| 摘要: |
| 建立高质量的生物样本库至关重要，低温保存过程中保护剂的种类和浓度对样本的冷冻效果有很大影响。本文以人乳腺细胞HBL-100为研究对象，在Me2SO含量10%的DMEM溶液中分别添加不同浓度海藻糖(0~0.3 mol/L)和不同体积分数FBS(20%~60%)。采用逐步降温法，先在﹣80 ℃冰箱中慢速降温4 h，再快速投入到液氮中(﹣196 ℃)，冻存7 d后，在37 ℃水浴锅中快速摇晃复温。分别用台盼蓝法、CCK法和24 h贴壁率法检测细胞的成活率，结果表明：与对照组相比，海藻糖和FBS对细胞的冷冻效果都有显著影响。当海藻糖浓度一定时，FBS体积分数越高，细胞的冷冻效果越好，但不添加海藻糖时，FBS的作用不明显；当FBS体积分数一定时，三种检测指标在海藻糖浓度为0.2 mol/L时最优，且高浓度海藻糖可能会抑制FBS作用。考虑成本等因素，最适宜冻存人乳腺细胞HBL-100的冻存液为10%Me2SO+40%~60%FBS+0.2 mol/L海藻糖。 |
| 关键词: 低温保存 存活率 海藻糖 胎牛血清 |
| DOI： |
| 投稿时间：2016-10-18
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| 基金项目:国家自然科学基金(50576059)资助项目。 |
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| Effects of Trehalose and Fetal Bovine Serum on Cryopreservation of HBL-100 Human Breast Cells |
|
Wang Meixia, Liang Wei, Ye Ping, Liu Baolin
|
| (Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology) |
| Abstract: |
| Establishing a high-quality biobank is very important, but the cryoprotective agent (CPA) type and concentration have a considerable influence on the cryopreservation effect. In this paper, HBL-100 human breast cells were treated with 10% Me2SO combined with trehalose (0-0.3 mol/L) and fetal bovine serum (FBS; 20%-60%) in Dulbecco’s modified Eagle’s medium (DMEM). Through gradual reduction of the temperature, the cells were cooled in a ﹣80 ℃ freezer for 4 h and then quickly transferred into liquid nitrogen. After storing for 7 days, the cells were shaken quickly at 37 ℃. The cell survival rate was assessed using the trypan blue test, cell counting kit (CCK) assay, and the attached rations after 24 hours. The results show that, compared with the control group, the effects of the trehalose and FBS are very obvious. In particular, when the trehalose concentration is constant, a higher FBS volume fraction yields a better effect. The FBS has no significant effect without trehalose. Further, when the FBS volume fraction is constant, the optimal effect is obtained for 0.2 mol/L trehalose. A high trehalose concentration may weaken the effect of the FBS. Considering the cost and other factors, the most suitable CPAs for the cryopreservation of HBL-100 human breast cells are 10% Me2SO + 40%-60% FBS + 0.2 mol/L trehalose. |
| Key words: cryopreservation viability trehalose fetal bovine serum |
