Search
Close this search box.

Liver-on-chip: keeping up with the technology

Introduction to the liver-on-chip technology

Liver-on-chip liver location microfluidics
Figure 1: Placement of the liver in the body.

The liver is involved in more than 300 vital functions [1]. Still, it is mainly known for being part of the digestive tract, which has the critical role of metabolizing xenobiotics and nutrients (carbohydrates and lipids). 

 

In toxicological studies (either fundamental or during clinical trials for drug development), the chemical of interest is constantly tested on the liver. Thus, it is important to have a convenient liver model. However, developing and using the current available models faces many challenges. Microfluidics, specifically, liver-on-chip was proposed as a solution. 

Issues and solutions

Today’s issue

Unfortunately, the predictions of the in-use models (in vitro or animal models) do not often correspond precisely to what is observed in humans. 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 [2].

 

The microfluidic device’s main challenge lies in the liver’s complexity. This organ is complex to model because of its numerous functions combined with a wide range of different kinds of cells and a specific architecture.

 

Among the different cell types, we find hepatocytes, Kupffer cells, and fibroblasts, which make the liver a versatile organ. Moreover, the other 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 the central vein. 

 

Different drugs are metabolized and cleared by cells in different zones. Adding a significant difficulty to the in vitro study of the liver, this phenomenon is highly associated with DILI. For all these reasons, a physiologically relevant liver model has emerged as an unmet need.

liver-on-chip different cells
Figure 2: Scheme of the different cell types present in the liver.

Hepatitis B's issue

Hepatitis B is a viral infection that attacks the liver and can cause acute and chronic diseases. An estimated 257 million individuals are infected worldwide, and over 750,000 people die of hepatitis B each year [3], 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, developing a therapy has an additional limitation. A liver-on-chip would accelerate this research.

Previous solutions

A lot of in-vitro model systems have already been developed for the liver. Their primary purpose was to investigate the potential adverse effects of chemicals and drugs. Liver tissue slices, perfused liver, and mainly immortalized cell lines and isolated liver cells [4] have been used. Despite a continuous increase in the application of these conventional in vitro models, they have not been satisfactory in entirely replacing animal models to predict human toxicity. 

 

They present a lot of problematic limitations, mainly a loss of viability, a limited throughput, and a decrease in liver-specific functionalities. To prevent those, cocultures of various cell types with hepatocytes (to avoid de-specializing the cells) and three-dimensional tissue constructs and 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 [4].

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. Implementing cocultures with different kinds of cells, hepatic zonation, and a clear tissue hierarchy seems to be the most promising technology nowadays. 
 
 
Many liver-on-chip use biophysically, preconfigured, or 3D bioprinted scaffolds to enable a 3D architectural reconstruction. However, these kinds of scaffolds also have limitations, and it would ideally be better to have this architectural reconstruction without needing a scaffold.
 

This architecture also helps the cells to live longer. Consequently, it allows for more extended studies, which can be significant, especially in identifying side effects that take longer to appear [5].

Technical characteristics of the liver-on-chip

Here, we present the liver-on-chip developed by Weng YS et al. [6], who succeeded in designing a device without scaffolding. Scaffold-based technologies have severe 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 physiological relevance of the extracellular matrix (ECM). 
 
 
The idea behind their device was to bypass the need for a scaffold by biomimicking the reconstruction of tissue hierarchy with the introduction of primary hepatic stellate cells (HSCs) to incorporate physiologically relevant ECM. The primary liver cells were isolated from male rats. They used micro-engineering to control the assembly of primary cells into an organotypic hierarchy.
 
 

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 create a culture chamber with a hydrophilic flow diverter to ensure a vertical cell anchorage. 

A peristaltic pump in the medium between the reservoir and the liver-on-chip mimicked the circulation between the liver and the body. 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.

liver-on-chip diagram principles
Figure 3: Schematic diagram of design principles. Multilayered PLCs were deposited on PDMS membrane to create a biological growing template and hexagonal coutour. From Weng YS et al., 2017, Scaffold-free liver-on-chip with multiscale organotypic cultures.

Study of multiple polarities of the liver-on-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. MRP2 is a liver transporter located on polarized apical canaliculi between hepatic cords and is responsible for drug excretion. MRP2 dysfunctions are associated with drug resistance and DILI [7, 8]. For its part, F-actin is a cortex cytoskeleton that seemed ideal for studying the dynamics of cell remodeling and assembly during organogenesis [9, 10, 11].

Results obtained with the liver-on-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 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 three days of perfusion, the primary liver cells were formed into round-shaper clusters on the growing template, and the flow removed some unhealthy cells, creating multiple cell clusters amid space in the device. 

 

These clusters were gradually connected, assembled, and organized into an organotypic architecture in one week. This could be identified as the typical sinusoid wall-like morphology and the fenestrated window-like nanostructure.

liver-on-chip liver lobule
Figure 4: scheme of the organization and form of a liver lobule.
The ECM was regenerated and remodeled, and a fiber-oriented texture was developed, 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 for further comparison, they also deposited PLCs on a 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 create a stable multilayered architecture after a culture longer than a week. 
 
