Objective    To study the formation and differentiation of mouse embryoid bodies (EBs) under simulating microgravity.      Methods    Mouse embryonic stem cells (ES cells) were dissociated with trypsin and seeded onto the rotary bioreactor simulating microgravity at a concentration of 1×10

6

·ml

-1

. The bioreactor was set to rotate at low speed at 37 ℃ in a humidified atmosphere containing 5% CO

2

. The formation and differentiation of EBs were observ

ed under microscope and by hematoxylin/eosin (HE) staining. LDH leakage analysis was performed. The EBs were seeded in 6-well plates and cultured for 3 to 5 days. The differentiation of EBs derived cells was examined by phase contrast microscope.     Results    A lot of multicellular aggregations were seen after 24-hour rotary culture. Phase-contract microscope and stereomicroscope showed that homogeneous EBs formed from these aggregations after 72-hour culture. The volume of EBs gradually increased

accompanied with the generation of new EBs. HE staining demonstrated the characteristics of multicellular differentiation of EBs cell-derived cells. Necrosis occurred in the center of some large EBs after 12-day cultivation

meanwhile the LDH leakage was increased. Blood vessels structure

endothelioid cells

fibroblasts

hepatocyte-like cells

and spontaneously beating cardiocyte-like cells  were observed under phase-contract microscope after EBs seeding on 6-well plates.      Conclusion    Under simulating microgravity

a great quantity of EBs were differentiated from mouse ES cells and formed quickly. These results reveal that rotary bioreactor should be a useful tool for ES cell research.