Development Of Three-dimensional Liver-sinusoid-on-a-chip Model Using Hepatocyte-like Cells From Human Induced Pluripotent Stem Cells

The liver,the largest internal organ of the body,plays a pivotal role in maintaining homeostasis of the organism.The liver sinusoid is a fenestrated capillary structure in the liver,and which supports the exchange of oxygen and nutrients,and exposes liver parenchymal cells to toxins and infectious agents.Since liver sinusoid becomes the primary target of wide range of liver diseases.However,their studies are particularly challenging owing to the lack of adequate models.For example,as a valuable tool in biomedical research,animal models are directly relevant to the human diseases because of specie variations.Despite that 2D cell models are characterized by their low cost and easy to culture,it cannot precisely reveal the structure,function,and response to drugs of human tissues.In addition,dynamic cultures of cells are another key factor for recapitulate the pathophysiological features of living human organs.Hence,the development of a high-fidelity model is needed.Meanwhile,hepatocytes are the major functional cells in the liver.In vitro PHHs are effective tools for studying liver pathophysiology,but its wide usage is limited by the shortage of available cells.For these reasons,differentiating from human induced pluripotent stem cells(hiPSC-HLCs),hepatocyte-like cells are an ideal alternative source of cells for in vitro models of hepatic tissue.In these contexts,we fabricated a bio-artificial human liver-sinusoid-on-a-chip by microfabrication techniques to faithfully replicate liver specific structure,microenvironment and functions.The device consists of a two parallel PDMS cell culture chambers separated by a 12 μm thick PET microporous membrane,and a pair of PMMA clamps tightened together to sandwich them.In the upper layer of the device,endothelial cells and Kupffer cells were seeded to mimic the in vivo sinusoidal capillary channels.The lower layer accommodates hiPSC-HLCs,which as an unlimited supply of human hepatocytes provide a solution to the challenges of the limited availability of human hepatocytes.In addition,generation of hepatocytes from hiPSCs can reduce the batch-to-batch variability of human hepatocytes.Importantly,the method to produce HLCs uses defined culture system,which avoids the presence of unknown factors.In summary,we developed a human liver-sinusoid-on-a-chip 3D culture platform with matured hiPSC-derived hepatocyte-like cells.By optimizing the basal medium,combination of small molecules,and initial cell-seeding density for multiple types of cell co-culture,we recapitulated the 3D structure and functions of the health liver sinusoid in vitro.This in vitro human liver-sinusoid-on-a-chip system may provide an experimental platform for physiologically relevant hepatic models,and might be potential for a wide range of applications in liver disease research and drug assessment in a high throughput fashion.