Study On Microfluidic-Based Bionic Liver Chips

The liver is one of the most important organs of the human body,with complex microstructures and unique microenvironments,performing a variety of important functions.Once the liver disease occurs,human health will be severely affected.Therefore,in order to prevent,diagnose,and treat liver diseases,it is necessary to deeply study physiological and pathological mechanisms of the liver.Currently,research related to physiological and pathological mechanisms of the liver is mainly based on animal models.Although animal models have advantages in simulating physiological environments,they are time-consuming,uncontrollable,unquantifiable,difficult to execute multi-parameter experiments,and cannot visually reveal changes in cell behaviors caused by liver lesions.The microfluidic-based liver chip is a novel tool that is rapidly developing in recent years and expected to replace animal models.It can achieve the purpose of simulating the liver by constructing liver tissue functional units on the chip and providing physiologically similar physical and chemical microenvironments.At present,the research of liver chips have made positive progress,but there are still many technical difficulties,such as how to achieve the three-dimensional(3D)ordered long-term co-culture of multiple types of cells,how to reconstruct highly biomimetic liver ultrastructure,how to accurately provide physiologically similar physical and chemical microenvironments and so on.Thus,the dissertation designs three kinds of liver chips to solve the above difficulties.The main contents are as follows:The first is a liver chip that mimics the blood flow of the sinusoids.In this chip,the pressure difference between two fluids flowing in a countercurrent manner on either side of the cell culture zone is employed to generate the convection across the cell culture zone to mimic sinusoid blood flow.The design greatly enhances the transport of nutrients in the cell culture zone and achieves high-density large-scale culture of hepatocytes.Here,the distributions of living and dead cells under various perfusion flow rates were observed and the oxygen concentration distribution in the cell culture zone was simulated,which confirmed that the mimicked sinusoid blood flow can promote the transport of substances and thereby improving cell culture.The main experimental results showed that the metabolic activity and detoxification function of hepatocytes worked well,and the density of hepatocytes cultured at 3-mm-thickness scale reached 7 × 107 cells/mL on Day 7.The second is a liver chip that reconstructs the tissue interfaces.In this chip,the tissue interfaces are constructed by mixing double-layer microspheres(the hepatocytes are located in the inner layer of the double-layer microspheres while the hepatic stellate cells are located on the outer layer)and the endothelial cells in the hydrogel,and a trivascular system(hepatic artery,hepatic vein and central vena cava)is designed to provide physiologically similar physiochemical microenvironments.The main experimental results showed that the vascularized liver tissue was formed in the cell culture area,and the concentrations of hepatocyte metabolite(urea)and synthetic(albumin)increased with the incubation time.This indicates that physiologically similar tissue interfaces and physiochemical microenvironments are conducive to maintaining liver function.The third is a hepatic lobule chip based on cell sheet.In this chip,the liver tissue interface is constructed by mixing the 3D hepatocyte-astrocyte sheet cultured in a culture dish and the endothelial cells in the hydrogel,and an oxygen sensor chip is added to adjust the dissolved oxygen concentrations in the culture medium from artery and vein inlets.The liver chip can simultaneously simulate the differences in oxygen concentration and nutrient concentration between the hepatic artery and vein,and the unique structure of the cell sheets is closer to the arrangement of cells inside the liver.The main experimental results showed that the hepatic plate structure,vascular network structure and bile duct structure could be observed on the liver chip;the damage of drug(acetaminophen)to co-cultured hepatocytes is significantly lower than the damage to hepatocytes cultured alone.This dissertation not only provides new ideas for the further bionic design of liver chips,but also provides powerful tools for drug testing,drug screening,and liver disease modeling,etc.,and thus has very important theoretical and practical values.