Objective:To investigate the effects of 3-D simulated microgravity culture on human hepatic stem cell proliferation and differentiation. Methods:The human hepatic stem cells,which were isolated from human fetal livers,were seeded in simulated microgravity 3-D Rotary Cell Culture System,conventional static culture and microcarrier culture system respectively. Cell morphology and growth pattern were observed under inverted phase contrast microscope and scanning electron microscope (SEM). Cellular proliferative activity and viability were assessed by monitoring the glucose consumption and lactate dehydrogenase activity in culture supernatants respectively. The specific transcripts of hepatic progenitors (ALB,EpCAM,CK19,AFP,HNF6,CYP3A4,CYP3A7) in different culture systems were detected by quantitative RT-PCR. Results:The hepatic stem cells,cultured in simulated microgravity 3-D culture system,efficiently adhered to the collagen-coated microcarriers and formed complex,multilayered 3-D aggregates. A massive extracellular matrix and microvillus were observed on the surface of 3-D aggregates under SEM. The glucose consumption in culture supernatants increased on day 9 and reached its peak level on day 17,whereas,the LDH activity maintained in low level. The RNA isolated from cells on day 21 demonstrated higher levels of EpCAM,ALB,CK19,HNF6 and CYP3A7,but no AFP or CYP3A4,compared with those from the cells in microcarrier culture system (P < 0.05). These results showed that hepatic stem cells proliferated more efficiently with more stable phenotypes in simulated microgravity 3-D culture system than in control cultures. Conclusion:Simulated microgravity 3-D culture system provides superior conditions for hepatic stem cells survival and proliferation, and should be useful for generating cells that can be used in bioartificial liver support systems or tissue engineered liver grafts.