Introduction:Recently reported data have suggested that only a small subset of cancer cells possess the capability to initiate malignancies. These observations were based on investigation of cells within the primary tumors displaying a distinct surface marker pattern. CD133 marker is a putative hematopoietic and neuronal stem cell marker, which is also considered to be a tumorigenic marker in brain, prostate and liver. Recent studies have shown that a small population of CD133-positive cells, indeed, exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. This study was aimed at isolating the cancer stem-like cells from hepatocellular carcinoma cell line HepG2 using three different methods: magnetic-activated cell sorting (MACS), spheroid culture (SC), and anti-tumor drug (ATD) resistant selection. Methods: HepG2 hepatocellular carcinoma cells were expanded to yield enough cells that could be used to isolate cancer stem-like cells by these three methods. For MACS, cancer stem-like cells were sorted using anti-CD133 monoclonal antibody. For the second method, cancer stem-like cells were enriched by selection of anti-tumor drug resistance property. Lastly, for the third method, three-dimensional (3D) culture was used to enrich for the cancer stem-like cells. The cells obtained by the three methods were expanded to obtain an adequate number of cells for confirmation of CD133 expression. Results: The expression of CD133 + cells in the three methods was found to be different. In the MACS method, the expanded CD133 + sorted cells cultured through 2 passages only contained 0.40 % CD133 + cells. In the 3D spheroid cell culture, of the population of cells there were 38.39 % that were CD133 + cells. Lastly, in the anti-tumor drug (doxorubicin at 150 nM) resistant selection, 66.22 % were CD133 + cells. Conclusion: This study shows that isolation of HepG2 derived CD133 + population by culture with doxorubicin (150 nM) yields the highest efficiency and purity of the 3 methods studied. P. Isolation of cancer stem-like cells from hepatocellular carcinoma cell lineHepG2 by methods of magneticactivated cell sorting, spheroid culture, and anti-tumor drug-resistant selection: A primary evaluation. Progress. Stem. Cell.; 7(1):279-289. 279 History •