Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Take a mouse embryonic stem cell suspension.

Centrifuge, remove the supernatant, and resuspend the cells in a neural differentiation medium.

Place droplets of the suspension on the inner side of a culture plate lid.

Add a buffer to the bottom plate to prevent the droplets from drying. Place the lid on the bottom plate, forming a hanging drop culture, and incubate.

Due to gravity, the cells cluster and form three-dimensional aggregates termed embryoid bodies or EBs.

Place the droplets into a plate containing the differentiation medium and incubate under agitation for the growth of EBs.

Centrifuge and remove the medium; resuspend the EBs in the differentiation medium supplemented with retinoic acid, transfer them to a culture plate, and incubate.

Retinoic acid enters the cells and triggers the formation of a transcription complex. The complex induces gene expression, triggering the differentiation into neural progenitor cells.

The differentiated EBs are ready for downstream applications.