New method for forming large embryoid bodies using the wall of the culture dish along with an analysis of their structural characteristics

Early embryonic stem (EES) cells, which were established from 2 cell stage embryos obtained from ddY mice, had similar characteristics as embryonic stem (ES) cells. These cells were maintained in an undifferentiated stage in growth media supplemented with leukemia inhibitory factor (LIF) and were capable of differentiating into triploblastic tissues under various growth factors. It has been known that normal sized embryoid bodies (EBs) are formed by removing LIF. In this study, large EBs gradually formed along the side wall of a culture dish, particularly at the boundary between the air and the growth medium when cells were cultured for a considerable period of time and without subculturing. We call this method the “wall adhesion culture” procedure. The method itself is simple and do not need any instruments except plastic dishes because only the side walls of the dishes were utilized. The mean thickness of the large EBs was about 1.5 mm 3 months after establishing the static culture. Their surface was covered with a monolayer of cells and they contained an eosinophilic cell matrix. By electron microscopy, some characteristic structures was observed, such as intracisternal A particles which were present inside the swelling of the rough endoplasmic reticulum. Since many tissues derived from ES cells are obtained through EBs, it is expected that efficient acquisition of sufficient quantities of these structures using the wall adhesion culture procedure will be a shortcut for using ES cells in regenerative medicine.