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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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Cryopreservation of A. thaliana T87 cells using commercial tube containers. The vials containing T87 cells in LSP solution were placed in a Mr. Frosty container and cooled at −30 or −80°C for 1–8 h (a). The vials containing T87 cells in LSP solution were placed in an EPS tube container (HS4283 or SD-14) and cooled at −30°C for 1–8 h (b). Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).
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pcs038-F5: Cryopreservation of A. thaliana T87 cells using commercial tube containers. The vials containing T87 cells in LSP solution were placed in a Mr. Frosty container and cooled at −30 or −80°C for 1–8 h (a). The vials containing T87 cells in LSP solution were placed in an EPS tube container (HS4283 or SD-14) and cooled at −30°C for 1–8 h (b). Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).

Mentions: To simplify the protocol, we assessed whether enclosing the sample vials containing cell–LSP mixtures in a commercial tube container would eliminate the need for programmed pre-freezing. First we tested a commercial freezing container (Mr. Frosty, Nalge Nunc), which is designed for cryopreservation of animal cells and holds a maximum of 18 vials. The vials with A. thaliana T87 cells suspended in LSP solution were placed in the container, which was then kept in a −30°C freezer for 6 h; the cell viability upon thawing was approximately 60% (Fig. 5a). However, when sample vials in the container were kept in a −80°C freezer for 8 h, as recommended for animal cells by the supplier, the cell viability was significantly low (<10%; Fig. 5a). Next, we tested two types of commercially available tube containers made of thick expanded polystyrene (EPS), HS4283 (W 210 mm×D 108 mm×H 67 mm for 50 vials, Heathrow Scientific) and SD-14 (W 175 mm×D 175 mm×H 81 mm for 64 vials, Maruemu Co. Ltd.). Samples in these containers were kept in a −30°C freezer for >4 h and had a significantly high viability, approximately 70%, upon thawing; this viability was comparable with that achieved with programmed pre-freezing (Fig. 5b). The cooling rate inside these containers was estimated to be −0.25°C min−1 under these conditions; therefore, the vials were likely to reach −30°C within 4 h.Fig. 5


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)

Cryopreservation of A. thaliana T87 cells using commercial tube containers. The vials containing T87 cells in LSP solution were placed in a Mr. Frosty container and cooled at −30 or −80°C for 1–8 h (a). The vials containing T87 cells in LSP solution were placed in an EPS tube container (HS4283 or SD-14) and cooled at −30°C for 1–8 h (b). Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

pcs038-F5: Cryopreservation of A. thaliana T87 cells using commercial tube containers. The vials containing T87 cells in LSP solution were placed in a Mr. Frosty container and cooled at −30 or −80°C for 1–8 h (a). The vials containing T87 cells in LSP solution were placed in an EPS tube container (HS4283 or SD-14) and cooled at −30°C for 1–8 h (b). Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).
Mentions: To simplify the protocol, we assessed whether enclosing the sample vials containing cell–LSP mixtures in a commercial tube container would eliminate the need for programmed pre-freezing. First we tested a commercial freezing container (Mr. Frosty, Nalge Nunc), which is designed for cryopreservation of animal cells and holds a maximum of 18 vials. The vials with A. thaliana T87 cells suspended in LSP solution were placed in the container, which was then kept in a −30°C freezer for 6 h; the cell viability upon thawing was approximately 60% (Fig. 5a). However, when sample vials in the container were kept in a −80°C freezer for 8 h, as recommended for animal cells by the supplier, the cell viability was significantly low (<10%; Fig. 5a). Next, we tested two types of commercially available tube containers made of thick expanded polystyrene (EPS), HS4283 (W 210 mm×D 108 mm×H 67 mm for 50 vials, Heathrow Scientific) and SD-14 (W 175 mm×D 175 mm×H 81 mm for 64 vials, Maruemu Co. Ltd.). Samples in these containers were kept in a −30°C freezer for >4 h and had a significantly high viability, approximately 70%, upon thawing; this viability was comparable with that achieved with programmed pre-freezing (Fig. 5b). The cooling rate inside these containers was estimated to be −0.25°C min−1 under these conditions; therefore, the vials were likely to reach −30°C within 4 h.Fig. 5

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