1. Introduction
Chinese hamster ovary (CHO) cells (Kao & Puck, 1968) play a key role as
heterologous hosts for the commercial-scale production of protein
pharmaceuticals (Greber & Fussenegger, 2007; Hacker, De Jesus, & Wurm,
2009; Omasa, Onitsuka, & Kim, 2010). The adaptivity of CHO cells to
high cell density processes has ensured their successful application for
this purpose. In the biopharmaceutical industry, a large number of
products have been introduced into the market using high cell density
processes such as batch and fed-batch cultures and different cell
culture strategies for reducing the production cost and improving
product quality (Pollock, Ho, & Farid, 2013; Tapia, Vazquez-Ramirez,
Genzel, & Reichl, 2016). Cell culture times are generally long (12 days
for fed-batch cultures) and advances are further prolonging culture
times; perfusion continuous bioprocessing (60 days) is widely recognized
as the next-generation biomanufacturing platform for achieving superior
product quality and reducing manufacturing costs (Konstantinov &
Cooney, 2015; Zydney, 2015).
A potential issue when using long-term cultures for therapeutic protein
production is the increasing cellular heterogeneity over time
(Altschuler & Wu, 2010). Cellular proliferation and gene expression are
critical cell processes but genomic mutations arise during DNA
replication (Furusawa, 2014) and RNA transcription (Bachl, Carlson,
Gray-Schopfer, Dessing, & Olsson, 2001) inherent to cell division and
gene expression, respectively. The limited knowledge regarding cellular
heterogeneity in high cell density processes poses a barrier to solving
this issue.
It has been proposed that insisting on “true” clonality is
scientifically unjustifiable (O’Callaghan et al., 2015; Wurm, 2013),
with cell line heterogeneity being included in the FDA “Challenges to
Potency Assay Development for CGT products” industry guidelines
(Guidance for Industry: Potency Tests for Cellular and Gene Therapy
Products) (FDA, 2011). We can only hope that the trend toward a modified
gene pool can be minimized by maintaining cell populations in stable
environments (Wurm, 2013). Deeper characterization such as single-cell
sequencing (Tang et al., 2009) of the heterogeneity within cell line
cultures would enable the study of cooperative and competitive
interactions between cell populations and the mechanisms underlying
related cell functions (Ben-David et al., 2018).
In this study, we performed single-cell transcriptome sequencing of
serum-free suspension-adapted CHO K1 cells in shake flasks. The degree
of cellular heterogeneity and the existence of subpopulations were
determined based on gene expression analysis and whole mitochondrial
genome sequencing. To facilitate comparisons of our data to those from
other single-cell transcriptome studies, we also performed single-cell
transcriptome sequencing of adherent CHO K1 cells in culture dishes.
Additionlly, we developed clonal markers to validate the clonality of
the cells based on their mitochondrial genome sequences.