Chen Aaron, Leith Matthew, Tu Roger, Tahim Gurpreet, Sudra Anish, Bhargava Swapnil
BioProcess Development, Seattle Genetics, Inc., Bothell, Washington, United States of America.
Clinical Manufacturing, Seattle Genetics, Inc., Bothell, Washington, United States of America.
PLoS One. 2017 Mar 6;12(3):e0173375. doi: 10.1371/journal.pone.0173375. eCollection 2017.
Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions.
基于台盼蓝染料排除法的市售自动细胞计数仪在工业细胞培养工艺开发和生产中被广泛使用，以提高通量并消除与手动血细胞计数器相关的主观解读中固有的变异性。使用这些细胞计数仪时，通常需要进行样品稀释以保持在测定测量范围内；然而，时间和稀释剂对细胞培养的影响尚未得到充分了解。本报告介绍了磷酸盐缓冲盐水作为稀释剂与自动细胞计数仪结合使用时对细胞活力的不利影响。细胞活力降低归因于自动细胞计数仪引入的剪切应力。此外，样品在磷酸盐缓冲盐水中孵育的时间长度也导致了观察到的细胞活力下降。最后，由于错误的活力测量可能会严重影响工艺决策和产品质量，本报告确定了几种替代稀释剂，它们可以随时间维持细胞培养活力，以确保准确反映细胞培养条件。
