Abstract
<jats:p>While geometric constraints shape tissue development, quantifying the resulting growth dynamics remains a central challenge in tissue engineering. Conventional methods often struggle to capture multi-scale kinetics without complex labeling or difficult single-cell tracking. Here, we analyze geometrically controlled growth of microtissues derived from human dermal fibroblasts using time-resolved, label-free brightfield microscopy, combined with optical flow and semi-automated deep learning mitosis detection. By extracting multi-scale flow fields and integrating them with tissue segmentation, we quantify directional tissue dynamics, separating flow into parallel and normal components relative to the local tissue contour. Applying this framework, we contrast the quiescent tissue interior with the advancing growth front where localized dynamics and cell proliferation drive expansion. Our results demonstrate that, compared to the bulk, the growth front exhibits higher fluctuations parallel to the tissue contour, positive mean normal flow, and significantly increased mitotic activity. Furthermore, evaluating flow divergence around mitotic events reveals distinct spatial behaviors: with the onset of mitosis, a contraction and subsequent expansion occurs in the vicinity of the dividing cells. Beyond the immediate cellular neighborhood, the broader regional dynamics remain consistent before and after mitosis onset, with net tissue expansion in proximity to the growth front and contraction within the tissue interior. By extracting continuous kinetic data from easily accessible, label-free brightfield imaging, this approach serves as a non-invasive, complementary tool for evaluating in vitro tissue morphogenesis and growth dynamics. This analytical framework can be expanded to study locally resolved tissue morphogenesis and growth kinetics in other microsystems, ranging from embryos to organoids.</jats:p>