Establishment Of 2D/3D HepaRG Cell Model And Its Application On Hepatotoxicity In Vitro

Method:In this experiment,HepG2 and HepaRG cells were taken as the research objects,and the enzymatic indexes,protein expression and urea secretion ability were evaluated comprehensively,so that more suitable cells were selected to construct 2D and3D spheroid models.The expression differences of 2D and 3D models at the gene level were studied by RT-PCR.Immunofluorescence and Westen blot were used to study the difference of 2D and 3D model at the protein level.In terms of toxicity experiments,the two models were initially validated by the negative drug aspirin?ASP?and the positive drug acetaminophen?APAP?,which showed that the model could predict drug hepatotoxicity.Then,the positive drugs troglitazone?TRO??nefazodone?NEF??tolcapone?TOL??isoniazid?ISO??Chlorpromazine?CPZ??Fluoxetine hydrochloride?FLX??Paroxetine?PAR??Trimipramine Maleate?TRM??Duloxetine hydrochloride?DUX?as well as the negative drug Folic Acid?FA?and so on,which had the risk of cholestasis,were selected to compare the advantages of the two models through CCK8 test and IC₅₀0 value,and to study their toxicity.It is proved that the spheroid model established by this subject can be used for the prediction of virto hepatotoxicityResult:Compared with HepG2 cells,the detection indexes of HepaRG cells were significantly up-regulated,so HepaRG cells were significantly better than HepG2 cells.In terms of model establishment,the drug metabolizing enzymes,transporters,nuclear receptors and major enzymes related to cholestasis in HepaRG spheroid models were significantly higher than those in 2D culture model.In the experimental phase of drug toxicity,both 2D and 3D cell models can be evaluated for hepatotoxicity to a certain extent,but 2D cell culture can only be administered in a single dose,and the 3D spheroid model is administered in a single dose and repeated administration,and this method of administration is closer to the clinical physiological administration mode.Comparison of IC₅₀0 values revealed that the 3D spheroid model showed more sensitive cytotoxicity in repeated dosing experiments.Conclusion:The 3D HepaRG spheroid model constructed in this study can be used for in vitro hepatotoxicity test and has good predictive ability in cholestasis hepatotoxicity.It can provide some research value for the study of cytotoxicity in vitro.