AIM:To develop the effective technology for reconstruc-tion of a liver organ in vitro using a bio-artificial liver.METHODS:We previously reported that a radial-flowbioreactor(RFB)could provide a three-dimensional high-density culture system.We presently reconstructed theliver organoid using a functional human hepatocellularcarcinoma cell line(FLC-5)as hepatocytes together withmouse immortalized sinusoidal endothelial cell(SEC)lineM1 and mouse immortalized hepatic stellate cell(HSC)line A7 as non parenchymal cells in the RFB.Two×10~7FLC-5 cells were incubated in the RFB.After 5 d,2×10~7A7 cells were added in a similar manner followed by an-other addition of 10~7 M1 cells 5 d later.After three daysof perfusion,some cellulose beads with the adherentcells were harvested.The last incubation period includedperfusion with 200 nmol/L swinholide A for 2 h and thenthe remaining cellulose beads along with adherent cellswere harvested from the RFB.The cell morphology wasobserved by transmission electron microscopy(TEM)andscanning electron microscopy(SEM).To assess hepato- cyte function,we compared mRNA expression for ureacycle enzymes as well as albumin synthesis by FLC-5 inmonolayer cultures compared to those of single-type cul-tures and cocultures in the RFB.RESULTS:By transmission electron microscopy,FLC-5,M1,and A7 were arranged in relation to the perfusionside in a liver-like organization.Structures resemblingbile canaliculi were seen between FCL-5 cells.Scanningelectron microscopy demonstrated fenestrae on SECsurfaces.The number of vesiculo-vacuolar organelles(WO)and fenestrae increased when we introduced theactin-binding agent swinholide-A in the RFB for 2h.Withrespect to liver function,urea was found in the medium,and expression of mRNAs encoding arginosuccinate syn-thetase and arginase increased when the three cell typeswere cocultured in the RFB.However,albumin synthesisdecreased.CONCLUSION:Co-culture in the RFB system can dra-matically change the structure and function of all celltypes,including the functional characteristics of hepato-cytes.Our system proves effective for reconstruction ofa liver organoid using a bio-artificial liver. AIM: To develop the effective technology for reconstruc- tion of a liver organ in vitro using a bio-artificial liver. METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed theliver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) lineM1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB.Two × 10 ~ 7 FLC-5 cells were incubated in the RFB.After 5 d, 2 × 10 -7 A cells were added in a similar manner followed by-other addition of 10 ~ 7 M 1 cells for 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period was included with 200 nmol / L swinholide A for 2 h and the nthe remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology wasobserved by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepato-cyte function, we compared mRNA expression for ureacycle enzymes as well as albumin synthesis by FLC-5 inmonolayer cultures compared to those of single-type cul- tures and cocultures in the RFB.RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusionside in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanningelectron microscopy demonstrated fenestrae on SECsurfaces. The number of vesiculo-vacuolar organelles (WO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h.Withrespect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate syn-thetase and arginase increased when the three cell types of cocultured in the RFB.However, albumin synthesis decreased. CONCLUSION: Co-culture in the RFB system can dra-matically change the structure and func tion of all celltypes, including the functional characteristics of hepato-cytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.