In Situ retrograde caval perfusion as an alternative anatomical route for murine liver decellularization

Liver decellularization enables the generation of tissue-specific extracellular matrix (ECM) scaffolds for investigating cell-matrix interactions and advancing hepatic tissue engineering. However, whole-organ decellularization in mice remains technically challenging due to the small caliber of hepatic vessels and the extensive vascular manipulation often required during conventional ex situ procedures. This study investigated in situ retrograde caval perfusion as an alternative anatomical route for murine liver decellularization and characterized the structural and biological properties of the resulting scaffolds. Following vascular access through the caudal vena cava, with cranial caval ligation and the portal vein maintained as an outflow route, murine livers were perfused with 0.5% sodium dodecyl sulfate for 4 h. The procedure generated translucent acellular scaffolds while maintaining gross hepatic morphology and a macroscopically identifiable vascular network. Histological and fluorescence analyses demonstrated extensive cellular and nuclear removal, with residual DNA levels below 50 ng/mg of dry tissue across all evaluated hepatic lobes. Histochemical and immunohistochemical analyses revealed the retention of collagenous structures, glycosaminoglycan-associated components, fibronectin, and collagen V, while scanning electron microscopy demonstrated an exposed three-dimensional fibrillar ECM network. Biological evaluation using AML12 cells and primary liver-derived cells demonstrated cellular attachment and association with the decellularized scaffolds, with greater proliferative activity observed in AML12 cells. Furthermore, late-passage primary liver-derived cells maintained albumin secretory activity during scaffold-associated culture, with increased albumin concentrations observed between days 3 and 7, whereas differences in urea production were not statistically significant. Collectively, these findings support the feasibility of in situ retrograde caval perfusion as an alternative anatomical approach for murine liver decellularization, enabling effective cellular removal while retaining selected structural ECM characteristics and supporting hepatic cell interactions. This approach provides a methodological basis for future investigations of hepatic scaffold recellularization and perfusion-based tissue engineering.