Department of Electrical and Computer Engineering, University of Manitoba, Winnipeg R3T 5V6, Canada.
Department of Microbiology, University of Manitoba, Winnipeg R3T 2N2, Canada.
Bioelectrochemistry. 2018 Dec;124:73-79. doi: 10.1016/j.bioelechem.2018.07.003. Epub 2018 Jul 5.
Nutrient depletion in fed-batch cultures and at the end of batch cultures is among the main causes of stress on cells and a trigger of apoptosis. In this study, we investigated changes in the cytoplasm conductivity of Chinese hamster ovary (CHO) cells under controlled starvation. Employing a single-cell dielectrophoresis (DEP) cytometer, we measured the DEP response of CHO cells incubated in a medium without glucose and glutamine over a 48-h period. Using the measured data in conjunction with numerical simulations, we determined the cytoplasm conductivity of viable and apoptotic cell subpopulations. The results show that a small subpopulation of apoptotic cells emerges after 24 to 36 h of starvation and increases rapidly over a short period of time, <12 h. The apoptotic cells have a dramatically lower cytoplasm conductivity, ∼0.05 S/m, than viable cells, ∼0.45 S/m. Viability of starvation cultures was measured by fluorescent cytometry, DEP cytometry, and trypan blue exclusion assays. DEP, Annexin V, caspase-8, and 7-AAD assays show a similar decline in viability after 36 h of starvation and indicate a very low viability after 48 h. Trypan blue exclusion assay fails to detect early-stage viability decline and estimates a much higher viability after 48 h.
补料分批培养和分批培养末期的营养耗尽是细胞应激和凋亡触发的主要原因之一。在这项研究中，我们研究了控制饥饿条件下中国仓鼠卵巢（CHO）细胞细胞质电导率的变化。我们使用单细胞介电泳（DEP）细胞仪，测量了在无葡萄糖和谷氨酰胺的培养基中孵育的 CHO 细胞在 48 小时期间的 DEP 响应。使用测量数据和数值模拟，我们确定了存活和凋亡细胞亚群的细胞质电导率。结果表明，在饥饿 24 至 36 小时后，会出现一小部分凋亡细胞，并且在很短的时间内迅速增加，<12 小时。凋亡细胞的细胞质电导率明显低于存活细胞，约为 0.05 S/m，而存活细胞的细胞质电导率约为 0.45 S/m。通过荧光细胞术、DEP 细胞术和台盼蓝排斥试验测量饥饿培养物的存活率。DEP、 Annexin V、caspase-8 和 7-AAD 试验表明，在饥饿 36 小时后存活率相似下降，并表明在 48 小时后存活率非常低。台盼蓝排斥试验无法检测早期存活率下降，并估计在 48 小时后存活率更高。
