Development of a matrix-supported 2.5-dimensional co-culture model: an in vitro platform for studying liver cell interaction and disease mechanisms.

Cell-cell crosstalk and the extracellular matrix are critical for maintenance of tissue-specific structure and functions. Conventional 2-dimensional (2-D) cultures provide analytical simplicity but lack spatial cues, whereas fully embedded 3-dimensional (3-D) systems introduce technical limitations for imaging and downstream molecular analyses. Here, we present a matrix-supported 2.5-dimensional (2.5-D) co-culture platform that combines the analytical advantages of 2-D culture with a more physiologically relevant environment for cells. It is simple and accessible, yet it incorporates the spatial organization of direct cell-cell and cell-matrix interactions, which is typical of 3-D models. As a proof of concept, we used the platform to model hepatic fibrosis by co-culturing hepatic stellate cells (LX-2) with a hepatocyte surrogate (HepG2). The cells cultured in 2.5-D co-culture displayed architectural features similar to those observed in fibrotic tissue and they showed a significant increase in fibrogenic gene expression following TGF-β1 stimulation compared to the monocultures. This system also enabled functional assessment of Δ40p53, a p53 isoform linked to fibrogenesis, revealing a clear association between its expression and collagen deposition during fibrogenesis. This platform provides a biomimetic in vitro platform for investigating cell-cell and cell-ECM signaling. By combining architectural complexity with experimental flexibility, the 2.5-D co-culture platform holds promise for mechanistic studies in fibrosis and tissue remodeling.