Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.
Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.
复制标题
DOI：
10.1371/journal.pone.0151529
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Valdez-Cruz NA
中科院分区：
文献类型：
--
作者：
Bedoya-López A;Estrada K;Sanchez-Flores A;Ramírez OT;Altamirano C;Segovia L;Miranda-Ríos J;Trujillo-Roldán MA;Valdez-Cruz NA
重组蛋白被广泛用作生物制药，但通过哺乳动物细胞培养生产它们是昂贵的。因此，非常需要提高生物过程的生产率。从37°C到28-34°C的温度降档（TDS）是延长电池生产寿命并提高其生产率（qp）的有效策略。在此，中国仓鼠卵巢（CHO）细胞培养物中的TDS（最初在37°C下生长，在指数生长期转换为30°C）导致重组人组织纤溶酶原激活剂（rh-tPA）的qp增加1.6倍。使用下一代测序（NGS）评估转录组学应答，以表征与TDS相关的细胞行为。与恒定37°C相比，在TDS培养物中观察到总共416个（q > 0.8）和3，472个（q > 0.9）差异表达的转录物，分别在TDS后24和48 h变化超过1.6倍。与TDS导致的延长的细胞存活一致，与细胞生长停滞相关的转录本差异表达，所述转录本控制细胞增殖而不激活DNA损伤反应。大多数上调的基因与线粒体能量代谢、线粒体生物发生、中枢代谢和避免凋亡细胞死亡有关。编码rh-tPA的基因没有差异表达，但在编码参与分泌机制，特别是糖基化的蛋白质的转录本中检测到波动。通过NGS的TDS引起的动力学过程进行了评估，在这个生物系统。
Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.