Adaptation for survival: phenotype and transcriptome response of CHO cells to elevated stress induced by agitation and sparging.

In this work, the response and adaption of CHO cells to hydrodynamic stress in laboratory scale bioreactors originating from agitation, sparging and their combination is studied experimentally. First, the maximum hydrodynamic stress, τ(max), is characterized over a broad range of operating conditions using a shear sensitive particulate system. Separate stress regimes are determined, where τ(max) is controlled either by sparging, agitation, or their combination. Such conditions are consequently applied during cultivations of an industrial CHO cell line to determine the cellular responses to corresponding stresses. Our results suggest that the studied CHO cell line has different threshold values and response mechanisms for hydrodynamic stress resulting from agitation or sparging, respectively. For agitation, a characteristic local minimum in viability was found after stress induction followed by viability recovery, while at highest sparging stress a monotonic decrease in viability was observed. If both stresses were combined, also both characteristic stress responses could be observed, amplifying each other. On the other hand, cellular metabolism, productivity and product quality did not change significantly. Transcriptome analysis using mRNA microarrays confirmed that separate adaptation mechanisms are activated in the different stress situations studied, allowing identification of these stresses using a transcriptome fingerprinting approach. Functional analysis of the transcripts was consequently used to improve our understanding of the molecular mechanisms of shear stress response and adaptation.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films.

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-ß and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.