ABSTRACT
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.
ABSTRACT
Objective To establish a hanging drop 3D cell culture model of human colon cancer cell (HT29) in 48-well cell culture plate,at the same time,through the comparison of several cell viability detection methods to determine the appropriate one for this cell culture way.Methods HT29 cells of 2 375,3 164,4 218,5 625,7 500 and 10 000/well were seeded in the bottom of the 48-well culture plate to form droplets.After 2 d of inversion culture,the cell spheroids were formed and incubated in medium for another 3 d.The volume of cell spheroids were measured,and the absorbance (A) values were detected through APH assay,MTT assay,MTT assay after digestion,CCK-8 assay and CCK-8 assay after digestion.The results were compared among different methods.Results After 5 d of culture,the cell spheroids were formed perfectly at the density of 2 375-10 000/well,and the volumes were in good linear with the original cell inoculation number at the density of 2 375-7 500/well.The A values of APH assay,MTT assay after digestion and CCK-8 assay after digestion increased with the increase of cell inoculation amount;But the cell ball digestion process was complex,and the cell viability was damaged.However,the A values of MTT and CCK-8 assay increased slowly.Conclusion The method of a hanging drop 3D cell culture model in 48-well culture plate combining with APH assay to detect cell viability is economical,accurate and easy to operate.
ABSTRACT
Objective To establish a hanging drop 3D cell culture model of human colon cancer cell (HT29) in 48-well cell culture plate,at the same time,through the comparison of several cell viability detection methods to determine the appropriate one for this cell culture way.Methods HT29 cells of 2 375,3 164,4 218,5 625,7 500 and 10 000/well were seeded in the bottom of the 48-well culture plate to form droplets.After 2 d of inversion culture,the cell spheroids were formed and incubated in medium for another 3 d.The volume of cell spheroids were measured,and the absorbance (A) values were detected through APH assay,MTT assay,MTT assay after digestion,CCK-8 assay and CCK-8 assay after digestion.The results were compared among different methods.Results After 5 d of culture,the cell spheroids were formed perfectly at the density of 2 375-10 000/well,and the volumes were in good linear with the original cell inoculation number at the density of 2 375-7 500/well.The A values of APH assay,MTT assay after digestion and CCK-8 assay after digestion increased with the increase of cell inoculation amount;But the cell ball digestion process was complex,and the cell viability was damaged.However,the A values of MTT and CCK-8 assay increased slowly.Conclusion The method of a hanging drop 3D cell culture model in 48-well culture plate combining with APH assay to detect cell viability is economical,accurate and easy to operate.