The 3R guidelines impose the replacement of animal testing with alternative models. Liver-on-chip represent a promising alternative for drug screening and toxicological assays. Albeit its increasing potential, the main challenge is the necessity of optimizations to mimic in a trustworthy way the functionalities and the microsomal environment of the liver cells. In this work, we developed a new liver-on-chip model including a relevant 3D matrix (porous hydrogel) for hepatic cell growth and function, namely BIOMIMESYS® Liver hydroscaffold. This 3D matrix is composed of liver ECM components. The HepG2/C3a cell line is cultured under flow for 20 days and compared to cells cultivated in static conditions (BIOMIMESYS® Liver in 96-well plates). The cells, in contact with the hydrogel, tend to assemble and form spheroids while attaching to the adhesion sites of the hydroscaffold. First of all, measurement has been done to ensure that an optimal flow is preserved with and without the gel. Then, the cell viability, physiology, spheroid integrity were studied by immunostaining of the tight junctions and the development of the actin network in addition to the albumin production and compared in the two conditions. These results have shown that the dynamic condition is associated with an increase in albumin production by 100-folds, a higher viability and a conservation of the integrity of the spheroids compared to the static condition. In addition, the cell growth caused an increase of the pressure in the biochips. These results highlight the tremendous role of the liver-on-chip model coupled with ECM matrix in the enhancement of cells functions and physiology in addition to the mimicking of the cellular microsome.