Rotary Suspension Culture Enhances the Efficiency, Yield and Homogeneity of Embryoid Body Differentiation

¹ The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University
² The Wallace H. Coulter Department of Biomedical Engineering, The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology/Emory University

^* To whom correspondence should be addressed. E-mail: todd.mcdevitt{at}bme.gatech.edu.

	Abstract

Embryonic stem (ES) cells hold great promise as a robust cell source for cell based therapies and as a model of early embryonic development. Current experimental methods for differentiation of ES cells via embryoid body (EB) formation are either inherently incapable of larger-scale production, or exhibit limited control over cell aggregation during EB formation and subsequent EB agglomeration. This report describes and characterizes a novel method for formation of EBs using rotary orbital motion that simultaneously addresses both concerns. EBs formed under rotary suspension conditions were compared to hanging drop and static EBs for efficiency of EB formation, cell and EB yield, homogeneity of EB size and shape, and gene expression. A 20-fold enhancement in the number of cells incorporated into primitive EBs in rotary versus static conditions was detected after the first 12 hours, and a 4-fold increase in total cell yield was achieved by rotary after 7 days. Morphometric analysis of EBs demonstrated formation and maintenance of a more uniform EB population under rotary conditions compared to hanging drop and static. Quantitative gene expression analysis indicated rotary EBs differentiated normally based on expression of ectoderm, endoderm and mesoderm markers. Increased levels of endoderm gene expression, along with cystic EB formation, indicated by histological examination, suggested that differentiation was accelerated in rotary EBs. Thus, the rotary suspension culture method can produce a highly uniform population of efficiently differentiating EBs in large quantities in a manner that can be easily implemented by basic research laboratories conducting ES cell differentiation studies.