Lung cancer is a very aggressive type of cancer with approximately 1.7 million deaths in 2018. The widely used monolayer cell culture model is no longer sufficient for representing the tumoral cells in situ. It lacks key components such as spatial organization, cell-cell or cell-matrix contact making it unfit for a good representation of the tumor. In this research, three patients' lung adenocarcinoma cell lines have been isolated. These cell lines have been characterized by evaluating proliferation, performing immunostainings and RT-qPCR. Then, they have been compared using monolayer cell culture, spheroids, and sections of the initial tumor. Firstly, this research showed that panexin is a good candidate for FBS replacement. Then, this work also showed that lung adenocarcinoma cell lines cultured in spheroids and monolayer have different expression patterns. Finally, we observed that isolated cell lines from the same tumoral fragment could diverge when passages were high.isolated cell lines from the same tumoral fragment could diverge when passages were high.