Comparative Transcriptomic Analyses of a Vero Cell Line in Suspension versus Adherent Culture Conditions

Abstract

The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling up challenging and operations very labor-intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested, but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show downregulation of the epithelial-to-mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, and the extracellular pathway. Moreover, a downregulation of the glycolytic pathway is balanced by an upregulation of the asparagine metabolism pathway, promoting cell adaptation to nutrient deprivation. A downregulation of the adherens junctions and the folate pathways alongside with the FYN gene are possible explanations behind the currently observed low-cell viability and long doubling time.

Figure 1

DESeq2 differential expression data quality control: volcano plot of DE genes with applied p value and log2 fold change cut-offs. FDR: false discovery rate; LogFC: log2 fold change.

Figure 2

Network of terms enriched by upregulated DE genes: (a) colored by cluster ID, where nodes that share the same cluster ID are typically close to each other; (b) colored by p value, where terms containing more genes tend to have a more significant p value. Each node represents one enriched term, and edges link similar terms (the edge thickness is proportional to the similarity between terms).

Figure 3

Network of terms enriched by downregulated DE genes: (a) colored by cluster ID, where nodes that share the same cluster ID are typically close to each other; (b) colored by p value, where terms containing more genes tend to have a more significant p value. Each node represents one enriched term, and edges link similar terms (the edge thickness is proportional to the similarity between terms).

Figure 4

Adherent and suspension comparative metabolic map. Blue and red arrows refer, respectively, to downregulated and upregulated reactions. Dashed gray arrows refer to nonsignificant dysregulations according to the Kolmogorov-Smirnov test with p value 0.01. Solid gray arrows refer to reactions with a variation lower than 20%.

Figure 5

GSEA bar chart with significantly enriched hallmark pathways highlighted (FDR < 0.05) (IL6: interleukin-6; STAT3: signal transducer and activator of transcription 3; HIF1A: hypoxia-inducible factor-1α; MYC: cellular myelocytomatosis oncogene; TGF: transforming growth factor; G2/M: Second growth phase/mitosis; E2F: eukaryote cellular transcription factor; FDR: false discovery rate).

Figure 6

Layout of key upregulated subnetworks and their top associated genes (network details in Table 1).

Figure 7

Layout of key downregulated subnetworks and their top-associated genes (network details in Table 2).