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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 the cooling rate on cell viability of cryopreserved A. thaliana T87 cells. Exponentially growing cells from 7-day-old cultures of T87 were mixed with LSP solution and incubated with shaking at room temperature for 30 min. The cells were cooled to −35°C at a rate of −2, −1 or −0.5°C min−1 using a programmable freezer. After reaching −35°C, the cells were kept at −35°C for 0, 30 or 60 min before freezing in liquid nitrogen. The cryopreserved cells were thawed and plated on filter paper on agar plate for re-growth (a). The viability of frozen cells was measured in three independent samples (b). Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).
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pcs038-F3: Effect of the cooling rate on cell viability of cryopreserved A. thaliana T87 cells. Exponentially growing cells from 7-day-old cultures of T87 were mixed with LSP solution and incubated with shaking at room temperature for 30 min. The cells were cooled to −35°C at a rate of −2, −1 or −0.5°C min−1 using a programmable freezer. After reaching −35°C, the cells were kept at −35°C for 0, 30 or 60 min before freezing in liquid nitrogen. The cryopreserved cells were thawed and plated on filter paper on agar plate for re-growth (a). The viability of frozen cells was measured in three independent samples (b). Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).

Mentions: To optimize pre-freezing conditions, the cells suspended in LS were cooled to −35°C at a rate of −0.5, −1 or −2°C min−1 using a programmable freezer. After reaching −35°C, cells were kept at −35°C for 0, 30 or 60 min, and then plunged into liquid nitrogen (Fig. 3). Cell viability was significantly high when cells were cooled at −0.5°C min−1, and the holding times at −35°C had no significant effect on cell viability when cells were cooled at −0.5°C min−1. In contrast, samples cooled at −2°C min−1 and immediately plunged into liquid nitrogen (duration at −35°C = 0 min) had much lower cell viability (Fig. 3). These results indicated that slower cooling resulted in higher cell viability and that the cooling at −0.5°C min−1 eliminated the need to hold cells at −35°C for 30 or 60 min, which was required when cells were cooled at −2 or −1°C min−1.Fig. 3


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 the cooling rate on cell viability of cryopreserved A. thaliana T87 cells. Exponentially growing cells from 7-day-old cultures of T87 were mixed with LSP solution and incubated with shaking at room temperature for 30 min. The cells were cooled to −35°C at a rate of −2, −1 or −0.5°C min−1 using a programmable freezer. After reaching −35°C, the cells were kept at −35°C for 0, 30 or 60 min before freezing in liquid nitrogen. The cryopreserved cells were thawed and plated on filter paper on agar plate for re-growth (a). The viability of frozen cells was measured in three independent samples (b). 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-F3: Effect of the cooling rate on cell viability of cryopreserved A. thaliana T87 cells. Exponentially growing cells from 7-day-old cultures of T87 were mixed with LSP solution and incubated with shaking at room temperature for 30 min. The cells were cooled to −35°C at a rate of −2, −1 or −0.5°C min−1 using a programmable freezer. After reaching −35°C, the cells were kept at −35°C for 0, 30 or 60 min before freezing in liquid nitrogen. The cryopreserved cells were thawed and plated on filter paper on agar plate for re-growth (a). The viability of frozen cells was measured in three independent samples (b). Values with the same letter are not significantly different according to Scheffé's F-test (P < 0.05).
Mentions: To optimize pre-freezing conditions, the cells suspended in LS were cooled to −35°C at a rate of −0.5, −1 or −2°C min−1 using a programmable freezer. After reaching −35°C, cells were kept at −35°C for 0, 30 or 60 min, and then plunged into liquid nitrogen (Fig. 3). Cell viability was significantly high when cells were cooled at −0.5°C min−1, and the holding times at −35°C had no significant effect on cell viability when cells were cooled at −0.5°C min−1. In contrast, samples cooled at −2°C min−1 and immediately plunged into liquid nitrogen (duration at −35°C = 0 min) had much lower cell viability (Fig. 3). These results indicated that slower cooling resulted in higher cell viability and that the cooling at −0.5°C min−1 eliminated the need to hold cells at −35°C for 30 or 60 min, which was required when cells were cooled at −2 or −1°C min−1.Fig. 3

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