Department of Electrical and Computer Engineering, University of Manitoba, Winnipeg, Canada.
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Biotechnol Bioeng. 2019 Nov;116(11):2896-2905. doi: 10.1002/bit.27115. Epub 2019 Jul 23.
The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na /K ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na /K ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.
在分批和补料分批培养中，监测细胞在营养限制期间的状态的能力对于优化生物工艺的生长条件非常重要。Na+/K+ATPase 泵的活性水平和细胞质离子浓度直接受到营养水平的影响，因此细胞质电导率可以用作无标记的细胞状态指标。在这项工作中，我们监测了中国仓鼠卵巢（CHO）细胞在营养剥夺和再引入期间细胞质电导率的变化。采用单细胞介电泳，在 48 小时内测量了细胞质电导率的变化。确定了细胞质电导率在营养再引入后恢复到正常水平的条件。此外，还使用不同 Na+/K+ATPase 泵抑制水平的细胞离子通量的数值模拟来预测营养剥夺的 CHO 细胞的最小电导率。该预测值接近观察到的最小实验细胞质电导率，对于能够在引入营养物质时将细胞质电导率恢复到正常存活水平的 CHO 细胞，该预测值接近观察到的最小实验细胞质电导率。通过将饥饿的细胞重新引入营养物质中 36 小时并用台盼蓝排斥测定法测量其增殖来验证饥饿细胞的恢复。我们得出结论，细胞质电导率可用作指示细胞是否处于可恢复状态的标记，使得营养物质的再引入导致细胞恢复到正常健康状态。
