Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.
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 cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS.
Department of Chemistry and Biochemistry, Suzuka National College of Technology, Shiroko-cho, Suzuka, Mie 510-0294, Japan. Electronic address: ogawa@chem.suzuka-ct.ac.jp.
Akiko
A
Ogawa
Department of Applied Chemistry and Biochemistry, University of Fukui, Bunkyo 3-9-1, Fukui, Fukui 910-8507, Japan.
Shinya
S
Mizui
Department of Applied Chemistry and Biochemistry, University of Fukui, Bunkyo 3-9-1, Fukui, Fukui 910-8507, Japan.
Yasuhito
Y
Chida
Department of Applied Chemistry and Biochemistry, University of Fukui, Bunkyo 3-9-1, Fukui, Fukui 910-8507, Japan.
Masafumi
M
Shimizu
Department of Applied Chemistry and Biochemistry, University of Fukui, Bunkyo 3-9-1, Fukui, Fukui 910-8507, Japan.
Satoshi
S
Terada
Fukui Prefectural Food Process, Maruoka-cho, Tsubonouchi 1-1-1, Sakai, Fukui 910-0343, Japan.
Takeshi
T
Ohura
Fukui Prefectural Food Process, Maruoka-cho, Tsubonouchi 1-1-1, Sakai, Fukui 910-0343, Japan.
Kyo-Ichi
K
Kobayashi
ELLE ROSE CO., Ltd., Sakai-cho Higashi 36-81,Sakai, Fukui 910-0033, Japan.
Saori
S
Yasukawa
ELLE ROSE CO., Ltd., Sakai-cho Higashi 36-81,Sakai, Fukui 910-0033, Japan.
Nobuyuki
N
Moriyama
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005615:Freezing; D005630:Fructans / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D006825:Hybridomas; D051379:Mice
D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D005630:Fructans; D004121:Dimethyl Sulfoxide
Chinese hamster ovary cells; Cryopreservation; Freezing stress; Fructan; Hybridoma; Serum-free