This article was written by Emilie Grandfils, and reviewed by Julie Cavallasca and Guilhem Velvé Casquillas.
Emilie Grandfils, master student at EPFL in life sciences, orientation neurosciences and neuroengineering.
Julie Cavallasca, scientific translator, studied languages and translation at Aix-Marseille University and Paris 7 Diderot University. She now works for for Elvesys and the NBIC Valley.
Dr. Guilhem Velvé Casquillas
Dr. Guilhem Velve Casquillas is the CEO of Elvesys and co-founded many innovative companies. He is a former cellular biology researcher at the Curie Institute and in microfluidics.
Liver-on-chip: keeping up with the technology
Introduction to the liver-on-chip
The liver is involved in more than 300 vital functions , but is mainly known for being part of the digestive tract, where it has the extremely important role of metabolizing both xenobiotics and nutrients (carbohydrates and lipids). In toxicological studies (either fundamental or during clinical trial for drug development), the chemical of interest is always tested on the liver. Unfortunately, the predictions of the in-use models (in vitro or animal models) do not often correspond exactly to what is consequently observed in humans. In fact, the liver toxicity (or so-called Drug-Induced Liver Injury, DILI) is one of the major causes that can halt the clinical phase in drug development, worsening the long delays of these drug tests .
The main challenge for the microfluidic device lies in the complexity of the liver itself. This organ is hard to model because of the complexity of its numerous functions combined with a wide range of different kind of cells and a specific architecture. Among the different cell types, we find hepatocytes, but also Kupffer cells and fibroblasts, which makes the liver a versatile organ. Moreover, the different functions of this organ are organized following the phenomenon of hepatic zonation: liver cells have specialized functions based on their position along the portal vein to central vein. This results in different drugs being metabolized and cleared by cells in different zones. This phenomenon, adding a big difficulty to the in vitro study of the liver, is highly associated with DILI. For all these reasons, a physiologically relevant liver model has emerged as an unmet need.
Hepatitis B’s issue
Hepatitis B is a viral infection that attacks the liver, and can cause both acute and chronic disease. An estimated 257 million individuals are infected worldwide and over 750 000 people die of hepatitis B each year , mostly from complications, like cirrhosis and hepatocellular carcinoma. An efficient vaccine is available, but there is no specific treatment for acute hepatitis B. As this disease only affects humans and chimpanzees, the development of a treatment suffers from an additional limitation. A liver-on-chip would accelerate this research.
A lot of in vitro model systems have already been developed for the liver. Their main purpose was to investigate the potential adverse effects of chemicals and drugs. Liver tissue slices, perfused liver, and mostly immortalized cell lines and isolated liver cells  have been used to this end. Despite a continuous increase in the application of these conventional in vitro models, they have not been sufficient to fully replace animal models to predict toxicity in humans. In fact, they present a lot of problematic limitations: mostly a loss of viability, a limited throughput and a decrease of liver-specific functionalities. To prevent those, co-cultures of various cell types with hepatocytes (to prevent the de-specialization of the cells) and three-dimensional tissue constructs, as well as bioartificial livers, are investigated. Despite these developments, most technologies fail to mimic the multifaceted physiology of the liver in long-term culture models, especially regarding the acetaminophen (APAP)-induced hepatotoxicity and the relevant zonal effects in the liver .
The liver-on-chip solution
Organ-on-a-chip technologies have been proposed as a new generation of in vitro models for drug candidate screening. By implementing co-cultures with the different kinds of cells, hepatic zonation and a clear tissue hierarchy, it seems the most promising technology nowadays. A lot of liver-on-chip uses biophysically, preconfigured or 3D bioprinted scaffolds to enable a 3D architectural reconstruction. However, these kinds of scaffolds also bring their limitations, and it would ideally be better to have this architectural reconstruction without the need for a scaffold.
This architecture also helps the cells to live longer. Consequently, it allows for longer studies, which can be significant, especially to identify side effects that take longer to appear.
Technical characteristics of the liver-on-chip
Here we decided to present the liver-on-chip developed by Weng YS and al.; , in which they succeeded in designing a device without scaffold. In fact, scaffold-based technologies have serious limitations, like the inherent stability of a scaffold and its unpredictable effects on signaling pathways. They had to overcome the different issues of the scaffold-free culture approaches, like the lack of zonation effect in long-term liver organoid culture, or the questioned physiological relevance of the extracellular matrix (ECM). The idea behind their device was to bypass the need for a scaffold, by biomimeting the reconstruction of tissue hierarchy with the introduction of primary hepatic stellate cells (HSCs), in order to incorporate physiologically relevant ECM. The primary liver cells were isolated from male rats. To control the assembly of primary cells into an organotypic hierarchy, they used micro-engineering.
