Construction And Application Of A Liver Lobule Chip With Hepatic Sinusoid Network

Liver is the largest substantial organ and digestive gland of human body,and also the main organ of drug metabolism.Currently,basic liver research is conducted in two ways:one is 2D cell culture,and the other is animal model.The emergence of organ chip provides a more accurate transformation platform for drug toxicity tests and disease model construction.In recent years,liver chips have developed rapidly in bionics.However,in terms of the structure and function of the liver chips,there is still a big gap between the current liver chips and the actual liver.As a result,the current liver chips are limited in application.In the design of liver chips,researchers usually regard portal vein and artery as the same blood vessel model.Although this scheme is easy to construct liver lobules in vitro,it cannot simulate physiologically differences of dissolved oxygen concentration in arterial and venous.In the construction of sinusoidal networks of the liver chip,there have been reported that sinusoidal networks were formed locally only under the induction of angiogenic factors.However,due to the interaction of various cells,it is still a huge challenge to construct a large-scale liver chip with sinusoidal networks in vitro.Accordingly,this study is based on microfabrication and microfluidic technology to construct a liver chip with hepatic sinusoidal network and apply it to drug testing and tumor growth.The main research contents are as follows:Firstly,the liver lobule chip(LLC)with hepatic sinusoidal network was constructed.In this study,a liver lobule chip was constructed by combining photolithography technology and mechanical micromachining technology.In the LLC,the portal vein(PV),artery(HA)and central vena cava(CV)fluid channels were independent of each other.The tissue culture area was designed with multiple hexagonal liver lobules,and the main blood vessel cavity was formed by a micropillar array.In order to make the PV and HA fluid dissolved oxygen concentration in the LLC reach a physiological level,this paper also designed an oxygen chip,which was based on the redox reaction of Na2SO3 and O2 to reduce the dissolved oxygen concentration of the culture medium.The LLC forms a self-assembled tissue interface without the assistance of a scaffold,including an orderly arrangement of hepatocytes,a bile duct formed by a depression between hepatocytes,and a radial hepatic sinusoidal network with a lumen.In addition,ultrastructures such as hepatocyte microvilli,Disse’s space and fenestrae were also formed.Primary mouse liver cells could maintain high metabolic activity in the LLC for at least 28 days.Secondly,the effects of oxygen concentration and flow rate on angiogenesis were studied.The results showed that cells could form better vascular morphology(that was,the number of sinusoids with lumens was the most,the average diameter of the lumens was relatively small,and the shape of the sinusoids were more similar to the physiological state)and maintain higher metabolic activity(the main indicators were albumin,urea,bile acid,CYP450,etc.)under the condition of physiological similarity dissolved oxygen concentration(HA:4.2 mg/L and PV:1.8 mg/L)and flow rate(50μL/min).The results showed that under physiologically similar dissolved oxygen conditions,the oxygen distribution in the liver chip was also closer to the physiological situation.Finally,the applications of LLC in drug hepatotoxicity testing and liver tumor modeling were demonstrated.In drug liver toxicity test,whether it was drug acute liver toxicity test or long-term liver toxicity test,the cell activity in LLC could better reflect the authenticity of drug liver toxicity.In terms of tumor modeling,this study simulated the growth of tumors(HepG2 cell sphere)in situ in LLC,and observed the growth of tumor cell clusters using a fluorescence microscope.The results showed that the presence of tumors would affect the angiogenesis and change the oxygen environment.Finally,the colonization of circulating tumor cells(colon cancer cell HCT116)was simulated,and it was found that the proliferation of circulating tumor cells in the early stage was not significant.This study not only proposed a design method of LLC with perfusable hepatic sinusoid networks,but more importantly,the constructed liver chip extended the application from drug screening to liver-related disease modeling.