A new flow chip in combination with multiphoton microscopy as a protocol for longitudinal 3D imaging of tissue calcification under shear stress

We present a novel miniaturized flow chip platform enabling thin‐layer smooth muscle cell (SMC) culture, continuous tangential perfusion, dual‐channel nutrient control, and longitudinal subcellular resolution 3‐D imaging via multiphoton microscopy. Using a case study of longitudinal imaging of vascular calcification, a key pathological process in cardiovascular disease typically challenging to dynamically model under physiological flow, we describe the platform and its potential. Within this system, immortalized vascular SMCs embedded in fibrin hydrogel on a textile‐reinforced scaffold are exposed to unidirectional flow (12 mL·min ⁻¹ ) of calcification medium for 4 days, with and without supplemental Mg. Fluorescent tracking of mineral deposition (mRuby‐labeled fetuin‐A) and cytoplasmic dyes (CellTracker Green) enable daily volumetric imaging. In Mg‐free conditions, rapid mineralization initiates at the textile scaffold and engulfs the construct by day 2, plateauing thereafter, yet viable cells remain postcalcification. In contrast, Mg‐supplemented constructs show markedly suppressed calcium phosphate deposition, progressing only slowly over the time course. These longitudinal findings align with known Mg‐mediated inhibition of hydroxyapatite formation and cellular osteogenic transition. Thus, this case study highlights a platform which can reproduce shear‐flow conditions, modulating media ionic composition, and performing time‐lapse 3‐D imaging of live cells within a flow environment. This versatile system not only has uses for further mechanistic studies of vascular calcification, screening of anticalcification therapies, and adaptation to endothelialized co‐cultures or excised natural tissues, but also has potential for longitudinal model studies in a broader context.