Studies On Model And Application Of The Integrated Discrete Multiple Organ Cells Co-culture (IdMOC) In Rat

According to statistics, of all50%of the drug candidate was eliminated because oftoxicity, of which about30%of the drug is eliminated because of security in late stagedevelopment process. Therefore, developing early toxicity screening methods is an urgentneed to reduce drug attrition rate in preclinical trials. Currently there is only single cell inexperiment, which is used to evaluate the specific effects of drugs on single cells. Thepoison has selectivity in the body organs. And the susceptibility and cytotoxicity of cells invarious organs may vary. While most drugs will be metabolite toxic or non-toxicmetabolites in vivo, but we use S9as activation system in vitro. Thirdly, it is limited whenwe don’t know the specific organ toxicity for toxicity testing. Therefore, our laboratory hasdeveloped the integrated discrete multiple organ cell culture (IdMOC) system of rats whichcan cover multiple organs. The initial verification showed better targeting in the targetorgan toxicity screening. However, the IdMOC system using the flyer plate has somedisadvantages, such as low flux, complex operation, more needed for test substances andso on. So we need to make futher improvements to address these issues.Objective1. To establish the integrated discrete multiple organ cell culture (IdMOC) model inrat by48well-plates2. To study the application of the IdMOC model in research new pollutants anddifferent scales nanoparticles.Methods1. Using primary hepatocytes, nerve astrocytes, pulmonary macrophages, cardiacmyocytes and renal cortical cells which are represent the five major organs. Each cellswere inoculated in the co-culture plates firstly, after each cells are adherent, then add upmore culture medium to make six holes communicate with each other.they are under thesame medium, the same under the test substance concentrations and the five organ cultureconditions, which are namely IdMOC system. Compare the growing proliferation ofco-culture and single cell culture conditions during7days by MTT assay. Take the toxicsubstances which have the specific organ toxicity of Paraquat, Carbon tetrachloride(CCl4)for the model, and the cytotoxicity was measured by MTT assay. Use the IC50software tocalculate the concentration for each cell in the50%inhibitory (IC50values). Then we can verify whether this model screening is suitable for screening the target organ.2. Take theErythromycin lactobionate, Streptomycin sulfate, Doxorubicin, and Busulfan for theIdMOC system, observe the morphological changes after24h, and determine the cellviability by MTT assay and calculate IC50values for each cell.3. Take the different sizesof Nano-TiO2(including5-10nm,60nm,90nm), different scales of Nano-SiO2(including15nm,50nm,100nm), different scales of Nano-ZnO (including30nm,50nanometers,100nm) for the model, observe the morphological changes after24h, and determine thecell viability by MTT assay and calculate IC50values for each cell.Results1. The separated methods of rat primary hepatocytes, neural astrocytes, pulmonarymacrophages, cardiac cells and renal cortex cell is stable, and survival rate of cells is high.The result of7days for five primary cells indicate that the growth and proliferation of fiveprimary cells co-cultured is good. The growth curves of single type of cell culture andco-culture are almost coincident. According to IC50values, the toxicity order of paraquat(methyl viologen) for five cells is: pulmonary macrophages> renal cortical cells>cardiomyocytes> astrocytes> hepatocytes. It is consistent with the animal test whichindicates lung is the most sensitive target organ. According to IC50values, the toxicityorder of CCl4for five cells is: hepatocytes> renal cortical cells> astrocytes>cardiomyocytes> pulmonary macrophages. It is consistent with the whole animal whichindicates that liver is the most sensitive target organ.2. According to IC50values, toxicity order of Erythromycin lactobionate for five cellsis: pulmonary macrophages> astrocytes> cardiomyocytes> kidney cortexcells>hepatocytes. The dose-response curve shows that its toxiciy has obviousdose-dependent for the five cells. Under the same dose, the survival of pulmonarymacrophages is lower than any other cells. The toxicity order of Streptomycin sulfate forfive cells is: pulmonary macrophages> kidney cortex cells> cardiomyocytes> astrocytes>hepatocytes. The dose-response curve shows that its toxiciy