Streamlined stable CHO cell line development using droplet microfluidics with image-based monoclonality assessment.

Rapid generation of monoclonal CHO cell lines remains a key bottleneck in mammalian cell line development (CLD), often requiring months of iterative screening and cloning. Here, we present an integrated droplet microfluidic workflow for high-throughput screening and isolation of antibody-producing ExpiCHO-S cells using a FRET-based secretion assay. The FRET assay was developed and optimized for picodroplet-based screening by evaluating probe configuration, donor-to-acceptor ratio, antibody concentration range, and signal saturation behavior. The platform enables picodroplet-based detection of secreted antibodies, sequential enrichment of antibody-producing cells, and image-verified single-cell isolation. Application of this approach yielded image-verified monoclonal antibody-producing clones that maintained consistent antibody production during the stability assessment performed in this study, including in the absence of selection pressure. By reducing the early clone-identification phase from DNA transfection to validated 96-well antibody-producing clones to approximately 5 weeks, this workflow addresses key limitations of conventional approaches and provides both a practical FRET assay-development framework and a scalable strategy for streamlining early CLD workflows in biomanufacturing.