Development and Maintenance of Serum-Free Embryoid Bodies
Transcription
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.
