Ogawa Yoichi, Suzuki Hideyuki, Sakurai Nozomu, Aoki Koh, Saito Kazuki, Shibata Daisuke
Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.
Cryo Letters. 2008 Sep-Oct;29(5):427-36.
We established a simple cryopreservation protocol for Arabidopsis T87 cells using an encapsulation-dehydration method. T87 cells were encapsulated into alginate beads containing 2 M glycerol and 0.4 M sucrose. Alginate beads containing T87 cells were dehydrated with silica gel for 2 h (to c. 0.7 g water per g dry weight followed by immersed in LN. After rewarming at 35 degree C for 3 min and 1-d incubation under continuous illumination at 22 degree C, cryopreserved T87 cells exhibited considerable regrowth. Exponentially-grown 7-d-old T87 cells regrew more vigorously (86% of control) than 14-d-old cells after cryopreservation without preculture in medium containing 0.3 M sucrose. Genetic stability of cryopreserved T87 cells was demonstrated by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and principal component analysis (PCA). Transformed T87 cells were cryopreserved using established protocols, and GUS expression was maintained within a 2-fold variance. These results indicate that cryopreservation of T87 cells is useful for comprehensive metabolomics research and for the large scale collection of transformed cultured cell lines for functional genomics research.
我们使用包埋脱水法为拟南芥T87细胞建立了一种简单的冷冻保存方案。将T87细胞包埋在含有2 M甘油和0.4 M蔗糖的藻酸盐珠中。含有T87细胞的藻酸盐珠用硅胶脱水2小时（至约每克干重0.7克水），然后浸入液氮中。在35℃复温3分钟并在22℃连续光照下培养1天后，冷冻保存的T87细胞表现出显著的再生长。在不含0.3 M蔗糖的培养基中预培养后，指数生长的7日龄T87细胞冷冻保存后的再生长比14日龄细胞更旺盛（为对照的86%）。通过气相色谱飞行时间质谱（GC-TOF-MS）和主成分分析（PCA）证明了冷冻保存的T87细胞的遗传稳定性。使用既定方案对转化的T87细胞进行冷冻保存，GUS表达保持在2倍差异范围内。这些结果表明，T87细胞的冷冻保存对于全面的代谢组学研究以及用于功能基因组学研究的转化培养细胞系的大规模收集是有用的。
