Development and Maintenance of Serum-Free Embryoid Bodies

Begin with a well containing neural progenitor cell or NPC aggregates in differentiating media-1 or DM-1.

These NPCs are precursors of neural lineage generated from human induced pluripotent stem cells or hiPSCs using DM-1 under controlled conditions.

Transfer the NPC aggregates onto a membrane insert placed inside a well containing DM-1 and remove excess media.

Change the medium to DM-2 and incubate.

DM-2 provides nutrients and differentiation factors that promote the maturation of NPCs into different neural cell types.

Undifferentiated NPCs transform into embryoid bodies, known as serum-free embryoid bodies or SFEBs, as the media lacks serum. These EBs resemble the morphology of a developing brain.

Transfer the insert into a well containing DM-3.

DM-3 contains growth factors essential for the maintenance and growth of SFEBs.

Incubate till SFEBs reach the desired size.

After two weeks of culturing the cells, prepare six-well plates with 40-micron cell culture inserts, and 1 milliliter of DM1 medium. Incubate these plates for at least four hours before adding the cultured cells.

Next, using a 200-microliter wide-mouth pipette tip, collect two-week-old cell aggregates with about 20