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High-throughput cryopreservation of plant cell cultures for functional genomics.

Ogawa Y, Sakurai N, Oikawa A, Kai K, Morishita Y, Mori K, Moriya K, Fujii F, Aoki K, Suzuki H, Ohta D, Saito K, Shibata D - Plant Cell Physiol. (2012)

Bottom Line: More than 100 samples were processed for freezing simultaneously.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.The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

View Article: PubMed Central - PubMed

Affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.

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.

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A schematic diagram of the simple protocol used for cryopreservation of suspension-cultured plant cells.
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pcs038-F1: A schematic diagram of the simple protocol used for cryopreservation of suspension-cultured plant cells.

Mentions: In preliminary experiments, we found that LS solution (2 M glycerol, 0.4 M sucrose), which was used as a protectant during cryopreservation of Citrus sinensis cells (Sakai et al. 1991), resulted in higher cell viability when cell solution mixtures were incubated at room temperature for up to 2 h, while without replacement of the culture medium mJPL3 with LS, no viable cells were recovered after cryopreservation. Thus, many cell samples could be handled in a high-throughput manner prior to freezing. Here, we optimized conditions for cryopreservation using a modified LS solution (see below) and a programmable freezer; we then modified these conditions to simplify the protocol. Using the simple protocol, only standard laboratory equipment, such as tube containers and a −30°C freezer, were required to process ≥100 cell samples for cryopreservation (Fig. 1).Fig. 1


High-throughput cryopreservation of plant cell cultures for functional genomics.

Ogawa Y, Sakurai N, Oikawa A, Kai K, Morishita Y, Mori K, Moriya K, Fujii F, Aoki K, Suzuki H, Ohta D, Saito K, Shibata D - Plant Cell Physiol. (2012)

A schematic diagram of the simple protocol used for cryopreservation of suspension-cultured plant cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3345369&req=5

pcs038-F1: A schematic diagram of the simple protocol used for cryopreservation of suspension-cultured plant cells.
Mentions: In preliminary experiments, we found that LS solution (2 M glycerol, 0.4 M sucrose), which was used as a protectant during cryopreservation of Citrus sinensis cells (Sakai et al. 1991), resulted in higher cell viability when cell solution mixtures were incubated at room temperature for up to 2 h, while without replacement of the culture medium mJPL3 with LS, no viable cells were recovered after cryopreservation. Thus, many cell samples could be handled in a high-throughput manner prior to freezing. Here, we optimized conditions for cryopreservation using a modified LS solution (see below) and a programmable freezer; we then modified these conditions to simplify the protocol. Using the simple protocol, only standard laboratory equipment, such as tube containers and a −30°C freezer, were required to process ≥100 cell samples for cryopreservation (Fig. 1).Fig. 1

Bottom Line: More than 100 samples were processed for freezing simultaneously.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.The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

View Article: PubMed Central - PubMed

Affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.

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.

Show MeSH
Related in: MedlinePlus