Abstract: Objective The mouse autologous tumor model H22 is more valuable for tumor immunological-related research. This paper aims to establish mouse hepatic tumor cell line (H22-luc2-tdT)that stably express the tandem-dimer tomato(tdTomato) and luciferase genes.Establish an in vivo imaging model of cell line derived transplanted tumors。Methods Using transplanted H22 tumor tissue, primary culture and continuous passage in vitro were performed to establish a continuous cell line. Cell proliferation, chromosome analysis, organoid culture, tumorigenicity, HE and ICH of aFP,CK7, CK15 were performed to charaterize the cell line. Then the luc2-tdTplasmid was transfected into H22 cells of P22, flow cytometry and in vitro/in vivo imaging were employed to screen and verify fluorescence expression. Mycoplasma detection and species verification of the established cell lines were performed. Results The H22 cells had been continuously passaged over 50 times. The cells of passsge 22(P22) were transplanted subcutaneously and intraperitoneally into C57 and Kunming mice, with a 100% tumor formation. The HE morphology of subcutaneous transplanted tumor were consistent with the original tumor. CK⁺/AFP⁺proved that it was of liver cancer origin. The H22 cells were hypo-triploid with a modal number of 40-44 chromosomes and telocentromeres, verifing its mouse origin. The latent phase for in vitro growth of H22 lasted from d0 to d3, while the exponential phaes d3 to d5, and reach plateou at d6. Successful transfection of H22 cells with the luc2-tdT were observed with in vitro/in vivo 100% fluorescence positivity, thus named H22-luc2-tdT. The transplanted tumor tissue of H22 cells could be primarily cultured to form organoids. The detection of Mycoplasma was negative, and its mouse origin confirmed by PCR. Conclusions H22 and H22-luc2-tdT cell lines are established and characterized, which can be used for the establishment and application of in vitro and in vivo liver cancer research and metastatic cell tracking. These cell lines are deposited at and can obtain from the National Biomedical Cell Resource Center(http://www.cellresource.cn).

Key words: mouse liver cancer cell line, fluorescence labeling, in vivoimaging, organoid