In order to establish a minimally invasive and safe liver regenerative technology, a technique for fabricating liver tissue possessing a vascular network was developed by subcutaneously transplanting a cell sheet composed of primary human hepatocytes and normal fibroblasts. However, differences in fibroblast characteristics owing to donor age may threaten the stability of liver tissue regenerated via this technology. Herein we describe the influence of fibroblasts from multiple donors on the fabrication of engineered human hepatocyte tissues in vitro and in vivo. Primary human hepatocytes were cultured with seven strains of fibroblasts derived from the skins of donors of various ages, ranging from a fetus (12 weeks) to the elderly (69 years). Engineered hepatocyte sheets were successfully harvested for all strains. At 2 weeks after the subcutaneous transplantation of the hepatocyte sheets into mice, the highest human albumin (hALB) serum concentration was noted in the mouse containing fibroblasts from a 12 year old (TIG- 118). Since the platelet-derived growth factor subunit B (PDGFB) gene expression of TIG-118 cells was significantly higher than that in the other cells, PDGFB may be considered to play an important role in the initial subcutaneous engraftment of primary human hepatocytes. Even though hALB concentration exhibited a parabolic tendency with age, there was no statistically significant difference noted within 6-8 weeks after transplantation. The present study demonstrates that this technology can produce consistent and stable hepatocyte sheets that exhibit long-term survival and liver-specific functionality in vivo regardless of the fibroblast donor age. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.