Résumé
This study was conducted to establish an in vitro 3D liver model and apply it to the drug liver toxicity evaluation. The 3D multicellular sphere model of HepaRG cells was established by hanging-drop technique for evaluation of liver function. The 3D liver model was used to test the hepatotoxicity of isoniazid and amiodarone hydrochloride compared to the 2D cell culture model. Our results showed that HepaRG cells formed a compact spheriod, and the level of cell albumin, urea and the CYP3A4 activity were significantly higher than that of 2D model. With the treatment of amiodarone hydrochloride in 2D and 3D model, the IC50 were 50 and 100 μmol·L-1, respectively. When the dose was less than 1 000 μmol·L-1, isoniazid had no hepatocyte toxicity in 2D model, while the IC50 in 3D model was 700 μmol·L-1. The LDH activities of both drugs in 3D model showed time-and dose-dependent correlation. The results suggest that this in vitro 3D hanging-drop liver model is good for testing liver functions with a high hepatic drug-metabolizing enzyme activity. Compared with the 2D model, the 3D liver model can accurately evaluate the liver toxicity of drugs. Our results demonstrated the importance of in vitro cell culture models for detection of in vivo-relevant adverse effects of drugs.
Résumé
Objective To evaluate the hepatocyte function and ultramicrostructure of marrow mesenchymal stem cells (MMSCs) derivation hepatocyte like cells. Methods MMSCs were isolated from fetal marrow. MMSCs were cultured and induced in vitro in 1% Matrigel as matrix, 2.5 ?mol/ml azacitidine (AZA) pretreatment for 10～12 h, hepatocyte growth factor (HGF) 10 ng/ml+fibroblast growth factor 4 (FGF4) 10 ng/ml+hepatocyte growth medium (HGM). Albumin (ALB) level in culture supernatant was determined with enzyme linked immunosorbent assay (ELISA), capability for dealing with ammonia into urea was detected with urease method, staining for glycogen of undifferentiated MMSCs and MMSCs derivated hepatocyte like cell at induction days 7, 14, 21 and 28 was conducted with periodic acid Schiff's (PAS) test, ultramicrostructure of MMSCs was observed by electromicroscope. Results Undifferentiated MMSCs did not produce ALB and urea. Following treatment with FGF4 and HGF, ALB and urea production by induced MMSCs increased in a time dependent manner. Glycogen storage was first seen by day 14, and maximum levels were seen after day 28, enhancement of glycogen staining for induced MMSCs showed that hepatocyte like cell had glycogen synthesis capability. Undifferentiated MMSCs possessed the construction features of juvenile cell. Induced MMSCs at day 28 had differentiated and had the polarity construction features of epithelioid cell. Conclusions The induced MMSCs has hepatocyte specific construction and functional features.