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

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.

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.

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.