tissue Eng. Part A 16, 759–766 (2010).
Kunz-Schughart, L.A., Kreutz, M. & Knuechel, R. Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int. J. Exp. Pathol. 79, 1–23 (1998).
Mueller-Klieser, W. Multicellular spheroids. A review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol. 113, 101–122 (1987).
Santini, M.T. & Rainaldi, G. Three-dimensional spheroid model in tumor biology. Pathobiology 67, 148–157 (1999).
Friedrich, J., Seidel, C., Ebner, R. & Kunz-Schughart, L.A. Spheroid-based drug screen: considerations and practical approach. Nat. Protoc. 4, 309–324 (2009).
Kundu, A.K., Khatiwala, C.B. & Putnam, A.J. Extracellular matrix remodeling, integrin expression, and downstream signaling pathways influence the osteogenic differentiation of mesenchymal stem cells on poly(lactide-co-glycolide) substrates. Tissue Eng. Part A 15, 273–283 (2009).
Holy, C.E., Shoichet, M.S. & Davies, J.E. Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period. J. Biomed. Mater. Res. 51, 376–382 (2000).
Bitar, M. et al. Effect of cell density on osteoblastic differentiation and matrix degradation of biomimetic dense collagen scaffolds. Biomacromolecules 9, 129–135 (2008).
Abbott, A. Cell culture: biology's new dimension. Nature 424, 870–872 (2003).
Handschel, J.G. et al. Prospects of micromass culture technology in tissue engineering. Head Face Med. 3, 4 (2007).
Cukierman, E., Pankov, R., Stevens, D.R. & Yamada, K.M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).
Weaver, V.M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245 (1997).
Sivaraman, A. et al. A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr. Drug Metab. 6, 569–591 (2005).
Kelm, J.M. & Fussenegger, M. Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol. 22, 195–202 (2004).
Estes, B.T., Diekman, B.O., Gimble, J.M. & Guilak, F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat. Protoc. 5, 1294–1311 (2010).
Kafienah, W., Al-Fayez, F., Hollander, A.P. & Barker, M.D. Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach. Arthritis Rheum. 48, 709–718 (2003).
Keller, G.M. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7, 862–869 (1995).
Potter, S.W. & Morris, J.E. Development of mouse embryos in hanging drop culture. Anat. Rec. 211, 48–56 (1985).
Wobus, A.M., Wolf, E. & Beier, H.M. Embryonic stem cells and nuclear transfer strategies. Present state and future prospects. Cells Tissues Organs 166, 1–5 (2000).
Kelm, J.M., Timmins, N.E., Brown, C.J., Fussenegger, M. & Nielsen, L.K. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 83, 173–180 (2003).
Burns, J.S., Rasmussen, P.L., Larsen, K.H., Schroder, H.D. & Kassem, M. Parameters in three-dimensional osteospheroids of telomerized human mesenchymal (stromal) stem cells grown on osteoconductive scaffolds that predict in vivo bone-forming potential. Tissue Eng. Part A 16, 2331–2342 (2010).
Meyer, U. & Wiesmann, H.P. Bone and Cartilage Engineering (Springer-Verlag, 2006).
Hildebrandt, C., Buth, H. & Thielecke, H. A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells. Tissue Cell 43, 91–100 (2011).
Jaiswal, N., Haynesworth, S.E., Caplan, A.I. & Bruder, S.P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J. Cell Biochem. 64, 295–312 (1997).
Kogler, G. et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J. Exp. Med. 200, 123–135 (2004).
Bonewald, L.F. et al. von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif. Tissue Int. 72, 537–547 (2003).
Daus, A.W., Goldhammer, M., Layer, P.G. & Thielemann, C. Electromagnetic exposure of scaffold-free three-dimensional cell culture systems. Bioelectromagnetics 32, 351–359 (2011).
Nyberg, S.L. et al. Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver. Liver Transpl. 11, 901–910 (2005).
Berahim, Z., Moharamzadeh, K., Rawlinson, A. & Jowett, A.K. Biological Interaction of 3d periodontal fibroblast spheroids with collagen-based and synthetic membranes. J Periodontol. 82, 790–797 (2011).
Miyagawa, Y. et al. A microfabricated scaffold induces the spheroid formation of human bone marrow-derived mesenchymal progenitor cells and promotes efficient adipogenic differentiation. Tissue Eng. Part A 17, 513–521 (2011).
Bartosh, T.J. et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc. Natl Acad. Sci. USA 107, 13724–13729 (2010).
Laib, A.M. et al. Spheroid-based human endothelial cell microvessel formation in vivo. Nat. Protoc. 4, 1202–1215 (2009).
Arufe, M.C. et al. Analysis of the chondrogenic potential and secretome of mesenchymal stem cells derived from human umbilical cord stroma. Stem Cells Dev. 20, 1199–1212 (2011).
Giovannini, S. et al. Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro. Eur. Cell Mater. 20, 245–259 (2010).
Conley, B.J., Young, J.C., Trounson, A.O. & Mollard, R. Derivation, propagation and differentiation of human embryonic stem cells. Int. J. Biochem. Cell Biol. 36, 555–567 (2004).
Wang, W. et al. 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials 30, 2705–2715 (2009).
Kelm, J.M. et al. A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. J. Biotechnol. 148, 46–55 (2010).
Kelm, J.M. & Fussenegger, M. Scaffold-free cell delivery for use in regenerative medicine. Adv. Drug Deliv. Rev. 62, 753–764 (2010).
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).
The authors declare no competing financial interests.
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)
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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