Development and Validation of a Scaffold‐Free Human Multilineage Spheroid Model for Early Stage Cholangiopathies Driven by Cholangiocyte Senescence

ABSTRACT Background and Aims Cholangiopathies, including primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), and post‐COVID‐19 cholangiopathy (PCC), involve chronic cholangiocyte injury, senescence, epithelial–stromal crosstalk, and progressive fibrosis. However, effective in vitro models to capture these interactions are limited. Here, we present a scaffold‐free 3D multilineage spheroid model, composed of hepatocyte‐like cells (HepG2), cholangiocytes (H69), and hepatic stellate cells (LX‐2), designed to recapitulate early fibrogenic responses driven by senescent cholangiocytes. Methods Cholangiocyte senescence was induced via ionising radiation prior to spheroid assembly. Fibrosis progression was assessed by mRNA expression of hepatic stellate cell (HSC) activation markers, immunofluorescence, and Masson's trichrome staining, in both direct HSC co‐culture and spheroids. To evaluate therapeutic potential, spheroids were treated with A‐1331852, a selective Bcl‐xL inhibitor known to induce apoptosis in senescent cells in PSC and PBC mouse models Drug efficacy was measured by apoptosis induction, reduction of fibrosis, and modulation of stromal activation. Results Senescent cholangiocytes induced robust HSC activation and promoted extracellular matrix (ECM) deposition, mimicking early fibrogenesis. Treatment with A‐1331852 selectively induced apoptosis in senescent cholangiocytes and activated HSCs, leading to a marked reduction in fibrosis, consistent with murine PSC and PBC models. Conclusions This 3D multilineage spheroid model offers a mechanistically relevant and scalable platform to study cholangiocyte senescence‐driven fibrosis. Its applicability to senolytic drug testing supports its use in preclinical screening and translational research across a spectrum of cholangiopathies.