Preparation of tirapazamine-resistant hepatoma cell line

ABSTRACT: The aim of the project was to obtain murine hepatoma MH22a [1] cell subline, resistant to anticancer agent 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), and examine the changes of the protein expression as compared with parental cells, with the main emphasis on the changes of redox proteins. MH22a cells obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia), were grown at 37°C in DMEM medium, supplemented with 10% fetal bovine serum and antibiotics in T25 flasks until reaching 70-80% confluence. The medium is then changed to an analogous medium with 5.0 μM TPZ, and, depending on cell viability, 50% of the medium with TPZ is changed 1-2 times a week, repeating that procedure for 3.5 months to form a stable monolayer. The resulting monolayer is inoculated 1:2 and further grown by changing 50% of the culture medium twice a week until re-inoculation. 20 passages were made over 2.5 months in total. We were unable to obtain a cell subline in a stepwise fashion by growing the cells in increasing concentrations of TPZ, because its concentration increase up to 10 μM caused cell death after 1 month. For analysis, one flask per each sample, 3 samples of parental cell line and 3 samples of TPZ-resistant cell line (between 15th and 20th passages), were selected. Cells were rinsed three times with PBS, detached with TrypLE Express (ThermoFisher Scientific, Waltham, MA, USA) and cell concentration and viability was determined with Trypan blue and Countess 2 (ThermoFisher Scientific, Waltham, MA, USA) automated cell counter. Cells were rinsed by centrifuging 500xg for 5 min and suspending in PBS three times. Finally, cells were flash frozen in liquid nitrogen and transferred to -86°C freezer for storage. [1] European Collection of Authenticated Cell Cultures (ECACC), Catalogue No. 96121721.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Hepatocyte

DISEASE(S): Liver Carcinoma

SUBMITTER: Frank Stein  

LAB HEAD: Narimantas Cenas

PROVIDER: PXD039876 | Pride | 2023-05-10

REPOSITORIES: Pride