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 of adherent CHO K1
cultures, 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. Indeed, the mitochondrial
genome sequencing method developed here enables 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.