Label-free Live-imaging Of Cellular And Matrix Changes Utilizing Computational 4-D Optical Coherence Tomography In An In Vitro 3D Calcific Aortic Valve Disease Model
Katherine Driscoll, Nichaluk Leartprapun, PhD, Justin Luo, PhD, Andrea Huang, Steven Adie, PhD, Jonathan Butcher, PhD.
Cornell University, Ithaca, NY, USA.
OBJECTIVE: Many patients only present Calcific Aortic Valve Disease (CAVD) symptoms after the valve is severely calcified. How this disease manifests in intermediate stages when a pharmacologic might be better effective is unknown. In-vitro experimentation allows for high-throughput mechanistic testing of CAVD cellular and matrix changes, but many of these approaches only include end-point analysis due to live-imaging limitations, especially in a more physiologically relevant 3D culture. The objective of this work is to develop a novel in-vitro 3D co-culture CAVD drug-testing platform for visualization of cellular and matrix changes in real-time, to better understand critical features of CAVD progression missed in endpoint-only experimentation. To accomplish this, we combined cutting-edge computational 4D-OCT imaging with our 3D tensioned valvular interstitial (VIC) and endothelial-cell (VEC) co-culture platform. METHODS: Longitudinal 3D-OCT images were taken of live VIC-VEC co-culture gels, and temporal speckle contrast was used to segment cells from collagen matrix. OCT datasets were taken either at D1 and D7, or D1, D3, D5, and D7-10. Macroscopic compaction of each gel was measured daily as final divided by initial area.
RESULTS: Our work demonstrates the ability to correlate bulk tissue compaction with label-free 4D‑OCT imaging of VIC and VEC morphology, collagen matrix changes, and features that resemble calcific nodules in the same sample over 7-10 days. At D7, gels treated with osteogenic media appeared to have more dense ECM aggregates inside the matrix than control gels. Utilizing OCT imaging, we observed novel osteoblast-like cellular “microtears” or “holes” created within the collagen matrix during remodeling.
CONCLUSIONS: The combination of cutting-edge computational 4D-OCT with our 3D VIC-VEC co-culture allows for label-free observation of CAVD-driven cellular and matrix changes, with future potential to identify when and what pathways may be effectively targeted with a medicinal therapeutic.
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