Comparison of HepaRG and HepG2 spheroids on morphology, cytochrome P450 enzymes and albumin expressions and sensitivity to drug hepatotoxicity / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To establish three-dimensional (3D) spherical tissue-like models for HepaRG cells and HepG2 cells and compare Ihe difference in morphology, functional protein expressions and drug hepatotoxicity tests to provide data for the selection of drug hepatotoxicity detection models in vitro. METHODS Two in vitro 3D hepatocyte spheroid models were constructed on HepaRG and HepG2 cells in logarithmic growth phase. The two types of cells were separately seeded in ultra-low attachment surface 96-well plates at a cell density of 100 cells per well, and cultured in a conventional manner without adding any inducer. The morphology of the spheroids on the 3∗ day (D3), D7, D14, D21 after seeding was observed under the microscope, and the average diameters of spheroids were caculated. Differential expressions of cytochrome P450 enzymes and albumin at mRNA and protein levels in the two models were studied with quantitative real-time PCR, immunofluorescence staining and Western blotting. Seven drugs (thiamine, fialuridine, acetaminophen, benzbromarone, cyclophosphamide, isoniazid and nefazodone) were selected for hepatotoxicity detection. After a single dose and repeated dose experiments, the cell inhibitory rate was measured and inhibitory concentration 50 (IC∗) was calculated. RESULTS The average diameters of the two models increased with time. The average diameters of HepaRG cell spheroids on D7, D14 and D21 after seeding were 317.5, 334.3 and 397.8 pm, respectively, which were smaller than those of HepG2 spheroids (P40, 0.87, >20 , 35.74 and 2.57 mmol • L"\ respectively. Under the single administration and partial drug repeated administration, the inhibitory effect on the suivival of 2 spheroid cells did not reach half of the inhibiory level. CONCLUSION HepaRG spheroids are superior to HepG2 spheroids in morphology control and functional protein expressions. They are more sensitive in drug hepatotoxicity detection in vitro. HepaRG spheroids are a better 3D spherical tissue-like model in vitro.