The extracellular matrix (ECM) in a liver-specific extracellular matrix (L-ECM) scaffold facilitates hepatocyte viability and maintains hepatocyte functions in vitro. However, whether an intact composition of ECM is required for an efficient ECM-based substrate design remains to be clarified. In this study, two L-ECM hydrogels, namely L-ECM I and L-ECM II, were prepared by pepsin solubilization at 4 °C and 25 °C, respectively. The solubility at 4 °C was 50% whereas that at 25 °C was 95%, thus indicating well-preserved L-ECM. Analysis confirmed higher ECM protein components (especially collagen) in L-ECM II, along with denser fiber network and larger fiber diameter. L-ECM II gel exhibited high compression strength and suitable viscoelastic properties. Furthermore, hepatocytes in L-ECM II showed higher expression of liver-specific functions in 3D culture and wider spread while maintaining the cell-cell contacts in 2D culture. Therefore, an intact L-ECM is important to realize effective substrates for liver tissue engineering.
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