Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytocluome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.