Breaking limitations of complex culture media: Functional non-viral miRNA delivery into pharmaceutical production cell lines
Breaking limitations of complex culture media: Functional non-viral miRNA delivery into pharmaceutical production cell lines
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DOI：
10.1016/j.jbiotec.2013.08.027
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发表时间：
2013-12-01
影响因子：
4.1
通讯作者：
Otte, Kerstin
中科院分区：
文献类型：
--
作者：
Fischer, Simon;Wagner, Andreas;Otte, Kerstin
MicroRNAs(MiRNAs)通过调节关键的细胞通路，是细胞工程的有希望的靶标。有效地将miRNAs导入细胞是可靠研究microRNA功能的前提。此前，核酸的非病毒传递已被证明与细胞类型和培养介质有关。由于中国仓鼠卵巢(CHO)和Cevec的羊水细胞生产(CAP)细胞在生物制药研究和生产中的重要性，我们以中国仓鼠卵巢(CHO)和Cevec的羊水细胞生产(CAP)细胞为宿主细胞，研究了成功传递小非编码RNA(NcRNAs)的转染剂及其在细胞内允许miRNAs和小干扰RNAs(SiRNAs)生物活性的能力。在本研究中，我们筛选了许多转染剂，以确定它们是否适合将miRNA模拟物成功地输送到CHO DG44和CAP细胞中。我们的研究表明，仅测定给定转染剂的转染率并不足以得出结论，说明其维持miRNA的功能的能力。我们可以证明，与观察到的几种试剂的高转染率无关，只有一种试剂适合将功能性miRNA模拟物有效地导入到复杂蛋白生产介质中培养的细胞中。我们通过证明siRNA介导的蛋白质水平和细胞表型的变化以及其上游miR-1调节因子双胞胎蛋白-1(twf-1)转录水平的降低，为转移的ncRNAs的功能提供了证据。此外，该过程可以被证明是可扩展的，这对生物技术应用具有重要意义。(C)2013爱思唯尔B.V.保留所有权利。
MicroRNAs (miRNAs) are promising targets for cell engineering through modulation of crucial cellular pathways. An effective introduction of miRNAs into the cell is a prerequisite to reliably study microRNA function. Previously, non-viral delivery of nucleic acids has been demonstrated to be cell type as well as culture medium dependent. Due to their importance for biopharmaceutical research and manufacturing, Chinese hamster ovary (CHO) and Cevec's Amniocyte Production (CAP) cells were used as host cell lines to investigate transfection reagents with respect to successful delivery of small non-coding RNAs (ncRNAs) and their ability to allow for biological activity of miRNAs and small interfering RNAs (siRNAs) within the cell. In the present study, we screened numerous transfection reagents for their suitability to successfully deliver miRNA mimics into CHO DG44 and CAP cells. Our investigation revealed that the determination of transfection efficiency for a given transfection reagent alone is not sufficient to draw conclusions about its ability to maintain the functionality of the miRNA. We could show that independent from high transfection rates observed for several reagents only one was suitable for efficient introduction of functional miRNA mimics into cells cultured in complex protein production media. We provide evidence for the functionality of transferred ncRNAs by demonstrating siRNA-mediated changes in protein levels and cellular phenotype as well as decreased twinfilin-1 (twf-1) transcript levels by its upstream miR-1 regulator. Furthermore, the process could be shown to be scalable which has important implications for biotechnological applications. (C) 2013 Elsevier B.V. All rights reserved.