This article describes a method for differentiating mouse embryonic stem cells into neural progenitor cells using a hanging drop culture technique. The process involves the formation of embryoid bodies (EBs) and their subsequent treatment with retinoic acid to induce differentiation.
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.
Begin by setting up the hanging drop cell culture. For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20 microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micropipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren't too close together to prevent them from merging.
Fill the plate with 5 to 10 milliliters of PBS, and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate, filled with 10 milliliters of differentiation medium. Then, place the plate on an orbital shaker set to low speed in the incubator. After another two days, centrifuge the cells at 200 times g for three minutes, and remove the supernatant.
To induce embryoid body differentiation into neural progenitor cells, resuspend the cells in differentiation medium with 5-micromolar retinoic acid. Incubate the plate for two days. Then, replace the least half of the medium with fresh medium by tilting the plate and pipetting it out.
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