Abstract
Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a protocol specifically developed for the reliable and reproducible MSCC of industrially relevant Chinese hamster ovary (CHO) suspension cell lines. We summarize critical experimental steps from the initial seed train up to the final MSCC experiment, with a special focus on pre-culture management and medium preparation, device inoculation, and the establishment of a constant medium perfusion.