Methods for Embryoid Body Formation: The Microwell Approach

Embyroid body (EB) formation is a key step in many embryonic stem cell (ESC) differentiation protocols. The EB mimics the structure of the developing embryo, thereby providing a means of obtaining any cell lineage. Traditionally, the two methods of EB formation are suspension and hanging drop. The suspension method allows ESCs to self-aggregate into EBs in a nonadherent dish. The hanging drop method suspends ESCs on the lid of a dish and EBs form through aggregation at the bottom of the drops. Recently, alternative methods of EB formation have been developed that allow for highly accurate control of EB size and shape, resulting in reproducibly produced homogeneous EBs. This control is potentially useful for directed differentiation, as recent studies have shown that EB size may be a useful determinant of the resulting differentiated cell types. One particular approach to generate homogeneous EBs utilizes nonadhesive microwell structures. The methodology associated with this technique, along with the traditional approaches of suspension and hanging drop, is the focus of this chapter.