The differentiation of embryonic stem cells (ESC) into tissue-specific cells utilizes either monolayer cultures or three-dimensional cell aggregates called embryoid bodies (EB). However, the generation of a large number of EB of controlled sizes can be challenging and labor intensive. Our laboratories have developed a simple, robust, ultra-rapid, and inexpensive design of Honeycomb Microwells for generation of EB. Here, we compare EB generated using (1) Honeycomb Microwells, (2) the commercially available AggreWell™400, and (3) the more traditional Hanging Drop method. We compared the efficiency, viability, quality, and control of EB sizes. Results indicate that the Honeycomb Microwell and AggreWell™400 efficiently generate small EB at approximately 500 cells per EB. However, the cone-bottomed AggreWell plate generates cone-shaped EB at 1000-2000 cells per EB. Moreover, the cone-shape correlates with a reduction in the formation of the primitive endoderm GATA-4+ cells (1% compared with 6-8% in spherical EB), but does not significantly affect mesoderm or ectoderm development. We conclude that the non-spherical EB shape correlates with a reduction in the development of primitive endoderm, and that use of these AggreWell plates should be avoided in deriving endoderm tissue products. Key words: Embryonic stem cells, embryoid bodies, Microwell, AggreWell, hanging drop, primitive endoderm, GATA-4.
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