 
This allowed them to conclude that the regular architecture of cells and ECM assembly in the liver-on-chip probably resulted from the vertical anchorage and the horizontal flow stimuli they implemented.

Moreover, for the MRP2 test, after one week of the liver-on-chip culture, the distribution of MRP2 was optically reconstructed and resembled 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. 


On day 3, the F-actin was expressed and polarized toward a junction of cell contacts and cell cortex, meaning 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 a liver lobule. In the control culture, the F-actin expression faded after the first week.

Liver function study

After one and two weeks, they decided to evaluate the albumin and urea synthesis of the liver-on-chip as feedback concerning the state of the liver’s specific functions. 

 

These two proteins are synthesized in the liver, indicating their activation level. In the liver-on-chip group, both albumin and urea synthesis were restored after one week. After two weeks, these concentrations slightly decreased but remained significantly higher than those observed in the control culture (conventional Petri dish culture).

Drug metabolism study

To evaluate the drug metabolism capacity and the device’s clearance, 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 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 ketoconazole as inducers and inhibitors of CYP, respectively [12]. 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 ketoconazole group.

liver-on-chip metabolic activities
Figure 5: Liver-specific functions and metabolic activities in long-term cultured LOC. a), b) Albumin and urea production in long-term cultured LOC; c) Metabolic activities of LOC. From Weng YS et al., 2017, Scaffold-free liver-on-chip with multiscale organotypic cultures.

Conclusion of the research

Despite a lack of scaffold, they succeeded in developing a liver-on-chip device, which seemed to recapitulate the main features of the liver architecture required for the phenomenon of hepatic zonation. This allowed us to overcome the earlier limitations that come with a scaffold. 
 
The results concerning the liver’s different essential functions were always much better than those 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 suitable but not sufficient for further studies on drug toxicology. Some adverse drug effects can appear after a more extended period.
 
 

Moreover, although the device helped achieve better results, it seems that the liver-specific activities were decreasing after two weeks, maybe meaning there was a loss of specialization. Thereby, some improvements can be made 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. 

 

This also represents a limitation to this paper since we would further want to use this device for diseases that sometimes affect only humans and chimpanzees (like hepatitis B). From a global view, replacing these cells with 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.

Further research

Once these improvements are made, many diseases, like hepatitis B, will benefit from this liver-on-chip technology. Other diseases have already been tested on this kind of microdevice. This is the case for Non-alcoholic fatty liver disease (NAFLD), which is the most common liver disorder in developed countries [13]. The most significant 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 identify this potential risk factor. Manuele Gori et al. successfully used a liver-on-chip approach to study this disease [14].

Another promising ongoing research was stated by Khazali et al. [15], who used liver-on-chip platforms to target liver metastases to improve patient outcomes.

 

Finally, the idea of connected liver-on-chip and gut-on-a-chip would provide a promising technology, giving a more global vision of how a xenobiotic is washed out of the body (first-pass effect).

Authors:
Emilie GrandfilsJulie Cavallasca and 

Guilhem Velvé Casquillas

With the support of the NMBP-RIA PANBioRA project
Grant agreement number: 760921

Contact: 

Partnership[at]microfluidic.fr

Emilie Grandfils
eu_funded_en
 
   
 logo-actions-marie-curie_0-300x223
 
References
  1. Natalie Angier, “Physiologie. Le foie, cet organe à tout faire”, Courrier international (The New York Times), 13 juillet 2017
  2. Navarro VJ, Senior JR. Drug-related hepatotoxicity. N Engl J Med. 2006;354:731–739.
  3. Hepatitis B Fact sheet N°204. who.int. July 2014. Retrieved 4 November 2014
  4. 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.
  5. “the cure – liver on a chip”, https://www.youtube.com/watch?v=2EJlRXvpnf8
  6. 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.
  7. M. Trauner, J. L. Boyer, Physiol. Rev. 2003, 83, 633
  8. A. Esteller. World J. Gastroenterol. 2008, 14, 5641
  9. E. M. Huisman, T. V. Dillen, P. R. Onck, E. V. Giessen, Phys. Rev. Lett. 2007, 99, 208,103
  10. L. Lanzetti, Curr. Opin. Cell Biol. 2007, 19, 453.
  11. J. D. Humphrey, E. R. Dufresne, M. A. Schwartz, Nat. Rev. Mol. Cell Biol. 2015, 15, 802.
  12. E. L. LeCluyse, R. P. Witek, M. E. Andersen, M. J. Powers, Crit. Rev. Toxicol. 2012, 42, 501.
  13. Rinella ME, June 2015, Nonalcoholic fatty liver disease: a systematic review.
  14. 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
  15. A. S. Khazali, A. M. Clark, A. Wells, June 2017, A Pathway to Personalizing Therapy for Metastases Using Liver-on-a-Chip Platforms
  16. Written by Emilie Grandfils, corrected by Julie Cavallasca, under the supervision of Dr. Guilhem Velve Casquillas