Institute for Advanced Studies in Biological Process Technology, University of Minnesota, 55108-6106, St. Paul, Minnesota, USA.
Cytotechnology. 1995 Jan;19(1):11-26. doi: 10.1007/BF00749751.
Because of the interest in understanding and optimizing secretion of proteins from mammalian cells, reliable and more reproducible methods are needed to monitor the external redox potential of animal cells in suspension culture. An improved off-line method was established that greatly reduces the typically long response time of redox electrodes in cell culture media and improves the standardization of redox probes. In addition, the dependence of medium redox potential on dissolved oxygen concentrations and pH was investigated using cell-free medium. Off-line as well as on-line redox potential measurements were then applied to spinner or bioreactor cultures of murine hybridoma cells. Serum containing or protein-free medium were used. The time dependence of the experimentally determined external redox potential was found to be affected not only by oxygen, pH, and medium composition. but to a significant extent by the rate of generation of reductants by hybridoma cells. The observed specific rate of medium reduction by generation of reductants (ΔmV h(-1) viable cell(-1)) decreased during exponential growth while cell number increased from 2×10(5) viable cells ml(-1) to 3.5×10(6) viable cells ml(-1). This rate, however, was essentially constant at -7.3 mV h(-1)±3.7 mV h(-1) per 10(10) viable cells during growth under conditions of constant dissolved oxygen tension and constant pH. Using these observations, the quantity of reductants synthesized and secreted into the medium by viable hybridoma cells was estimated to be approximately 1.3 mole h(-1) per 10(10) viable hybridoma cells. The time course of specific monoclonal antibody secretion rate did not correlate with changes in the external oxidation/reduction potential in either serum containing or protein-free medium.
由于人们对理解和优化哺乳动物细胞分泌蛋白质的兴趣，需要可靠且更具可重复性的方法来监测悬浮培养动物细胞的外部氧化还原电位。建立了一种改进的离线方法，该方法大大缩短了细胞培养介质中氧化还原电极的典型长响应时间，并提高了氧化还原探针的标准化程度。此外，使用无细胞培养基研究了介质氧化还原电位对溶解氧浓度和 pH 的依赖性。然后将离线和在线氧化还原电位测量应用于鼠杂交瘤细胞的旋转瓶或生物反应器培养。使用含血清或无蛋白的培养基。实验确定的外部氧化还原电位的时间依赖性不仅受氧气，pH 和培养基组成的影响，而且在很大程度上还受杂交瘤细胞产生还原剂的速率的影响。观察到的通过还原剂生成（ΔmV h(-1) 活细胞(-1))使介质还原的特定比速率在指数生长期间降低，而细胞数从 2×10(5)个活细胞 ml(-1)增加到 3.5×10(6)个活细胞 ml(-1)。然而，在恒定溶解氧分压和恒定 pH 条件下生长时，该速率基本上保持恒定，为-7.3 mV h(-1)±3.7 mV h(-1) 每 10(10)个活细胞。根据这些观察结果，估计活杂交瘤细胞合成并分泌到培养基中的还原剂的量约为 1.3 毫摩尔 h(-1) 每 10(10)个活杂交瘤细胞。在含血清或无蛋白的培养基中，特异性单克隆抗体分泌速率的时间过程与外部氧化还原电势的变化均无相关性。
