The liver, a crucial organ responsible for various metabolic functions, including protein and hormone synthesis and detoxification, plays a vital role in maintaining overall health. However, traditional in vitro models have limitations in replicating human organ functions accurately. Enter organ-on-a-chip technology—a promising and innovative alternative to static two-dimensional (2-D) cultures—revolutionising the way we study human organs in vitro.

By utilising microfluidic devices, organ-on-a-chip technology introduces dynamic elements like perfusion and shear stress, allowing the co-culture of multiple cell types that make up human organs and facilitating three-dimensional (3-D) cell-cell communication. These advancements offer a more reliable and physiologically relevant in vitro model of the functional units of human organs.

In the context of liver disease research, organ-on-a-chip technology provides an unprecedented opportunity to cultivate human liver cells under relevant physiological stresses. This capability allows researchers to delve deep into the pathophysiology of liver diseases, paving the way for improved drug development and disease modelling.

Pathophysiology of liver diseases

Drug-induced liver injuries

The liver is responsible for drug metabolism and therefore is susceptible to drug-induced liver injuries (DILI). In recent years, organ-on-a-chip technology has emerged as an effective and transformative platform for recreating DILI scenarios in vitro. By incorporating 3D culture and perfusion, organ-on-a-chip systems successfully mimic the liver tissue’s microarchitecture. The continuous fluid flow in these setups provides essential mechanical stimuli, ensuring consistent oxygen delivery and cell polarization, thereby maintaining high functionality for long-term studies. With the added advantage of perfusion, organ-on-a-chip offers greater flexibility to investigate immune-related toxicity issues by introducing circulating immune cells into the system.

In a study by Deng et al., a liver sinusoid-on-a-chip comprising four different types of transformed cell lines – HepG2 cells (hepatocytes), LX-2 cells (hepatic stellate cells), EAhy926 cells (liver sinusoidal endothelial cells), and U937 cells (Kupffer cells) was developed. They showed that the device maintained synthetic and secretory functions, and preserved enzymatic activities, as well as sensitivity of drug metabolism. This model was designed to replicate liver blood flow and biliary efflux, enabling researchers to explore drug hepatotoxicity and drug-drug interactions.

Moreover, organ-on-a-chip technology has the unique capability to emulate organ complexity and facilitate multi-tissue crosstalk. A study by Schimek et al. co-cultured bronchial MucilAir cultures with human liver spheroids derived from HepaRG cells and primary human hepatic stellate cells. This setup allowed them to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. Cultivated in a closed circulatory perfusion system for 14 days, the cells demonstrated viability and tissue homeostasis. Interestingly, the study revealed that a single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B₁ impaired the functionality of bronchial MucilAir tissues in monoculture. However, when the tissues were co-cultured with liver spheroids, a protective effect was observed, indicating successful crosstalk in this new human lung-liver co-culture. Such setups hold great promise for determining the effects of exposure to inhaled substances on a systemic level.

NAFLD/NASH

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the most rapidly growing liver conditions worldwide. Its primary causes are linked to obesity, type II diabetes, dyslipidemia, and insulin resistance. NAFLD progresses through distinct stages, starting with a buildup of fat in liver cells, leading to a simple fatty liver (steatosis). Subsequently, the liver becomes inflamed, giving rise to a more severe form known as non-alcoholic steatohepatitis (NASH). Over time, persistent inflammation triggers fibrosis, causing scarring of liver tissues and nearby blood vessels. In the most severe cases, after years of inflammation, NAFLD can culminate in cirrhosis—a state of permanent liver damage and eventual liver failure.

The pathophysiology of NASH is complex, involving various cell types and environmental and stromal effects. Addressing this complexity is essential to understand the disease better and develop effective treatments.

Organ-on-a-chip technology is an exceptional platform for recreating NAFLD/NASH scenarios. Freag et al. developed a liver-on-a-chip model comprising human primary hepatocytes (HCs), Kupffer cells (KCs), hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs). This sophisticated system was exposed to metabolic and inflammatory stressors, mimicking the conditions seen in NASH patients. By subjecting the liver-on-a-chip to a lipotoxic environment, the researchers observed the gradual development of NASH phenotypic characteristics. Interestingly, they also demonstrated that these disease manifestations could be reversed by elafibranor, a drug under study for the therapy of NASH, showcasing the potential of this powerful platform to study disease pathogenesis and develop novel anti-NASH drugs.

Alcoholic Liver diseases

Excessive alcohol consumption can take a toll on the liver, leading to alcohol liver disease (ALD), a spectrum of conditions ranging from fatty liver to hepatitis and cirrhosis. Understanding the intricate processes involved in ALD is crucial for developing effective treatments.

