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.

Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans

“…Continuous culturing of transgenic plant cells frequently resulted in the loss of transgene functions or reduced cell viability. Recently, cryopreservation procedures for various plant cells have been developed [ 27 , 28 ]. We thus attempted to establish a procedure for sodium alginate-based cryopreservation of F .…”

“…Cryopreservation of F . koreana wildtype and transgenic cells was tested under various conditions using sodium alginate based on previous reports with various modifications [ 27 , 28 ]. U18i-CPi-Fk was centrifuged at 100 x g for 5 min, and was then resuspended in Gamborg’s B-5 medium containing 2% (w/v) sodium alginate, with cell density of 5 x 10 6 / ml.…”

Improving Pharmaceutical Protein Production in Oryza sativa

“…Arabidopsis cell lines have been cryogenically frozen at −30 °C or −80 °C and then thawed out, and the cell activity remained similar to unfrozen cells [103]. A similar experiment was also performed in transgenic rice expressing recombinant hCTLA4Ig and cells recovered from cryopreservation banking had cell viability of 88% [102].…”

A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation

“…Several methods can be employed to overcome recalcitrance during cryopreservation. These include one-or two-step freezing protocols and the use of various cryoprotectants (to minimize ice crystal formation and cell damage) or modification of cryopreservation protocols, e.g., by using: the most effective but least toxic vitrification solutions; pre-culture treatment involving sugars, abscisic acid or proline; progressive osmotic dehydration at high sucrose concentrations; and increased cooling/warming rates [3][4][5][6][7]. Sometimes, these protocols are lengthy, and this restricts their use to small-scale cryopreservation of plants/cell lines.…”

“…Sometimes, these protocols are lengthy, and this restricts their use to small-scale cryopreservation of plants/cell lines. As a result, a much simpler method would be most welcome [6]. The effective cryopreservation of ferns has been described for the gametophytes of 18 species [5,[8][9][10] and the young sporophytes of a single species of epiphytic fern, namely Platycerium ridleyi H. Christ.…”