has obvious dose-dependentfor the five cells. The toxicity order of Doxorubicin for five cells is: astrocytes> pulmonarymacrophages> kidney cortex cells> hepatocytes> cardiomyocytes. The dose-responsecurve shows that its toxicity has obvious dose-dependent for the five cells. Under the samedose, the survival of pulmonary macrophages and astrocytes are lower than other cells,which indicates Doxorubicin is the maximum toxic to pulmonary macrophages and astrocytes. The toxicity order of Busulfan for five cells is: astrocytes> cardiomyocytes>pulmonary macrophages> kidney cortex cells> hepatocytes. The dose-response curveshows that in addition to the hepatocytes, its toxicity has obvious dose-dependent for theother cells. The cell viability decreased with the increased concentration especially withastrocytes.3. In addition to5-10nm Nano-TiO2, the toxicity order of Nano-TiO2for five cells is:pulmonary macrophages> astrocytes> cardiomyocytes> hepatocytes, renal cortex cells. Itis consistent with the animal test which indicates lung is the most sensitive target organ.Different scales Nano-TiO2showed significant dose-dependent toxicity of each cell. Thetrend of dose-response curve of each cell is similar for each scales. The sequence oftoxicity for Nano-TiO2are:5-10nm>60nm>90nm.According to IC50values, the toxicity order of Nano-SiO2for five cells is: pulmonarymacrophages> astrocytes> cardiomyocytes> hepatocytes, kidney cortex cells. It isconsistent with the animal test which indicates lung is the most sensitive target organ.Different scales Nano-SiO2showed significant dose-dependent toxicity of each cell. Asthe concentration increases, the cytotoxicity increased. The trend of dose-response curveof each cells is similar between100nm with50nm Nano-SiO2; But15nm Nano-SiO2curve is steeper than the other two scales, and cell viability rate decreased quickly with theincreasing concentration. The sequence of toxicity for Nano-SiO2is:15nm>50nm>100nm. The results preliminary indicate that, target organ of Nano-SiO2is the lung.According to IC50values, the toxicity order of Nano-ZnO for five cells is:cardiomyocytes> astrocytes> hepatocytes> renal cortical cells, pulmonary macrophages. Itis consistent with the animal test which indicates heart is the most sensitive target organ.Different scales Nano-ZnO shows significant dose-dependent toxicity of each cell.As theconcentration increases, the cytotoxicity increased. The trend of dose-response curve ofeach cell is similar for each scale. With the scales declined, the cytotoxicity increased. Thesequence of toxicity for Nano-ZnO is:30nm>50nm>100nm. The results preliminaryindicate that, target organ of Nano-ZnO is heart.Conclusions1. The establishment of IdMOC system in48-well plates is successful. It indicatesthat the model has a good prospect in targeted screening and research of toxicitycharacteristics, through the application of paraquat, CCl4, and three species of nanoparticles.2. Erythromycin whose more common side effects are irritation effects,gastrointestinal reactions; less common allergic reactions, ototoxicity and cardiac toxicity,kidney toxicity, that belongs to machrolides. Erythromycin is the maximum toxic topulmonary macrophages, followed by astrocytes. The main toxicity of streptomycin is thedamage of vestibular, cochlear nerve, kidney as well as trigger allergic reactions.Streptomycin shows the maximum toxic to pulmonary macrophages in the study.Doxorubicin is one of anthracycline drugs, which can cause bone marrow suppression andcardiac toxicity. Doxiorubicin is the maximum toxic to astrocytes, followed by pulmonarymacrophages. Busulfan’s side effects are pulmonary fibrosis and bone marrow suppressionon human. We found the busulfan is toxic to astrocytes and cardiomyocytes obvious.3. Nano-TiO2of each scale has the most toxicity to pulmonary macrophages. With thescales declined, the cytotoxicity increased, sensitive organ cells also increased. The resultspreliminarily indicate that, target organ of100nm and50nm Nano-TiO2is the lung, whilethe target organs of5-10nm Nano-TiO2are lung and CNS.Nano-SiO2of each scale shows remarkable toxic to cardiomyocytes. With the scaledeclined, the cytotoxicity increased. The results indicate that, target organ of Nano-ZnOmay be heart.Nanopaticles have significant scale effects for the primary cells. With the scaledeclined, the toxicity increased.The IdMOC system of rat can be used for screening and research the specific toxicityand characteristics of nanoparticles.