Using HepG2, LX-2, EAhy926, and U937 cells arranged in a physiological distribution under perfusion, Deng et al., demonstrated that not only the HepG2 cells have improved activities but also maintained high liver functions, including albumin synthesis and urea secretion. This model successfully recreated the damage process of hepatic non-parenchymal cell lines induced by alcohol. Furthermore, it shed light on the intercellular communication between different types of hepatic cells during ALD, meticulously measuring multiple biomarkers, such as Ve-cadherin, eNOS, VEGF, and α-SMA.

In a separate study, Nawroth et al. unveiled a liver-on-a-chip in a tri-culture configuration. This configuration combined primary hepatocytes cultured in the upper channel with primary LSECs and KCs, effectively recapitulating early events in alcohol-induced steatosis. Upon short-term exposure to ethanol, ALD-like hepatocytes were observed, showcasing increased lipid accumulation and ROS production. The researchers also demonstrated the potential for ALD recovery through alcohol abstinence, as well as the worsening of the phenotype with co-exposure to alcohol and bacterial endotoxin.

Infectious liver diseases

The human liver, a vital organ involved in crucial metabolic processes, is susceptible to various viral infections, including Hepatitis A, B, C, D, and E. These infections can trigger liver inflammation and damage, posing significant public health concerns. While Hepatitis A and E are commonly transmitted through contaminated water or food, Hepatitis B, C, and D primarily spread through blood and sexual contact.

Viral hepatitis can be classified into two categories: acute hepatitis and chronic hepatitis. Acute hepatitis is often self-limiting, with the body successfully fighting off the infection. However, chronic hepatitis poses more severe risks, as long-term infections can lead to complications like cirrhosis, liver failure, and liver cancer. Notably, Hepatitis B virus (HBV) infection is the most prevalent form of chronic viral hepatitis in Singapore.

Numerous studies have already harnessed the power of organ-on-a-chip technology to explore viral hepatitis. For instance, Ortega-Prieto et al. designed a liver-on-a-chip containing primary human hepatocytes and primary Kupffer cells within a microfluidic platform. This model successfully recapitulated all stages of the HBV life cycle, offering a valuable tool to study immune responses against viral infections.

Additionally, Kang et al. developed a human-liver-sinusoid-on-a-chip model, where primary human hepatocytes were co-cultured with immortalized bovine aortic endothelial cells in a dual microfluidic setup. This model allowed the study of HBV replication, showcasing the cells’ sustained morphology and viability for up to 26 days—a significant achievement for long-term studies of persistent viral diseases.

Summary

With organ-on-a-chip technology at our disposal, the possibilities for advancing liver disease research and enhancing our understanding of complex diseases are truly remarkable. By harnessing the potential of microfluidic chips, we can take significant strides toward improving human health and revolutionising medical research.

References

• Deng, J., Chen, Z., Zhang, X. et al. A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells. Biomed Microdevices 21, 57 (2019). https://doi.org/10.1007/s10544-019-0414-9
• Deng J, Zhang X, Chen Z, et al. A cell lines derived microfluidic liver model for investigation of hepatotoxicity induced by drug-drug interaction. Biomicrofluidics. 2019;13(2):024101. Published 2019 Mar 7.  
• Freag MS, Namgung B, Reyna Fernandez ME, Gherardi E, Sengupta S, Jang HL. Human Nonalcoholic Steatohepatitis on a Chip. Hepatol Commun. 2020 Nov 29;5(2):217-233. doi: 10.1002/hep4.1647
• Guo, X.; Yin, X.; Liu, Z.; Wang, J. Non-Alcoholic Fatty Liver Disease (NAFLD) Pathogenesis and Natural Products for Prevention and Treatment. Int. J. Mol. Sci. 2022, 23, 15489. https://doi.org/10.3390/ijms232415489
• Kang, Y.B.; Rawat, S.; Duchemin, N.; Bouchard, M.; Noh, M. Human Liver Sinusoid on a Chip for Hepatitis B Virus Replication Study. Micromachines 2017, 8, 27. https://doi.org/10.3390/mi8010027
• Nawroth JC, Petropolis DB, Manatakis DV, et al. Modeling alcohol-associated liver disease in a human Liver-Chip. Cell Rep. 2021;36(3):109393. doi:10.1016/j.celrep.2021.109393
• Ortega-Prieto, A.M., Skelton, J.K., Wai, S.N. et al. 3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection. Nat Commun 9, 682 (2018). https://doi.org/10.1038/s41467-018-02969-8
• Schimek K, Frentzel S, Luettich K, Bovard D, Rütschle I, Boden L, Rambo F, Erfurth H, Dehne EM, Winter A, Marx U, Hoeng J. Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies. Sci Rep. 2020 May 12;10(1):7865. doi: 10.1038/s41598-020-64219-6
• Way, G.W.; Jackson, K.G.; Muscu, S.R.; Zhou, H. Key Signaling in Alcohol-Associated Liver Disease: The Role of Bile Acids. Cells 2022, 11, 1374. https://doi.org/10.3390/cells11081374