A Gravity-driven Microfluidic Platform for High-throughput Culture and Inflammatory Stimulation of Human Articular Chondrocytes.

Articular cartilage is a load-bearing connective tissue whose structural integrity depends on a highly specialized extracellular matrix and the activity of resident chondrocytes. In vivo, chondrocytes are continuously exposed to mechanical stimuli, including fluid-induced shear stress associated with synovial fluid movement, which plays a critical role in regulating cell phenotype and matrix homeostasis. Osteoarthritis (OA) is a degenerative joint disease marked by extracellular matrix breakdown, chondrocyte dysfunction, and inflammation. Despite its high prevalence, effective disease-modifying therapies remain unavailable, in part due to the lack of in vitro models that recapitulate both the biochemical and biomechanical environment of native cartilage. This article describes a pumpless, gravity-driven microfluidic platform for modelling inflammatory OA-like conditions under dynamic flow, based on and expanding upon our previously published work. This system is based on gravity-driven flow generated by a rocker platform, eliminating the need for external pumps and enabling scalable, parallelized operation. Defined microchannel geometries allow spatially resolved exposure of cells to varying shear stress levels within a single device. Human articular chondrocytes are cultured under dynamic conditions and stimulated with interleukin-1β to induce inflammatory responses. The protocols presented here include preparation and extracellular matrix coating of the microfluidic chips, measurement of flow dynamics under rocker-induced motion, long-term dynamic culture of chondrocytes under gravity-driven flow, and induction of OA-like inflammatory conditions followed by evaluation of chondrocyte responses. The platform supports assessment of cellular responses, including viability, metabolic activity, expression of major extracellular matrix markers (collagen type II and aggrecan), and secretion of inflammatory mediators. This approach provides a reproducible in vitro model that integrates dynamic cell culture and inflammation while offering a scalable alternative to conventional static cultures and externally actuated organ-on-chip systems. The method enables efficient investigation of chondrocyte inflammatory responses and has potential applications in OA disease modelling and drug screening. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extracellular matrix coating and operation of a gravity-driven microfluidic chip for long-term culture of human articular chondrocytes. Support Protocol 1: Measurement of gravity-driven flow rate in microfluidic chips under rocker-induced tilt. Basic Protocol 2: Seeding and long-term dynamic culture of human articular chondrocytes under gravity-driven flow. Basic Protocol 3: Induction of osteoarthritis-like inflammatory conditions using IL-1β under gravity-driven flow and analysis of chondrocyte responses.