The device consisted of a micro-patterned hydrophobic polydimethylsiloxane (PDMS) membrane with a depth of 150 µm that was fabricated for multilayered cell depositions.
The cells deposited were primary liver cells, forming a biological growing template on the collagen-coated PDMS membrane. The cell-loaded PDMS membrane was enclosed to form a culture chamber with a hydrophilic flow diverter, to ensure a vertical cell anchorage. The circulation between the liver and the body was mimicked by a peristaltic pump in the medium between the reservoir and the liver-on-chip. The culture chamber was hexagonal, to simulate the portal vein function, and the inlets were located at each corner of the chamber. A flow was introduced radially from six discrete inlets into the culture chamber. To mimic the flow from the portal vein to the central vein, an outlet was positioned in the center of the culture, which received the flow from the inlets. The flow may pass through the structure corresponding to the hepatic cord radially, which can simulate biomimetic radial flow in the liver lobule. With the help of a scanning electron microscope (SEM), they further investigated the progressive morphogenesis of the LOC.
Study of multiple polarities of the liver-on-a-chip
To further understand the development of multiple polarities in the liver-on-a-chip, they used MRP2 to investigate functional polarization, and F-actin for structural polarization. In fact, MRP2 is a liver transporter that is located on polarized apical canaliculi between hepatic cords, and is responsible for drug excretion. MRP2 dysfunctions are associated with drug resistance and DILI  . For its part, F-actin is a cortex cytoskeleton, that seemed ideal for studying the dynamics of cell remodeling and assembly during organogenesis   .
Results obtained with the liver-on-a-chip
Global viability and organotypic architecture
After a week, the liver-on-chip culture showed good cell viability. The analyses of the different images of the culture clearly revealed the hepatic cords-like architectures in the liver-on-chip on Day 7. The features observed resembled the lobule of a living liver. With 3 days of perfusion, the primary liver cells were formed into round-shaper clusters on growing template, and the flow had removed some unhealthy cells, creating multiple cell clusters amid empty space in the device. In one week, these clusters were gradually connected, assembled, and organized into an organotypic architecture, which could be identified to the typical sinusoid wall-like morphology and the fenestrated window-like nanostructure.
Concerning the ECM, it was regenerated, remodeled and developed a fiber-oriented texture, which was an excellent result for a first scaffold-free culture. On day 7, this structure transformed from a scaffold-free state to one that connected, aligned and stabilized the PLCs in a multilayered fashion. As a control used to further comparison, they also deposited PLCs on hydrophobic PDMS membrane without a flow (“static PDMS group”). In this control group, the PLCs formed a spheroid structure, which did not stay attached to the PDMS and failed to form a stable multilayered architecture after a culture longer than a week. This allowed them to conclude that the stable architecture of cells and ECM assembly in the liver-on-chip probably resulted from the vertical anchorage and the horizontal flow stimuli that they implemented. Moreover, for the MRP2 test, after one week the liver-on-chip culture, the distribution of MRP2 was optically reconstructed and resembling a liver tubule. MRP2 was expressed in each cell and was also polarized, connected and reassembled into a nanonetwork across an apical domain of connected hepatocyte cords. Concerning the F-actin, on day 3, it was expressed and polarized toward a junction of cell contacts and cell cortex, meaning that there was cell-cell contact and membrane integration. F-actin polarized to the peripheral cortex of cells and developed a 3D intracellular skeletal network, resembling to a liver lobule. In the control culture, the F-actin expression faded after the first week.
Liver functions study
After one and two weeks, they decided to evaluate the albumin and urea synthesis of the liver-on-chip, as a feedback concerning the state of the liver’s specific functions. These two proteins are synthesized in the liver, indicating its level of activation. In the liver-on-chip group, after one week, both albumin and urea synthesis were restored. After two weeks, these concentrations underwent a slight decrease, but still remained significantly higher than the concentration observed in the control culture (conventional Petri dish culture).
Drug metabolism study
To evaluate the capacity of drug metabolism and clearance of the device, they decided to quantify the activity of cytochrome P450 3A4 (CYP 3A4). CYP 3A4 is one of the most important enzymes involved in the metabolism of xenobiotics. They found that its activity was successfully maintained for weeks in the liver-on-chip, and that this activity was a lot higher than the one observed in the control cultures (both Petri dish culture and static PDMS culture). They also evaluate the metabolic dynamics of the device by applying rifampin and ketoconzazole as an inducer and inhibitor of CYP, respectively . After 12h of dosing, on day 14, the metabolic activity of the liver-on-chip was increased in the rifampin group and reduced in the ketoconzazole group.
