Microsphere size effects on embryoid body incorporation and embryonic stem cell differentiation Richard L. Carpenedo, 1 * Scott A. Seaman, 1 * Todd C. McDevitt 1,2 1 The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, Georgia 2 The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia Received 14 July 2009; revised 28 September 2009; accepted 22 October 2009 Published online 8 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32710 Abstract: Differentiation of pluripotent embryonic stem cells (ESCs) in vitro via multicellular spheroids called embryoid bodies (EBs) is commonly performed to model aspects of early mammalian development and initiate dif- ferentiation of cells for regenerative medicine technolo- gies. However, the three-dimensional nature of EBs poses unique challenges for directed ESC differentiation, includ- ing limited diffusion into EBs of morphogenic molecules capable of specifying cell fate. Degradable polymer micro- spheres incorporated within EBs can present morphogenic molecules to ESCs in a spatiotemporally controlled man- ner to more efficiently direct differentiation. In this study, the effect of microsphere size on incorporation into EBs and ESC differentiation in response to microsphere- mediated morphogen delivery were assessed. PLGA microspheres with mean diameters of 1, 3, or 11 lm were fabricated and mixed with ESCs during EB formation. Smaller microspheres were incorporated more efficiently throughout EBs than larger microspheres, and regardless of size, retained for at least 10 days of differentiation. Retinoic acid release from incorporated microspheres induced EB cavitation in a size-dependent manner, with smaller microspheres triggering accelerated and more complete cavitation than larger particles. These results demonstrate that engineering the size of microsphere delivery vehicles incorporated within stem cell environ- ments can be used to modulate the course of differentia- tion. Ó 2010 Wiley Periodicals, Inc. J Biomed Mater Res 94A: 466–475, 2010 Key words: embryoid body; embryonic stem cell; micro- sphere; differentiation; retinoic acid INTRODUCTION Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of developing blas- tocysts with the inherent ability to differentiate into all somatic cell types. 1–4 The pluripotent nature of ESCs has attracted interest in these cells as a renew- able cell source for tissue engineering and other cell based therapies, as well as for in vitro drug screening and studies in mammalian development. 5–7 Differen- tiation of ESCs can be induced using a variety of methods, including monolayer culture, 8–10 co-culture with stromal cells, 11–13 and aggregation into multicel- lular spheroids called embryoid bodies (EBs). 1,3,14 The use of EBs for ESC differentiation is particularly common because of their ability to differentiate to cells from all three germ lineages and similarities to embryonic development; however, the microenviron- ment within EBs is complex and dynamic, resulting in spontaneous and heterogeneous differentiation events. 15 Efforts to control and direct differentiation of ESCs comprising EBs have traditionally focused on the addition of soluble morphogens to the culture media. 16,17 While soluble treatment can improve the efficiency of directed differentiation to specific cell types, a high degree of heterogeneity is still com- mon. Studies investigating the diffusion of molecules into tumor spheroids 18–21 and EBs 22–24 have shown that diffusion into cell spheroids is hindered by spheroid size, extracellular matrix (ECM) content, and tight cell junctions. The inability of morphogens Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Correspondence to: T. C. McDevitt; e-mail: todd.mcdevitt@ bme.gatech.edu Contract grant sponsor: National Science Foundation; contract grant number: CBET 0651739 Contract grant sponsor: Undergraduate Research Schol- ars program; contract grant number: GTEC, NSF EEC- 9731463 Contract grant sponsors: Presidential Undergraduate Research Award, George Family Foundation fellowship Ó 2010 Wiley Periodicals, Inc.