Bioluminescence-based biosensing of NF-κB activity for osteoarthritis drug screening in a cartilage-on-chip model

Osteoarthritis (OA) is a chronic joint disease lacking disease-modifying treatments, wherein targeted drug discovery is hindered by the limited physiological relevance of conventional in vitro assays. Contrary to the traditional view of OA as a purely degenerative joint disorder, emerging research highlights the involvement of inflammatory pathways and the existence of distinct molecular endotypes. Within this context, NF-κB activation is acknowledged as a central driver of inflammatory and catabolic processes in joint tissues making it a key therapeutic target. Bioluminescent reporter assays enable dynamic, pathway-specific investigations, but are rarely applied to organ-on-chip (OoC) models due to low-throughput bioluminescence imaging constraints. Here, we present a scalable approach for medium-throughput bioluminescence readout in a cartilage-on-chip model of OA using an NF-κB-NanoLuc reporter cell line. Two widely available imaging systems were repurposed: a small animal bioluminescence imager (Pearl Trilogy) and a Western blot chemiluminescence imager (FluorChem), accommodating 24 and 32 chips per run, respectively. Both enabled signal detection for 1 h following a single-time substrate addition, with FluorChem delivering superior signal-to-noise and signal-to-background ratios. NF-κB activation by IL-1β and its inhibition by NF-κB inhibitors were quantified in both 2D and 3D cultures, yielding consistent trends across formats. Differences in inhibitor potencies between 2D and 3D highlighted the value of using physiologically relevant OoC models for OA drug screening. This study demonstrates how standard laboratory imagers can be repurposed as off-the-shelf biosensing platforms for OoCs, providing a scalable, in situ strategy to overcome throughput bottlenecks and expand the utility of bioluminescence-based reporters in drug discovery workflows. • Repurposed imagers allow medium-throughput sensing of bioluminescence in Organ-on-Chips (OoCs). • NF-κB-NanoLuc reporter enables dynamic signalling readout in 3D cartilage-on-chip. • Trends of NF-κB inhibitor responses are consistent in 2D and 3D culture formats. • Inhibitor potency differences in 3D highlight the value of OoCs in drug screening. • Integrated biosensing in OoCs advances osteoarthritis endotype-relevant drug screening.