Engineering a Perfusable Biomimetic Vascularized Liver on a Chip

Introduction:: The liver is one of the largest internal organs in the body. It conducts hundreds of essential biological functions such as protein synthesis, carbohydrate and lipid metabolism, xenobiotic metabolism, and toxin removal. Remarkably, the liver is uniquely able to regenerate when it is damaged under constant insults. Pharmaceutical drugs pass through the liver where they are metabolized to become active or inactive. Consequently, drug-induced liver damage is one of the major reasons for the withdrawal of approved drugs. Although small animal models have proven to be valuable for drug toxicity screening, the physiology and biology of these models do not phenocopy human physiology and biology. As such, there is a tremendous interest in creating in vitro human liver models that recapitulate many aspects of human liver biology and physiology. From widely used 2D platforms to more complex in vitro microfluidic-based models, these systems have assisted the field to gain valuable understanding in liver biology and drugs screening. However, these microfluidic-based platforms fall short of incorporating true vascular bed (a network of capillary vessels), thereby missing a crucial architectural component of the liver. In fact, liver vascular endothelial cells (ECs) are known to regulate functional and structural organization of the liver. Here, in this study, to overcome the shortcomings of current in vitro liver models, we present a biomimetic perfusable vascularized human liver on a chip where we incorporate capillary vessels in the chip to better model liver functions and probe the cellular communication between hepatocytes and endothelial cells.

Materials and Methods:: To engineer a perfusable vascularized human liver on a chip, we utilized engineered endothelial cells which we re-expressed a vasculogenic transcription factor ETV2. Upon re-expression of ETV2, the engineered ECs were intermingled with primary human hepatocytes in a microfluidic device. The device is fabricated with two large biomimetic vessels to represent an arteriole and a venule. The engineered ECs formed an inter-connected network of capillary vessels that span the interstitial space between the arteriole and venule vessels. These capillary vessels also connected the arteriole and venule vessels. After the device was connected to a peristaltic pump to simulate blood circulation from arteriole to capillary to venule, we used this system to investigate long term function of hepatocytes in both homeostasis and under toxin exposure. We utilized single cell sequencing to investigate how human hepatocytes re-educate endothelial cells in the vascularized human liver-on-a-chip.


Results, Conclusions, and Discussions:: Results and Discussion: After the engineered ECs and primary human hepatocytes were embedded inside the interstitial hydrogel compartment in the devices, the ECs quickly vascularized the human primary hepatocytes. We found that hepatocyte functions were greatly enhanced when they were in co-culture with vascular vessels as compared to hepatocytes in monoculture. To investigate the impact of shear forces on hepatic functions, we varied the flow rates and found an optimal flow condition that ensured long-term functions of primary human hepatocytes as assessed by protein synthesis (albumin, alpha-1-anti-trypsin), urea cycle, and CYP3A4 metabolism. Interestingly, not all hepatic functions were dependent on shear stress. To model hepatic toxicity, we dosed the vascularized liver on a chip with an over-the-counter drug called Acetaminophen (a hepatotoxin also known as Tylenol) and observed diminished hepatic functions. Concurrently, we observed endothelial toxicity to Acetaminophen. Finally, we extended the co-culture for several weeks and performed single cell sequencing. We observed endothelial cell-re-education in several enriched endothelial cell clusters where liver-sinusoidal endothelial cell genes began to re-express or get enriched in coculture with hepatocytes.

Conclusions: Utilizing engineered vascular endothelial cells with a robust capacity to form capillary vessels and a microfluidic platform, we engineered a perfusable vascularized human liver on a chip where the vascular network arborized and surrounded primary human hepatocytes. We showed that this system enabled us to extend hepatocyte functions for several weeks. Because the system is fully vascularized, this system is a valuable platform to enable studies to reveal hepatic function dependency on intravascular shear forces. Additionally, toxin delivered through the vascular network enabled us to assess endothelial cell and hepatocyte toxicity in coculture. More importantly, we demonstrated that there were intercellular communications between hepatocytes and endothelial cells during coculture. Altogether, this innovative and versatile vascularized human liver on a chip will allow us to unravel the molecular cross-talks between different cellular compartments in the human liver and provide molecular insights in liver biology to further improve strategies to engineer more complex vascularized liver tissues for transplantation.


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References (Optional): : [1] Palikuqi, B., Nguyen, D.H.T., et al. (2020) “Adaptable and hemodynamic human vasculogenic endothelial cells for organogenesis and tumorigenesis”. Nature. 585: 426-432.