Abstract
3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or “mesoid bodies” (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.
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Acknowledgments
This work was supported by a grant to R.S.V. by the Department of Biotechnology (DBT-BT/PR11268/MED/12/416/2008). P.V. and P.S. wish to thank the Council of Scientific and Industrial Research, India, for senior research fellowship. We would like to thank Shanthi Devanathan of Department of Metallurgy, IITM for her valuable help in scanning electron microscopy experiments.
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Supplementary Fig 1
A Flow cytometry for MSC markers CD29, CD90 and Sca-1 after 8 passages of BMSC cultures. B CFU assay of RCCS MB (at 16RPM, row 1 and 27RPM, row 2) transferred to TCP and stained with crystal violet (In triplicates) showing colony formation after 72h. C BrdU incorporation in 27RPM RCCS MB transferred to TCP and expanded as monolayer showing proliferating cells. D Triglyceride accumulation is clearly demonstrated in the inverted image showing the considerable difference in numbers of cells committed to adipogenesis by RCCS ‘priming’ as compared to static. E Densitometry analysis of SQRT-PCR of CEBPα gene shows highest expression in R4S4* condition (that is, primed with RCCS for 4 d followed by AIM induction for 4 d under static conditions). CEBPα gene expression is higher than in S8* (static AIM induction for 8 d) condition (ACTB1 values were used to normalize data). F Densitometry analysis of western blots of ACTA1 and ACTB1 proteins under MB and TCP conditions following treatment with 5-azacytidine (ACTB1 values were used to normalize data) (GIF 73 kb)
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Vidyasekar, P., Shyamsunder, P., Sahoo, S.K. et al. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells. In Vitro Cell.Dev.Biol.-Animal 52, 204–217 (2016). https://doi.org/10.1007/s11626-015-9971-2
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DOI: https://doi.org/10.1007/s11626-015-9971-2