Study On The Formation Of Hepatic Sinusoids Faciliated By Microneedle Array In A Liver Acinus Chip

The liver is one of the most important organs in the human body,performing many functions that are vital to life.To study the mechanism of liver functions,the hepatotoxicity of drugs and the liver-related diseases,animal models and cell culture methods were usually used by researchers.However,the interspecies differences affect the reliability of animal models while traditional two-dimensional and even three-dimensional cell cultures can form only occasionally very simple liver microstructures and rarely generate self-organized perfusable blood vessels,let alone complicate vascular networks;in addition,for the emerging organoid technologies,there are also some limitations in the formation of perfusable vascular networks.Currently,the organ-on-a-chip technology has been developed rapidly,which can replicate physiologically similar microstructures and microenvironments,compared with traditional organ modeling methods.However,the construction of a highly biomimetic liver-on-a-chip is a challenge,and there is still a large gap between the reported liver chips and the liver,especially in the formation of hepatic sinusoids.In this study,therefore,a rapid,controllable,and large-scale hepatic sinusoid construction method was presented based on a self-developed microneedle array,and then the formation of hepatic sinusoids was realized on a liver acinus chip with a dual blood supply and three vessels.The main research contents are as follows:First,a liver acinus chip with dual blood supply and three vessels was designed and fabricated.The liver acinus chip includes a triangular prismatic cell culture area,portal vein,hepatic artery,and central vein.To facilitate the formation of hepatic sinusoids,a microneedle array was prepared based on 3D printing technology.By the microneedle demolding method,primary hepatic sinusoids were formed in the photocurable cell culture area of the liver acinus chip,which agreed with the expected design size and distribution.To provide physiologically similar oxygen concentrations for the liver acinus chip,the oxygen chip previously designed by our group was also optimized.The results showed that the improved oxygen chip more stably produced the dissolved oxygen concentration close to the physiological value.Then,flow simulations were performed for the liver acinus chip culture system.Here,a hybrid flow model was established by combining the finite element simulation method and the flow resistance simulation method,and then the flow in the whole culture system was simulated.The simulation results showed that the primary hepatic sinusoids constructed by the microneedle array demolding technology significantly reduced the flow resistance in the cell culture area and finally enhanced the flow in the acinus,and the larger the size of the formed hepatic sinusoid,the higher the flow in the acinus.In addition,the flow direction and rate in the cell culture area could be precisely controlled by means of adjusting the inlet flow rate of the liver acinus chip,the outlet pressure of the liver acinus chip and the size of the microneedle array.In this study,when the radius of hepatic sinusoid was 150 μm,under the inlet flux and outlet pressure close to human physiological conditions,the flow in the cell culture area was biomimetic.The experiment results confirmed the validity of the flow rate and flow direction in the cell culture area.Finally,the performance of the liver acinus chip was validated and the application of the liver acinus chip was demonstrated.Here,the performance of liver acinus chip was confirmed mainly in terms of cell metabolism,cell viability and liver microstructure.The results showed that the primary hepatic sinusoids constructed by microneedle demolding could effectively enhance the metabolism of hepatocytes,increase the viability of liver cells,and promote the formation of hepatic microstructures,especially the self-assembly formation of the secondary hepatic sinusoids.Moreover,when the radius of microneedle was 150 μm,the performance of the liver acinus chip was optimal.In terms of application,the effects of the concentration gradients of blood oxygen and glucose on cell metabolism and the effects of the drug acetaminophen on cell activity were preliminarlily studied in this paper.The results showed that compared with two-dimensional cell culture or three-dimensional cell culture on the chip,the hepatic sinusoids constructed by the microneedle array could facilitate the formation of oxygen and glucose concentration gradients in the liver acinus,and the cultured cells showed better metabolism ability and drug sensitivity.This study reported an approach for the construction of hepatic sinusoids in a liver chip.The novelty of this study is that,by demolding a self-developed microneedle array,primary hepatic sinusoids are formed in a photocurable cell culture area,and then secondary hepatic sinusoids are self-assembled in the presence of primary hepatic sinusoids and flows.This work paves an avenue for the construction of hepatic sinusoids and the fabrication of large-scale liver bioreactors.