Application Of 3D Hepatic Plate-Like Liver Model For Hepatotoxicity Evaluation

Research objectives:The drug induced liver injury has become the main reason for the withdraw of drug in the premarket stage.Therefore,to reduce the cost for developing a new drug,it is of a great value to develop a new in vitro model with excellent ability to forecast drugs'potential hepatotoxicity.Nowadays,the hepatocytes cultured in vitro have been widely used in monitoring the drugs' potential liver toxicity,of which the model in where hepatocytes are developed in 2D is very popular for its easy establishing procedure and low cost.However,a great deal of researches indicate that the hepatocytes developed in this state tend to lose its unique physiology function due to the change of morphology,the epithelial Mesenchymal transformation and so on,which may result in the loss of polarity of hepatocytes.Previous study developed a three-dimensional hepatic plate like coculture system with bionic advantage,which has significantly enhanced liver specific functions.This coculture system is optimized by replacing C3A with L-02 cell line,which has a more dominant bile secretion function and a more active cytochrome P450 enzyme system.We try to figure out its advantage as an in vitro model for hepatoxicity evaluation over other coculture system when exposed to statin and some known hepatotoxin such as Amiodarone.Research methods:1.Establish three-dimensional hepatic plate like coculture system,three-dimensional hybrid coculture system,sandwich coculture system and traditional two dimension coculture system.2.Hepatocyte morphology observation,hepatocyte viability and function detection.3.The detection of hepatocyte polarity with RT-qPCR?IF and western blot.4.Compare the sensitivity of different coculture system to different drugs when exposed to statin and some known hepatotoxin such as Amiodarone and so on.Research outcomes:1.Compared with other coculture systems,the three-dimensional hepatic plate like coculture system has the best performance from the perspective of tissue morphology maintenance,and its cell viability is also the best from the perspective of cell viability;2.Compared with other coculture systems,three-dimensional hepatic plate like coculture system tends to exhibit better function,which is implied by more urea and albumin synthesis.In the aspects of gene expression,many genes such as ALB and CPSI have higher expression compared to other coculture system.3.The three-dimensional hepatic plate like coculture system exhibit significantly improved metabolic and biotransformation functions compared to other coculture systems,which has higher gene expression of phase ?.metabolic enzyme,phase ?metabolic enzyme and nuclear receptor;4.Compared to other coculture system,L-02 cells in the three-dimensional hepatic plate like co-culture model showed a better polar phenotype.Gene expressions associated with liver polarity such as RAC1 gene are significantly elevated in Three-dimensional hepatic plate like coculture system,moreover,the expression level of multi-resistance related protein 2(MRP2)is higher than that in other coculture systems.5.Three-dimensional hepatic plate like coculture system exhibits better ability to predict drug hepatotoxicity.Research conclusion:Three-dimensional hepatic plate like coculture system can significantly improve hepatocyte vitality and polarity,compared with other co-culture system.Moreover,hepatocyte function has close relation with correct polarity which has great influence on bile secretion.This research provides strong evidence for the establishment of bionic liver structure in three dimension in vitro which can be used for drug development,liver polarity research in vitro,liver tissue engineering and so on.