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bioRxiv · 10.64898/2025.12.11.693844

Accelerating CHO-K1 Cell Line Development by Reducing Suspension Adaptation with a Microplate Agitation Culture System

Lin, S.-P.·Lin, C.-N.·Wang, W.-R.·Tsai, C.-H.
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Abstract

Stable and productive CHO cell lines are essential for biopharmaceutical manufacturing, yet early expansion steps are often constrained by prolonged period required for suspension adaptation. Single-cell cloning (SCC) ensures monoclonality and regulatory compliance, but cells transitioning from static to suspension culture frequently exhibit variable recovery, which prolongs timelines and increases process variability. To address this challenge, mixing-based microplate culture systems have been developed to improve early expansion efficiency. The C.NEST platform provides controlled pneumatic mixing and environmental monitoring that facilitates earlier adaptation to suspension conditions. At the 96-well and 24-well stages, this system allows cells to establish stable growth under suspension-like environments, thereby shortening the adaptation period following transfer to shaking culture. In this study, we applied C.NEST to the SCC workflow for developing CHO-K1 stable cell lines. Integrating C.NESTs controlled mixing reduced adaptation time, enhanced the consistency of clone expansion, and improved the ability to identify high-yield clones. These findings highlight the potential of C.NEST to streamline cell line development workflows by accelerating early suspension adaptation and improving clone selection reliability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693844v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ca76bforg.highwire.dtl.DTLVardef@3a3079org.highwire.dtl.DTLVardef@447e01org.highwire.dtl.DTLVardef@ac8d6f_HPS_FORMAT_FIGEXP M_FIG C_FIG C.NEST mixing shortens suspension adaptation, accelerates clone expansion, and enhances early-stage screening. HighlightO_LIThe C.NEST microplate agitation culture system accelerates early CHO-K1 cell line development. C_LIO_LIControlled pneumatic mixing improved oxygen transfer and medium homogeneity, promoting stable growth during early expansion. C_LIO_LIEarly mixing shortens suspension adaptation by approximately one week. C_LIO_LIMixing cultures enabled more accurate clone performance assessment, revealing high-producing outliers. C_LIO_LIC.NEST provides a scalable and reproducible solution for integrating mixing-based culture into single-cell cloning workflows. C_LI

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Lin, S.-P., Lin, C.-N., Wang, W.-R., Tsai, C.-H.. 2025-12-15. Accelerating CHO-K1 Cell Line Development by Reducing Suspension Adaptation with a Microplate Agitation Culture System. https://doi.org/10.64898/2025.12.11.693844

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