To clinically apply bioartificial livers (BALs), an effective liver cell cryopreservation method is required for a stable cell supply. In this study, we performed tissue-engineered construct (TEC) cryopreservation of fetal liver cells (FLCs) in which FLCs cultured within a porous polymer scaffold were cryopreserved. Growth and albumin secretion in TEC-cryopreserved FLCs after thawing were compared to freshly isolated FLCs (control experiments). The effect of preculture duration prior to cryopreservation (0-3 weeks) on these functions was also examined. In the three-dimensional cultures, the TEC-cryopreserved FLCs with preculturing showed constant growth, and this growth was comparable to controls. On the contrary, the TEC-cryopreserved FLCs without preculturing did not proliferate after thawing. Albumin secretion of TEC-cryopreserved FLCs with preculturing rapidly increased up to day 12 and high secretory activity comparable to controls was maintained thereafter in FLCs with 1- or 2-week preculturing, suggesting this as an appropriate preculture duration. Compared to conventionally cryopreserved FLCs, growth and albumin secretion in the TEC-cryopreserved FLCs were significantly higher, indicating their usefulness as a potent cell source for BALs. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.