Promoters play a pivotal role in integrating and processing the signals related to transcription initiation. Strong natural viral promoters, such as hCMV or SV40E, have been routinely employed to achieve a high rate of gene expression in ubiquitously used Chinese hamster ovary (CHO) cells. However, viral promoters are susceptible to epigenetic silencing and lack precise regulation levers. This has paved the way to more sensible control elements: endogenous, inducible, and synthetic promoters. In this review we summarize and discuss the use of natural viral, mammalian, and endogenous promoters, as well as recent advances in synthetic promoters and inducible systems for protein expression in CHO cells. Not only the level of transcription, but its long-term stability is crucial for recombinant protein production. Epigenetic chromatin-modifying elements, such as ubiquitously acting chromatin opening elements (UCOEs), matrix attachment regions (MARs), insulators and stabilizing anti-repressors (STARs) significantly improve transcription levels over extended cultivation time and are also discussed here. This review provides up-to date information to facilitate the choice of a suitable promoter and adjacent chromatin-modifying elements to maximize transgene expression as well as ensure long-term expression stability in CHO cell culture.

Exposure of Chinese hamster ovary cells (CHO) to highly concentrated feed solution during fed-batch cultivation is known to result in an unphysiological osmolality increase (>300 mOsm/kg), affecting cell physiology and morphology. Extending previous observation on osmotic adaptation, the present study investigates for the first time potential effects of hyperosmolality on CHO cells on both population and single-cell level.We intentionally exposed CHO cells to hyperosmolality of up to 545 mOsm/kg during fed-batch cultivation. In concordance with existing research data, hyperosmolalityexposed CHO cells showed a nearly triplicated volume accompanied by ablation of proliferation. On the molecular level, we observed a strong hyperosmolality-dependent increase in mitochondrial activity in CHO cells compared to control. In contrast to mitochondrial activity, hyperosmolality-dependent proliferation arrest of CHO cells was not accompanied by DNA accumulation or caspase-3/7-mediated apoptosis. Notably, we demonstrate for the first time a formation of up to eight multiple, small nuclei in single hyperosmolality-stressed CHO cells. The here presented observations reveal previously unknown hyperosmolality-dependent morphological changes in CHO cells and support existing data on the osmotic response in mammalian cells.

Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume—almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.

Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added to replenish nutrients and regulate pH values. Some effects of hyperosmolality, such as increased cell size and growth inhibition, have been elucidated by previous research, but the impact of hyperosmolality and the specific effects of the added osmotic-active reagents have rarely been disentangled. In this study, CHO cells were exposed to four osmotic conditions between 300 mOsm/kg (physiologic condition) and 530 mOsm/kg (extreme hyperosmolality) caused by the addition of either high-glucose-supplemented industrial feed or mannitol as an osmotic control. We present novel single-cell cultivation data revealing heterogeneity in mass gain and cell division in response to these treatments. Exposure to extreme mannitol-induced hyperosmolality and to high-glucose-oversupplemented feed causes cell cycle termination, mtDNA damage, and mitochondrial membrane depolarization, which hints at the onset of premature stress-induced senescence. Thus, this study shows that both mannitol-induced hyperosmolality (530 mOsm/kg) and glucose overfeeding induce severe negative effects on cell growth and mitochondrial activity; therefore, they need to be considered during process development for commercial production.

Chinese hamster ovary (CHO) is the most commonly used host cell line for therapeutic protein production. Their exposure to highly concentrated feed solution during fed-batch cultivation can cause an unphysiological osmolality increase (>300 mOsm/kg) affecting cell physiology, morphology, and proteome. In a companion article "Hyperosmolality in CHO Culture: Effects on Cellular Behavior and Morphology" we show that hyperosmolalities of up to 545 mOsm/kg force cells to ablate proliferation and gradually increase their volume, almost triplicating it. CHO cells also exhibit a significant hyperosmolality-dependent mitochondrial activity increase. To get a deeper insight into molecular mechanisms involved in these processes, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed vs. control CHO cells. Our analysis revealed key differentially expressed proteins mediating mitochondrial activation, oxidative stress amelioration, and cell cycle progression. We also discovered a previously unknown strong regulation of proteins altering cell membrane rigidity and permeability. Among others, we detected three members of septins, filamentous proteins forming diffusion barriers in the cell, to be highly upregulated in response to hyperosmolality. Taken together, our observations correlate well with the recent CHO-based fluxome and transcriptome studies and expose new unknown targets involved in response to hyperosmotic pressure in mammalian cells.