title: High-throughput cryopreservation of plant cell cultures for functional genomics.
abstract: Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.
authors.affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ogawa
authors.forename: Nozomu
authors.initials: N
authors.lastname: Sakurai
authors.forename: Akira
authors.initials: A
authors.lastname: Oikawa
authors.forename: Kosuke
authors.initials: K
authors.lastname: Kai
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Morishita
authors.forename: Kumiko
authors.initials: K
authors.lastname: Mori
authors.forename: Kanami
authors.initials: K
authors.lastname: Moriya
authors.forename: Fumiko
authors.initials: F
authors.lastname: Fujii
authors.forename: Koh
authors.initials: K
authors.lastname: Aoki
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Daisaku
authors.initials: D
authors.lastname: Ohta
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shibata
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005615:Freezing; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D023281:Genomics / Q000379:methods*; D005966:Glucuronidase / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D059828:Plant Cells / Q000187:drug effects / Q000378:metabolism*; D011392:Proline / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D011392:Proline; D005966:Glucuronidase