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These immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. -

[RF84750] THLE-2 and THLE-3 cells express phenotypic characteristics of normal adult liver epithelial cells. They are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. [RF84750] THLE-2 and THLE-3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. [RF84750] Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells.

[RF84750]  A culture submitted to the ATCC in May of 1989 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline. The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative. The cured cell line is available as ATCC CRL-2706. The original patent deposit is available as ATCC CRL-10149

1. Remove and discard culture medium.
2. Add a fresh 0.05% trypsin-EDTA solution  (2.0 – 3.0 mls for a T75 flask) to cover the cells, rinse and remove most of the trypsin solution leaving a thin coating behind..
3. Allow the culture to sit at room temperature (or 37°C) until the cells detach and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Neutralize the trypsin with 0.1% soybean trypsin inhibitor and aspirate cells by gently pipetting. Remove the dissociation agent by gentle centrifugation (150 x g to 400 x g for 8-12 minutes) and resuspend cells in fresh complete growth medium. 
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C. 
7. A 0.05% Trypsin-EDTA solution (GIBCO cat# 25300-054) is recommended for dissociation. Neutralize with a 0.1% Soybean Trypsin inhibitor solution (ATCC 30-2104).

Harris CC, et al. Human liver epithelial cells. US Patent 5,759,765 dated Jun 2 1998

Pfeifer AM, et al. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. Proc. Natl. Acad. Sci. USA 90: 5123-5127, 1993. PubMed: 7685115