This article describes the process of differentiating mouse embryonic stem cells into neural progenitors. The protocol includes cell detachment, resuspension, and plating onto gelatin-coated surfaces to promote adherence and growth.
Begin with a culture of mouse embryonic stem cells.
Wash the cells with a buffer to remove media and cell debris.
Add an enzyme cocktail to detach cells from the flask surface.
Add media to stop the enzymatic activity and collect the cells in a tube. Centrifuge and discard the supernatant containing enzymes.
Resuspend the cells in serum-free neuronal culture media.
Seed the cells onto gelatin-coated multi-well plate wells.
Incubate. The embryonic stem cells adhere to the gelatin-coated surface. Media constituents, including nutrients, growth factors, and hormones, facilitate cell proliferation and form a monolayer.
At this cell density, the embryonic stem cells produce and release optimal levels of growth factors and cytokines.
These molecules trigger intracellular signaling pathways, facilitating the differentiation of embryonic stem cells into neural progenitors.
Mix the gelatin with 500 milliliters of warm PBS and cover the bottom of the cell culture container with just enough of the solution to coat the surface. While the gelatin is coating, rinse the subconfluent culture of mouse embryonic stem cells two times in PBS. Then, add 1 milliliter of dissociation reagent to the culture.
After two to five minutes, tap the vessel to dislodge the monolayer into a single-cell suspension and collect the culture in a total of 10 milliliters of medium and cell dissociation reagent.
Transfer the cells into a 15-milliliter conical tube and spin them down in a centrifuge. Then, after careful aspiration of the supernatant, resuspend the pellet in 10 milliliters of pre-warmed N2B27 pipetting against the side of the tube to avoid creating bubbles.
Now, count the cells recording the concentration and resuspend them at the appropriate number of cells per unit of volume per well in pre-warmed N2B27 according to the table. Then, aspirate the gelatin from the culture vessel and plate the cells without swirling the container.
Place the cultures in a humid incubator at 37 degrees Celsius and 5% carbon dioxide, replacing the medium every one to two days with fresh N2B27.
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