Formation of Embryoid Bodies with Controlled Sizes and Maintained Pluripotency in Three-Dimensional Inverse Opal Scaffolds

Embryoid bodies (EBs) are aggregates of cells derived from embryonic stem (ES) cells, which can serve as a good model system to investigate molecular and cellular interactions in the earliest stages of embryo development. Current methods for producing EBs mainly rely on the use of hanging drops or suspensions in non-tissue culture treated plates, microwells, and spinner flasks. The capability of these methods is limited in terms of size uniformity and distribution as well as scalability. Here, a new platform based on three-dimensional alginate inverse opal scaffolds with uniform pores is presented, where uniform EBs with controllable sizes could be produced in the pores and then recovered after disintegration of the scaffolds. The size of the EBs could be readily controlled by varying the culture time and/or by using scaffolds with different pore sizes. The EBs maintained their viability and undifferentiated state, and they were able to differentiate into specific lineages upon stimulation.