A Perfused Liver Cell Spheroid Culture Model For Hepatotoxicity Screening

BackgroundThe liver damage caused by the drug itself and/or its metabolites is called drug-induced liver injury.It is also known as drug-induced liver disease.It can be clinically manifested as various acute and chronic liver diseases.The light can be restored after discontinuation of the drug,and severe cases can be endangered life.Because of the serious clinical consequences it may cause,drug-induced liver damage is an important cause of the withdrawal of many drugs.In the past 60 years,about 25%of drugs were withdrawn due to hepatotoxicity,which greatly increased drug development.The cost and risk.Therefore,in the development of new drugs,it is important to test whether the drugs and their metabolites produce damage to the liver.The conventional method of toxicity testing in animal experiments not only consumes a lot of manpower and financial resources,but also has a long experimental period.Due to species differences and individual differences,the experimental results do not reflect the true toxicity of the drug.The in vitro cell model was used for drug hepatotoxicity screening because of its advantages such as short experimental cycle,good repeatability,easy operation,and high throughput.Although in vitro liver cell culture models are increasingly used for drug absorption,metabolism,distribution,excretion(ADME)and toxicity testing.However,the traditional two-dimensional cell culture model in vitro is still not enough to completely replace animal models for predicting the toxic effects of drugs in humans.Due to the lack of sufficient function and structure of cells in vitro culture,it is difficult to provide sufficient accurate prediction results in terms of ADME and toxicity.Three-dimensional culture increases the contact between cells and cells and promotes the expression and maintenance of functions and phenotypes during cell culture in vitro;thus,it can provide more accurate in vitro prediction results.Cell ball culture is a commonly used three-dimensional culture model.The cultured cell spheres have a characteristic concentration gradient of oxygen and nutrients from the surface of the cell sphere to the center,which is compatible with the oxygen and nutrition of the liver lobules from the portal area to the central vein.The distribution of material concentration gradient characteristics;previous results showed that cell-cultured hepatocyte-like cells have better functions.However,due to the low oxygen supply from the central cells,the cell spheres are prone to central cell necrosis.Continuous perfusion culture can simulate the shear force produced by the body fluid flowing in the body at the same time.It can promote the functional expression and maintenance of the cells.Continuous perfusion culture can slow the hypoxia of the center of the cell sphere.However,there are few reports on the combination of spheronization and continuous perfusion culture as drug screening cultures.For this purpose,this project combines two culture methods to establish a new drug screening model and verify its feasibility as a drug screening model.ObjectionEstablish a drug screening model that combines pedogenic culture with continuous perfusion culture and evaluate the accuracy of drug screening.Methods and materialsThe hepG2 cells were seeded in a agarose mold and allowed to stand for 24 hours to pellet the cells.The cell pellet was transferred into the tissueflex reactor along with the mold and the flow rate was set to 1 ml/24 h.The control group was set to statically cultured cell pellets,which were changed 1 ml every 24 h.The morphology of the cell spheres was observed on days 1,3,5,and 7 respectively,and the life-and-dye staining and CCK-8 cell growth curve were plotted.RNA was extracted and RT-qPCR was used to detect the expression of CYP enzyme in the cells.According to the previous results,the drug intervention experiment was conducted on the fifth day of culture to determine two hepatotoxicity-negative drugs,and six liver toxicity-positive drugs were used to determine their IC50.SPSS software was used for statistical analysis.Results1.Morphological observations showed that the cells cultured with tissueflex for continuous perfusion culture were more compact and the cell spheres grew faster,but both reached the plateau stage on the 6th day and did not increase.The cell sphere growth curve determined by CCK-8 showed that the dynamic cell viability was higher than static,and both reached the highest on the 6th day.On the sixth day,the cells started to apoptosis due to the excessive cell diameter.2.Live and live staining showed that there were fewer dead cells inside the dynamic cell,and the cell survival rate was higher;3.Drug experiments indicate the IC50 of durgs in perfusion condition was lower than static.Conclusion1.Continuous perfusion of cultured hepatocyte cells can better express and maintain hepatocyte drug metabolism;2.Continuous perfusion culture of hepatocyte cells can more accurately predict hepatotoxicity of drugs,which is a good drug screening model.