Organoid chip based automatic system for long-term quantifying ciliary beating under drug intervention

Background: The beating of motile cilia plays an important role in many physiological processes. In recent years, the precise monitoring of ciliary beating has provided valuable insights into cilia-related diseases and their therapies. However, a gap persists between homogeneous in vitro cell models and heterogeneous in vivo ciliary systems. Human-derived ciliary organoids (HDCOs) show promise in bridging this gap but face technical barriers: limited numbers of HDCOs cannot meet multi-well-plate demands, and long-term tracking accuracy is compromised by organoid overlapping/displacement during culture. Results: A multifunctional microfluidic chip (MOCiB-Chip) was developed, featuring a three-layer structure (fluidic layer, porous membrane, capture layer) and two independent regions for multi-concentration experiments. It integrates single-HDCO immobilization, culture, in situ observation, and on-chip drug mixing. Combined with an independently developed analysis program, Ciliary Beating Frequency Analysis Software (CBFAS), and a fluid control module, the automatic quantifying system for ciliary beating (AuCilia) was established. It achieved 7-day monitoring of human bronchial HDCOs and evaluated roflumilast at 0–100 nM: roflumilast at 10 nM increased beating frequency by 81.5 %, 1 nM by 27.8 %, while 100 nM caused cessation by day 2. cAMP measurements confirmed roflumilast's mechanism, validating the system. Significance: This strategy eliminates HDCO loss via on-chip integration of all procedures, resolving the conflict between limited samples and multi-condition tests. It improves tracking accuracy by avoiding organoid displacement/overlapping, overcoming multi-well-plate limitations. The system's low cost and high reproducibility accelerate drug screening (e.g., roflumilast) and enable precise study of cilia physiology, laying a key technical foundation for cilia-related disease research and clinical translation.