Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial‐mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter
Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial‐mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter
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DOI：
10.1002/btpr.3159
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发表时间：
2021-04
影响因子：
2.9
通讯作者：
T. Hirano;S. Adachi;N. Ichimura;Akira Kasai;Masashi Kobayashi;T. Okuda;R. Ogawa;G. Kagiya
中科院分区：
文献类型：
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作者：
T. Hirano;S. Adachi;N. Ichimura;Akira Kasai;Masashi Kobayashi;T. Okuda;R. Ogawa;G. Kagiya
中国仓鼠卵巢（CHO）细胞常作为宿主用于重组蛋白生产（RPP）。虽然RPP已被证明是成功的，但仍有进一步改进的迫切需要。环烯烃聚合物（COP）是一种应用广泛的塑料材料，具有蛋白质吸收率低等特点。我们将其作为RPP细胞培养的原料，看看COP是否合适。在Moloney小鼠白血病病毒长末端重复序列（MMLV‐LTR）的控制下，建立了表达人红细胞生成素（hEPO）基因的重组CHO细胞系。当细胞在由COP制成的培养皿中培养时，细胞附着在底部，然后开始漂浮并形成球体。RNASeq数据分析表明，在培养后不久，受体酪氨酸激酶激活触发了上皮-间充质转化（EMT）。这与hEPO转录增加相一致。细胞漂浮后，虽然EMT标记基因表达下降，但hEPO表达持续增加。当纤维连接蛋白作用于COP培养皿表面时，细胞漂浮受到抑制，hEPO表达降低。然后我们用m - β cd处理细胞，一种破坏脂质筏的药物，消除筏中的分子。这促进了细胞的漂浮和球形形成，同时hEPO表达增强。这些结果表明，细胞和COP表面之间的相互作用可能触发EMT和随后的事件，这两个事件都激活了MMLV‐LTR启动子。因此，利用COP培养细胞，可以建立一个有效的RPP体系，并具有高效纯化蛋白质的优势。
Chinese hamster ovary (CHO) cells are frequently used for recombinant protein production (RPP) as a host. While the RPP has been proven successful, there is still a compelling need for further improvement. Cyclo olefin polymer (COP) is a plastic material widely utilized due to its properties including its low protein absorption. We applied this as a raw material for RPP cell culture to see if the COP is suitable. A recombinant CHO cell line expressing the human erythropoietin (hEPO) gene under the control of the Moloney murine leukemia virus‐long terminal repeat (MMLV‐LTR) was established. When the cells were cultured in a dish made from COP, the cells attached to the bottom, and then started to float and form spheroids. RNASeq data analysis suggested the epithelial‐mesenchymal transition (EMT) was triggered with receptor tyrosine kinase activation shortly after cultivation. It coincided with the hEPO transcription increase. After the cell floating, though EMT marker gene expression subsided, a hEPO expression increase sustained. When fibronectin was applied to COP dish surface, the cell floating was suppressed and hEPO expression decreased. We then treated cells with MβCD, a drug that destroys the lipid raft, eliminating molecules in the raft. This facilitated cell floating and spheroid formation coincided with hEPO expression enhancement. These results suggest interactions between a cell and COP surface might trigger the EMT and the subsequent event, both of which activated the MMLV‐LTR promoter. Thus, employing COP for culturing cells, a potent RPP system could be established with its advantage for efficient protein purification.