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首页> 外文期刊>Biotechnology Progress >CHO Cells Engineered for Fluorescence Read out of Cell Cycle and Growth Rate in Real Time
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CHO Cells Engineered for Fluorescence Read out of Cell Cycle and Growth Rate in Real Time

机译：CHO细胞在实时读出细胞周期和生长速度的荧光

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摘要

For efficient production of recombinant proteins by mammalian cells in a bioreactor, optimal growth rates are required and represent the most important process parameter. We present the first successful attempt to monitor the growth behavior and cell cycle state of a mammalian production relevant cell line under bioreactor cultivation conditions up to 1.2 l, utilizing a fluorescent read-out without the need of additional staining or marking. For this purpose, we developed two new production relevant cell line derivatives (CHO-K1 FUCCI CM & CHO-K1 FUCCI CN) and corresponding analytical methods. The approach is easily scalable, applicable to mammalian recombinant protein production cell lines, and it allows for real-time monitoring using appropriate fluorescence probes. It is based on the Ubiquitination-based Cell Cycle Indicator (FUCCI) system developed by Miyawaki et al. CHO-K1 was chosen as a model cell line due to its close relationship to several production cell lines. 1 We defined a new process parameter ired, a quantitative and numerically robust representation of the cell cycle distribution, and demonstrate it to be linearly correlated with the cell cycle state and inversely related to the real time growth rate. Detection of growth rate limitations is possible earlier than using cell-count-based approaches. Analytics were compatible with bulk fluorescence methods, using a plate reader as well as a flow cytometer. For future real time applications in industry scale bioreactors we recommend the use of on-line or at-line fluorescence probes. (C) 2017 American Institute of Chemical Engineers
机译：为了在生物反应器中通过哺乳动物细胞有效生产重组蛋白，需要最佳的生长速率并表示最重要的过程参数。我们介绍了在生物反应器培养条件下监测哺乳动物生产相关细胞系的生长行为和细胞周期状态，其在生物反应器培养条件下，其利用荧光读出而不需要额外的染色或标记。为此，我们开发了两种新的生产相关细胞系衍生物（CHO-K1 FUCCI CM＆CHO-K1 FUCCI CN）和相应的分析方法。该方法很容易可扩展，适用于哺乳动物重组蛋白质生产细胞系，并且允许使用适当的荧光探针实时监测。它基于Miyawaki等人开发的泛素型的细胞周期指示器（FUCCI）系统。由于其与几种生产细胞系密切相关，因此选择CHO-K1作为模型细胞系。 1我们定义了一种新的过程参数，具有细胞周期分布的定量和数值鲁棒表示，并证明其与细胞周期状态线性相关并与实时增长速率相反。比使用基于细胞计数的方法可能早期检测生长速率限制。分析与批量荧光方法相容，使用板读取器以及流式细胞仪。对于工业规模生物反应器的未来实时应用，我们建议使用在线或在线荧光探针。 （c）2017美国化学工程师研究所

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