A multi-well plate microfabricated with highly hydrophilic polymer, Cell-able facilitates 3-dimensional (3D) culture of primary hepatocytes and induces long-term well-functioning culture, comparing to conventional monolayer. We report here the improved outcome using novel culture medium specialized for human hepatocytes and mouse fibroblasts as feeder cells. [Methods] Primary human hepatocytes were isolated from surgically resected liver tissue (Etical permission No.385 and 396) or purchased as cryopreserved hepatocytes (Xenotech). Hepatocytes (2x10^4/well) were seeded on 96-weell type Cell-able that had been precultured with mouse fibroblasts (JCRB9019 or ATCC CCL-163). Cells were cultured with RM101 (Transparent) based on Williams medium E containing 1% fetal bovine serum. Hepatocyte functions were evaluated by testosterone metabolism and transportation of tritiated-taurocholic acid and carboxy-dichlorofluorescein diacetate (CDF-DA). [Results and Discussion] Hepatocytes cultured on Cell-able with RM101 showed 1.55 to 3.43 fold higher activity of testosterone 6-beta hydroxylation of other 5 media (3 commercially available and 2 previously reported media) examined on day 7 and the superiority increased thereafter until day 21. Activity of testosterone 6-beta hydroxylation was maintained well for 54 days. Noticeably, the cells on day 54 increased the activity from 0.15 to 0.80 fmol/cell/min in response to rifampicin. Time course changes in taurocholate uptake were 24.1 fmol/cell (day of isolation), 2.84 (day 3), and 6.83 (day 7), while those of monolayer hepatocytes were 0.49 (day 3) and not detectable on day 7. The increase of the activity from day 3 to 7 on Cell-able was thought to be due to maturation of spheroid structure. The evidence of bilially efflux was examined by cofocal microscope using CDF-DA. The clear fluorescent spots were detectable intercellular area of the spheroids. When calcium ion was withdrawn from the culture medium, the intensity and sharpness of fluorescent spots became weaker and vaguer, suggesting the formation of small bile duct-like structure between hepatocytes. The present results indicate that Cell-able culture system suits for the examination of a sequence of reactions of DMPK research.