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The accumulation of metabolic wastes in cell cultures can diminish product quality, reduce productivity, and trigger apoptosis. The limitation or removal of unintended waste products from Chinese hamster ovary (CHO) cell cultures has been attempted through multiple process and genetic engineering avenues with varied levels of success. One study demonstrated a simple method to reduce lactate and ammonia production in CHO cells with adaptation to extracellular lactate; however, the mechanism behind adaptation was not certain. To address this profound gap, this study characterizes the phenotype of a recombinant CHO K-1 cell line that was gradually adapted to moderate and high levels of extracellular lactate and examines the genomic content and role of extrachromosomal circular DNA (eccDNA) and gene expression on the adaptation process. More than 500 genes were observed on eccDNAs. Notably, more than 1000 genes were observed to be differentially expressed at different levels of lactate adaptation, while only 137 genes were found to be differentially expressed between unadapted cells and cells adapted to grow in high levels of lactate; this suggests stochastic switching as a potential stress adaptation mechanism in CHO cells. Further, these data suggest alanine biosynthesis as a potential stress-mitigation mechanism for excess lactate in CHO cells.
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Microevolutionary dynamics of eccDNA in Chinese hamster ovary cells grown in fed-batch cultures under control and lactate-stressed conditions
Abstract Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr ® 250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.
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- PAR ID:
- 10410948
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41598-023-27962-0
