Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform- sized EBs, the size of which can be controlled by changing the cross-sectional size of rnicrochannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.