基于电容测定的罗汉果细胞超低温保藏快速方法

Rapid cryopreservation for Siraitia grosvenorii cells based on cells’ capacitance detection

摘要:

建立了一种基于活细胞电容值定量测定的植物细胞超低温保藏的快速评价方法，优化了罗汉果细胞超低温保藏方法。通过采用活细胞传感仪测定冻存后细胞的存活率并结合细胞生活力 (细胞线粒体活性/TTC) 对罗汉果细胞的低温保藏过程进行优化，确定了罗汉果细胞较为适宜的冷冻保护剂组分为基本培养基中添加10%的蔗糖和10%的DMSO。预处理剂的考察实验表明，采用0.2 mol/L蔗糖的预处理剂处理细胞时冻存后细胞存活率和细胞活力较高；采用0.2 mol/L蔗糖预处理剂处理细胞时，随着预处理时间的增加，细胞存活率先增加后降低，预处理时间为9 h时，细胞存活率和细胞活力最高。保藏后的细胞复苏实验结果表明：细胞存活率与采用活细胞电容值得到的细胞存活率具有很好的一致性，同时经过冻存的细胞复苏培养后，仍保留了原始细胞的形态和合成甜苷V的特性，说明该冻存方法适用于罗汉果细胞的超低温保藏。因此基于活细胞传感仪测得的电容值进行细胞冻存过程细胞活性的快速评价方法具有较好的可行性和可靠性。

Abstract:

A rapid quantitative evaluation method for Siraitia grosvenorii cells was successfully developed based on plant cells’ capacitance value detected by a viable cell mass monitor and the cryopreservation of S. grosvenorii suspension cells was optimized. The survival rate of S. grosvenorii cells was quantitatively measured by viable cell mass monitor and 2, 3, 5-triphenyltetrazolium chloride (TTC). An optimum cryoprotectant recipe is that the growth medium contained 10% sucrose and 10% DMSO. The experimental results also showed higher cell survival rates and cell viabilities were achieved when suspension cells were treated with pretreatment of 0.2 mol/L sucrose. With the increase of concentration of sucrose, however, the cell survival rate was decreased. And the cell survival rate represented a bell shape with the increase of pretreatment time. The highest cell survival rate and cell viability were obtained with the 9 h’ s pretreatment. In addition, there was a good correlation between the cell survival rate measured by cell recovery test and that measured by viable cell mass monitor, while there were no significant differences in the cell morphology and the ability of mogrosides V production by S. grosvenorii cells cultured in suspension after cryopreservation. Therefore, the evaluation method developed based on the viable cell mass monitor has a good feasibility and reliability.