Aims: Liver transplantation is an effective approach to end-stage liver disease. Shortage of donor liver and increased waiting time for liver transplantation necessitate the development of an organ culture system by which livers can be cultured and maintained ex situ for a prolonged period of time. The aim of this work is to test whether cell culture condition in vitro could be used to culture whole livers ex situ without the use of erythrocytes. Methods: Eight castrated male land race/farm young porcine livers were exposed to 30 min warm ischemia and 30 min cold perfusion. Livers were isolated and connected to an ex situ liver culture system using a standard culture medium RPMI 1640 supplied with 10% of fetal calf serum and sufficient dissolved oxygen under a normothermic condition for 6 hours. Metabolic biomarkers, bile and urea production, hepatic cell viability, and histology analysis of biopsies were performed and analyzed. Results: Dissociated porcine hepatic cells survived and grew in vitro under the standard RPMI 1640 culture medium. When the same RPMI 1640 medium supplemented with 10% of FCS and sufficient oxygen was used to culture livers ex situ, over 98% of liver cells were viable for at least 6 hours during ex situ whole organ culture based on the results from biochemical assays. Conclusions: Our data demonstrate that the liver culture system established in this work can be used to culture whole livers ex situ in the absence of erythrocytes.