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Molecular Dynamics of Immortalized Chicken Fibroblasts from Adherent Cultivation to Carrier-Free Suspension Culture

ABSTRACT: This study aimed to elucidate the internal mechanisms of biological changes in immortalized chicken embryo fibroblasts (DF-1) by transitioning them from adherent to suspension culture. Initially, we determined the optimal growth conditions necessary for suspension cell culture. The adherent DF-1 cells (2D) were then adapted to suspension culture, resulting in short-term (3D_E) and long-term (3D_L) suspension cells. Growth metrics were monitored throughout the suspension phase. Subsequently, RNA-seq analysis was conducted to identify key genes and signaling pathways that exhibited distinct changes in 2D, 3D_E and 3D_L cells. The transcriptome data guided further investigations into the transdifferentiation of 2D, 3D_E, and 3D_L cells into lipids and their collagen secretion capabilities.

ORGANISM(S): Gallus gallus

PROVIDER: GSE269288 | GEO | 2026/07/01

REPOSITORIES: GEO

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Project description:<p>Human embryonic kidney 293 (HEK293) cells have been successfully adapted from adherent to suspension culture and have been widely applied in both scientific research and the pharmaceutical industry. However, the alterations in cells during the adaptation have not been well described, which raise some uncertainties and concerns regarding the underlying changes and cell behavior.</p><p>In this work, we adapted adherent HEK293 to suspension culture with desirable cell growth and high production titers for recombinant adenoviral vectors, and cells at several stages throughout the process were characterized. First, we obtained three strains of suspension cells from adherent parental HEK293 cells by gradually phasing out fetal bovine serum in original Dulbecco’s modified essential medium with a simultaneous medium replacement with four serum-free suspension culture media, and one strain was chosen as the preferred candidate for further studies due to its satisfying cell conditions and adenoviral vector productivity. Slower cell growth rate, lower glucose uptake, increased lactate production, weaker cell-surface adhesion, and prolonged S phase in the cell cycle were observed in suspension cells compared to their adherent counterparts. We further performed transcriptomics, proteomics, and metabolomics analysis to identify key switches in cells. A total of 2476 differential genes were found, including 1218 up-regulated genes and 1258 down-regulated genes in suspension cells. A similar and correlated pattern was observed in the proteomic study: an almost balanced up-down regulation between suspension and adherent cells, and 702 differentially expressed metabolites were identified by untargeted metabolomics. By virtue of enrichment analysis on differentially expressed genes, proteins and metabolites, we summarized that HEK293 adherent cells survived and adapted to suspension culture by structural remodelling, metabolic network reconstruction and inherent stress resistance. Additionally, we identified claudin7 as a key player involved in suspension transformation in both transcriptomic and proteomic aspects. Our results provide a molecular enlightenment for the mechanism of suspension adaptation and new directions for the rational design of genetically engineered HEK293-derived cell lines for viral-vectored vaccine production.</p>

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Molecular Dynamics of Immortalized Chicken Fibroblasts from Adherent Cultivation to Carrier-Free Suspension Culture

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