Establishment And Application Of Three-dimensional Liver Inflammation Model In Vitro

Liver inflammation is one of the important factors that increase the toxicity of various drugs.However,before the drug is marketed,it is usually not evaluated clinically.Therefore,the establishment of a suitable and reliable human in vitro liver-like model is critical to the reasonable evaluation of drug toxicity.In this study,a spherical plate in vitro 3D culture technology was used.Three humanized cells were selected as HepaRG cells,hepatic stellate cells(HSC),and liver Kupffer cell(KC)cells for in vitro cell co-culture.The number of HSC is the same,and the number of different KC cells is adjusted simultaneously to establish three in vitro 3D co-culture models,named 0% KCs,10% KCs(simulating human normal liver)and 40% KCs(simulating human liver inflammation).The model is established.It was evaluated for liver-specific functions and cell morphology for 17 days after completion.The results show that the 3D liver pellets cultured using a spherical ultra-low-absorption culture plate have the spheres centered and the spheres can be formed quickly.They do not need to be transferred to a new culture plate and can be easily treated with drugs.The three in vitro 3D co-culture models established have gradually increased with time,and the maximum diameter can reach 500-600?m;all can maintain high levels of albumin,total bile acid secretion,and urea in 17 days.synthesis.In short,the 3D spheres we established in vitro can retain the specific functions of the liver for a long time.Then we evaluated the function of KC cells in the 3D sphere.Lipopolysaccharide(LPS)is a kind of bacterial endotoxin,which can stimulate the body to produce interleukins and tumor necrosis factors,etc.,thereby causing inflammatory reactions.Therefore,we selected three models induced by LPS at a concentration of 1 ?g / mL for 24 hours,and detected the secretion levels of three pro-inflammatory cytokines IL-6,TNF-?,IL-1?,and liver drug enzymes(CYP3A4,CYP2B6)and P glycoprotein(Pgp)gene expression analysis.Before and after LPS induction,there were no significant changes in the three cytokines in the 0% KCs model;the 10% KCs model increased slightly;the 40% KCs model increased significantly,among which TNF-? and IL-6 increased significantly.This illustrates the key role of KC,a liver non-parenchymal cell,in the inflammatory response in co-culture models.RT-pcr results showed that after inflammation induced by LPS,the expressions of CYP3A4 and CYP2B6 were all down-regulated,resulting in a decrease in activity and a decrease in drug clearance.P-glycoprotein gene expression is relatively up-regulated,resulting in resistance in the model.Therefore,in the following drug toxicity evaluation experiments,we chose two models of 10%KCs and 40% KCs as the experimental basis to explore the characteristics of different hepatotoxic drugs under the state of liver inflammation.We selected four hepatotoxic drugs: acetaminophen,isoniazid,amiodarone hydrochloride,and phenytoin sodium,and tested the release of lactate dehydrogenase(LDH)of these four drugs in the LPS-induced inflammation state.,Cell viability(ATP)detection and the secretion of three cytokines.The results showed that the 40%KCs model had higher cytokine release,increased LDH,and decreased ATP levels.Therefore,the 40% KCs model is significantly more cytotoxic under LPS-induced inflammation.It proves that adding different proportions of KC cells is critical.In the future,the proportion of different proportions of cells can be adjusted to evaluate the characteristics of various types of drugs under human diseases,providing a new direction for safety evaluation before new drugs are marketed in the future.In addition,we also used these two models to explore the treatment of hydrocortisone(HC)for four hepatotoxic drugs.The results showed that before HC was added,the cytokine release concentration and LDH toxicity were dose-dependent.After HC treatment,the three inflammatory factors and LDH toxicity release were significantly reduced.It shows that HC can reduce the inflammatory response by inhibiting the synthesis of inflammatory factors,and has certain anti-inflammatory effects on both models.In summary,we have established a valuable in vitro 3D liver model that is stableand capable of reproducing the physiological functions of the liver in vitro and can be used to study the characteristics of different types of hepatotoxic drugs in the state of liver inflammation.Drug liver toxicity provides a new platform.