The 3R guidelines recommend the replacement of animal testing with alternative models. One of the proposed solutions is organ-on-chip technology in which liver-on-chip is one of the promising alternatives for drug screening and toxicological assays. Albeit its increasing potential, the main challenge in this field is the necessity to achieve relevant in vivo like functionalities of the liver tissue in a best optimized micro environment of the cells. In this framework, we have investigated the development of hepatic cells under dynamic conditions inside a 3D hydrogel scaffold embedded in a liver organ on chip. The hydroscaffold, so call Biomimesys@Liver, is made of hyaluronic acid and composed of extra cellular matrix components (galactosamine, collagen type I and IV) with RGDs active sites for the 3D cell adherence that ensures further 3D colonization. The design of the biochip allows the dynamic culture of the hepatic cells. The HepG2/C3a cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregate and then proliferated forming 3D like spheroids. The cell viability, physiology, spheroid integrity and albumin production were studied and compared to a static condition. The results have shown a 3D spheroid organization of the cells up to large organoid formations, high viability, an important increase of the albumin production and finally an enhancement of the HepG2 cell line metabolism. Overall, the data demonstrated the potential of the Biomimesys@Liver hydroscaffold for organ on chip cultures. More particularly, these results highlight the tremendous role of the liver-on-chip model coupled with ECM matrix in the enhancement of cells functions and physiology.