Differentiate ES cells into cystic embryoid bodies

Day -1  Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.

Day 1  Trypsinized the cells as for normal passaging until the colonies lift off. Try to keep the loosely connected clumps of cells together by gentle handling. Then directly plate the cells 1:3 into bacterial grade Petri dishes in LIF free ES cell medium.

Day 3  Aspirate the medium carefully. Avoid sucking up too many of the aggregates. Then add new medium.

Day 5  Aspirate as in Day 3 and replace the medium. At this stage the differentiation the aggregates are called simple embryoid bodies.

Day 7  Change the medium

Day 9  Change the medium

Day 11  Change the medium

Day 13  Change the medium

Day 15 By this day a high percentage of the embryoid bodies will have developed fluid filled cavities. The structures are called cystic embryoid bodies