Isolation of senescent cells by iodixanol (OptiPrep) density gradient-based separation
- Cell Prolif. 2019 Nov;52(6):e12674. doi: 10.1111/cpr.12674.
- 1. Center for Translational Medicine, International Clinical Research Center (FNUSA-ICRC), Brno, Czech Republic.
Objectives: Chemotherapeutic drugs induce senescence in Cancer cells but, unlike replicative senescence or oncogene-induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy-induced senescent cells requires their isolation from a mixed population of adjacent senescent and non-senescent Cancer cells.
Materials and methods: We have developed and optimized a rapid iodixanol (OptiPrep)-based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)-induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh-7) in vitro.
Results: After cellular exposure to DXR, we used iodixanol gradient-based centrifugation to isolate and re-plate cells on collagen-coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR-induced senescent HCC cells, as confirmed by proliferation arrest assay, and β-galactosidase and DNA damage-dependent γH2A.X staining.
Conclusions: Analysing pure cultures of chemotherapy-induced senescent versus non-responsive Cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.