Single‐cell transcriptome analyses reveal heterogeneity in suspension cultures and clonal markers of CHO‐K1 cells. Issue 2 (20th November 2020)

• Record Type: Journal Article
• Authors: Ogata, Norichika; Nishimura, Akio; Matsuda, Tomoko; Kubota, Michi; Omasa, Takeshi
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 2(2021)
• Page Start: 944
• Page End: 951
• Publication Date: 2020-11-20
• Subjects: Chinese hamster ovary (CHO) cells -- heterogeneity -- heteroplasmy -- monoclonality -- single‐cell transcriptome sequencing; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27624
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Cell‐to‐cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum‐free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle‐dependent subpopulations and cell clusters, which typically appear in other single‐cell transcriptome analyses, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle‐dependent heterogeneity. Whole mitochondrial single‐cell genome sequencing showed cell‐to‐cell mitochondrial genome variation and heteroplasmy within cells. The mitochondrial genome sequencing method developed here is potentially useful for the validation of cell clonality. The culture time‐dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose. Abstract : Single‐cell transcriptomes of high cell density suspension CHO K1 cell cultures were sequenced. Cellular transcriptome heterogeneity increased with culture time. Cell cycle‐dependent subpopulations and cell clusters, which typically appear in other single‐cell transcriptome analyses, were not found in these suspension cultures. Single‐cell resolution mitochondrial genomic heterogeneity revealed cell‐to‐cell mitochondrial genome variation and heteroplasmy within cells, which is potentially useful for the validation of cell clonality.