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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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Effect of cryoprotection on the viability of cryopreserved A. thaliana T87 cells. T87 cells from 7-day-old cultures were incubated in LS solution at room temperature for 0–120 min with or without shaking. Cells were then cooled to −35°C at −0.5°C min−1. Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. NA, not available. No significant difference was observed among values by Kruskal–Wallis test (P = 0.194).
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pcs038-F2: Effect of cryoprotection on the viability of cryopreserved A. thaliana T87 cells. T87 cells from 7-day-old cultures were incubated in LS solution at room temperature for 0–120 min with or without shaking. Cells were then cooled to −35°C at −0.5°C min−1. Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. NA, not available. No significant difference was observed among values by Kruskal–Wallis test (P = 0.194).

Mentions: We examined the effects of LS solution on cell viability of A. thaliana T87 cells, which were subjected to cryopreservation under strictly controlled cooling conditions using a programmable freezer. Cells in exponential phase were suspended in LS solution and then kept for 0, 30, 60, 90 or 120 min at room temperature with or without shaking; cells were cooled at a rate of −0.5°C min−1 down to −35°C (i.e. pre-freezing). Cooled samples were then plunged into liquid nitrogen. Cell viability was defined as the ratio of the percentage of viable cells after freezing to that of unfrozen cells as shown in the Materials and Methods. Incubation in LS solution for up to 120 min with or without shaking showed high cell viability of 45–55% without a significant difference at P < 0.05 (Fig. 2). As 0 min of incubation with LS gave almost the same viability as 30–120 min, it is likely that the cooling rate of the cells with LS (−0.5°C min−1) is slow enough to protect the cells for cold damage without incubation at room temperature. As incubation in LS for up to 120 min is enough to prepare >100 sample vials, LS solution is suitable for high-throughput cryopreservation of Arabidopsis cells.Fig. 2


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)

Effect of cryoprotection on the viability of cryopreserved A. thaliana T87 cells. T87 cells from 7-day-old cultures were incubated in LS solution at room temperature for 0–120 min with or without shaking. Cells were then cooled to −35°C at −0.5°C min−1. Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. NA, not available. No significant difference was observed among values by Kruskal–Wallis test (P = 0.194).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

pcs038-F2: Effect of cryoprotection on the viability of cryopreserved A. thaliana T87 cells. T87 cells from 7-day-old cultures were incubated in LS solution at room temperature for 0–120 min with or without shaking. Cells were then cooled to −35°C at −0.5°C min−1. Data are presented as the mean ± SD of three independent experiments, each of which was examined using three vials. NA, not available. No significant difference was observed among values by Kruskal–Wallis test (P = 0.194).
Mentions: We examined the effects of LS solution on cell viability of A. thaliana T87 cells, which were subjected to cryopreservation under strictly controlled cooling conditions using a programmable freezer. Cells in exponential phase were suspended in LS solution and then kept for 0, 30, 60, 90 or 120 min at room temperature with or without shaking; cells were cooled at a rate of −0.5°C min−1 down to −35°C (i.e. pre-freezing). Cooled samples were then plunged into liquid nitrogen. Cell viability was defined as the ratio of the percentage of viable cells after freezing to that of unfrozen cells as shown in the Materials and Methods. Incubation in LS solution for up to 120 min with or without shaking showed high cell viability of 45–55% without a significant difference at P < 0.05 (Fig. 2). As 0 min of incubation with LS gave almost the same viability as 30–120 min, it is likely that the cooling rate of the cells with LS (−0.5°C min−1) is slow enough to protect the cells for cold damage without incubation at room temperature. As incubation in LS for up to 120 min is enough to prepare >100 sample vials, LS solution is suitable for high-throughput cryopreservation of Arabidopsis cells.Fig. 2

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