This article outlines a protocol for differentiating neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) into serum-free embryoid bodies (SFEBs). The method involves a series of media changes and careful handling of cell aggregates to promote neural differentiation.
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 microliters of medium. Transfer four to six aggregates onto each insert with minimal solution. Remove any excess.
During this step, it's important to work quickly and with a steady hand. While removing medium from around the SFEBs, you may see the SFEBs move across the solution due to surface tension. It's OK to reposition them after the medium is removed.
Once all the aggregates are loaded onto inserts, change the medium to 1 milliliter of DM2 and continue culturing the cells. Every other day replace 3/4ths of the medium with fresh medium. After 14 days of culture, start adding DM3 medium to the cultures. Continue the culturing for 16 more days to grow the SFEBs.
繁體中文
