AIM: To develop the effective technology for reconstruction 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 highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 107 FLC-5 cells were incubated in the RFB. After 5 d, 2 x 107 A7 cells were added in a similar manner followed by another addition of 107 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB.RESULTS: By transmission electron microscopy, FLC-5,M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (WO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium,and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased.CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.