Further application of the liver-on-chip device
Conclusion of the research
Despite a lack of scaffold, they succeeded in developing a liver-on-chip which seemed to recapitulate the main features of the liver architecture, required for the phenomenon of hepatic zonation. This allowed to overcome the limitations mentioned earlier that come with a scaffold. The results concerning the different important functions of the liver were always a lot better than the ones obtained with traditional Petri dish culture and static PDMS culture. On the other hand, most of the results were collected after just one week, which is already good, but not sufficient for further studies about drug toxicology. In fact, some adverse drug effects can appear after a longer period. Moreover, although the device helped to achieve better results, it seems that after two weeks, the liver specific activities were decreasing, maybe meaning that there is a loss of specialization. Thereby, some improvements can be made, also regarding the coculture, in which we could add other cells involved in the liver, like Kupffer cells. The primary hepatic stellate cells were isolated from rats, which also represent a limitation to this paper, since we would further want to use this device for diseases sometimes affecting only humans and chimpanzees (like hepatitis B). From a global view, to replace these cells by human hepatocytes would allow for more correct studies. Another way of improvement would be to recreate the bile ducts,which have not been investigated yet, despite their central position in the liver.
Once these improvements have been made, a lot of diseases, like the hepatitis B, will benefit from this liver-on-chip. Other diseases have already been tested on this kind of microdevice. This is the case for the Nonalcoholic fatty liver disease (NAFLD), which is the most common liver disorder in developed countries . The biggest complication coming from NAFLD is hepatocellular carcinoma, which is ranked as the third highest cause of cancer-related death. An early diagnosis of NAFLD would help the identification of this potential risk factor. Manuele Gori and al., successfully used a liver-on-chip approach to study this disease .
Another promising ongoing research was stated by Khazali AS and al.,  by using liver-on-chip platforms to target liver metastases in order to improve the patient’s outcome.
Finally, the idea of a connected liver-on-a-chip, together with a gut-on-a-chip, would provide a promising technology, giving a more global vision about the way a xenobiotic is washed out the body (first-pass effect).
 Natalie Angier, “Physiologie. Le foie, cet organe à tout faire”, Courrier international (The New York Times), 13 juillet 2017
 Navarro VJ, Senior JR. Drug-related hepatotoxicity. N Engl J Med. 2006;354:731–739.
 Hepatitis B Fact sheet N°204. who.int. July 2014. Retrieved 4 November 2014
 Toxicol Res (Camb). 2013 Jan 1;2(1):23-39. Epub 2012 Nov 23. In vitro models for liver toxicity testing. Soldatow VY, Lecluyse EL, Griffith LG, Rusyn I.
 “the cure – liver on a chip”, https://www.youtube.com/watch?v=2EJlRXvpnf8
 Weng YS, Chang SF, Shih MC, Tseng SH, Lai CH, 2017 Jul 21. doi: 10.1002/adma.201701545. Scaffold-Free Liver-On-A-Chip with Multiscale Organotypic Cultures.
 M. Trauner, J. L. Boyer, Physiol. Rev. 2003, 83, 633
 A. Esteller. World J. Gastroenterol. 2008, 14, 5641.
 E. M. Huisman, T. V. Dillen, P. R. Onck, E. V. Giessen, Phys. Rev. Lett. 2007, 99, 208,103.
 L. Lanzetti, Curr. Opin. Cell Biol. 2007, 19, 453.
 J. D. Humphrey, E. R. Dufresne, M. A. Schwartz, Nat. Rev. Mol. Cell Biol. 2015, 15, 802.
 E. L. LeCluyse, R. P. Witek, M. E. Andersen, M. J. Powers, Crit. Rev. Toxicol. 2012, 42, 501.
 Rinella ME, June 2015, Nonalcoholic fatty liver disease: a systematic review.
 Manuele Gori, Maria Chiara Simonelli, Sara Maria Giannitelli, Luca Businaro, Marcella Trombetta, and Alberto Rainer, 2016, Investigating Nonalcoholic Fatty Liver Disease in a Liver-on-a-Chip Microfluidic Device
 A. S. Khazali, A. M. Clark, A. Wells, June 2017, A Pathway to Personalizing Therapy for Metastases Using Liver-on-a-Chip Platforms.
Written by Emilie Grandfils, corrected by Julie Cavallasca, under the supervision of Dr. Guilhem Velve Casquillas