Drug-induced liver injury (DILI)

Qualifying primary cells prior to running a PhysioMimix® Organ-on-a-chip assay is key to a successful experiment and should not be overlooked. Firstly, starting with primary hepatocyte cells, we can culture several different lots in our Multi-chip Liver plates to ensure good three-dimensional (3D) tissue formation, physiologically relevant tissue functionality, metabolic activity, and extended longevity. Once we have identified primary hepatocyte lots that perform well i.e., within our pass/fail criteria, we co-culture them with Kupffer cells, test their response to an inflammatory stimulus, and examine the general tissue health and function over an 8 to 14-day period.

For more detailed information about the cell validation process and its importance, please read this recent Blog: Mirror, mirror on the wall, which is the best cell of them all? 

Our common endpoints are cited above; however, this is by no means an exhaustive list of detectable endpoints for the assay. As we can extract and sample a large volume of media from each chip (up to 1 mL per chip from Multi-chip Liver-12 plates and up to 0.25 mL per chip in the Multi-chip Liver-48 plates), many additional endpoints can also be measured in parallel to deliver detailed mechanistic insights into a drug’s toxicity profile. For example, a Luminex assay panel may contain 20 or more analyte readouts. Clinical biomarkers, such as AST/ALT, can also be detected offering advantages over traditional in vitro cultures. Furthermore, the scaffold containing the liver microtissues can be removed for additional endpoint analysis, such as staining for microscopy, or extraction of protein/RNA/DNA for screening.

The PhysioMimix DILI assay’s ability to detect clinical measurements, such as ALT/AST, relates to the relatively large microtissues that are cultured in our Multi-chip Liver plates. Other systems are limited by the small amount of cells/supernatant that is available for sampling from their assay. Our cell-to-media volume ratio is key to accessing a wide range of metabolites and biomarkers. The PhysioMimix Liver-12 plate Liver-on-a-Chip model comprises up to 6 x 10⁵ primary human hepatocytes that are perfused by a large volume of media (up to 1.6 mL). This provides the sensitivity required for the reliable quantification of clinical markers that are notoriously challenging to measure in other in vitro models.

For more information about how the PhysioMimix DILI assay provides greater assay sensitivity and the benefits of this approach for de-risking DILI predictions, please read the following Blog: De-risking drug-induced liver injury through the predictive power of organ-on-a-chip. 

Spheroids are limited to some assays as they only contain 1,000 to 3,000 cells. They are typically cultured under static cell culture, which leads to necrotic cores – cell death at the center due to a lack of nutrients and oxygen, after just a few days. Even when perfused, these models tend to apply a gravity-driven perfusion mechanism, so, cells at the center continue to suffer poor access to nutrients and oxygen compared to the cells on the outer edge of the structure. This limits their longevity. Spheroids also suffer from loss of function over time (de-differentiation) and display comparatively low metabolic activity. Our PhysioMimix OOC systems culture larger-scale 3D liver microtissues, which mimic the architecture of the liver sinusoid. We maintain microtissue viability, phenotype, and metabolic activity for over 30 days by perfusing individual microtissues via fluidic flow. Although most of our customers prefer to capture their DILI data over 8-day experiment periods the PhysioMimix DILI assay allows for longer-term assessments of toxicity, the detection of clinically-translatable biomarkers, and more complex biological interactions to be studied if required (Long et al., 2016).  

For more information about how the PhysioMimix DILI assay provides greater assay sensitivity and the benefits of this approach for de-risking DILI predictions, please read the following Blog: De-risking drug-induced liver injury through the predictive power of organ-on-a-chip.

Yes, you can. It usually takes 4 days to induce fatty liver diseases such as Non-alcoholic fatty liver disease (NAFLD), (also known as metabolic dysfunction-associated steatotic liver disease – MASLD),  or non-alcohol steatohepatitis (NASH), (also known as Metabolic dysfunction-associated steatohepatitis – MASH) – in our tri-culture Liver-on-a-chip model. Inducing acute or chronic DILI takes a bit longer, usually around 14 days. Diseased 3D Liver microtissues can be cultured and treated with drugs or compounds of choice for 14 days or more.

To learn more about our fatty liver diseases and DILI model, download Application Note: Microphysiological System for Studying Fatty Liver Diseases and its Impact on Drug-Induced Liver Injury.

Co-culture models containing primary human hepatocytes (PHH) and human non-parenchymal cells (Kupffer cells) are used to assess innate immune-mediated toxicological responses as standard, increasing the assay sensitivity versus primary PHH cultures.  

Peripheral immune cells can also be added into the media that perfuse the liver microtissues to assess adaptive immune responses. However, we do not have a fully validated SOP for this application yet. Contact us for more information, or to register your interest. 

Yes, the MPS model provides a viable alternative to animal models, where human translatability is known to be poor. New modalities, such as antibodies, RNA therapies and cell-base therapies, are difficult to test in animals as they are specific to humans, and animal models often lack the correct targets or pathways to produce useful results. Our human-relevant DILI model is well suited for testing the interaction of these new therapeutics with the liver.

For more information, please read our Application Note: Human liver microphysiological system for predicting the drug-induced liver toxicity of differing drug modalities.

We have tested our MPS models against in vivo models to reiterate that some interspecies differences can lead to discrepancies between the predictions observed in out humanized assay compared to in vivo animal studies. Look out for our preclinical animal MPS comparative datasets in 2024.  

Using reference compounds (IQ MPS Consortium DILI validation set) our human PhysioMimix DILI Assay (using the Multi-chip Liver-12 plate) delivers 100% sensitivity, 85%, accuracy and 100% precision. As well as giving “yes/no” predictions for DILI positive and negative compounds, the model gives multi-parametric detail to the nature and extent of DILI severity. Clinical markers such as ALT can be directly compared to clinical ULN (upper limit of normal) metrics. Together, this demonstrates the power of the PhysioMimix DILI assay to predict and quantify DILI risks.

Up to six Multi-chip Liver-12 plates can be run simultaneously by one PhysioMimix OOC Controller unit, providing 72 samples per run for investigative studies that require deep insights into mechanism of action. 

Alternatively, we have recently developed a miniaturized Liver-48 plate for use in a higher throughput setting such as lead optimization. Currently, three Liver-48 plates can be simultaneously run using one PhysioMimix Single-organ HT controller unit, providing 144 samples/run capacity.  

In the liver, hepatocytes experience very low (near zero) shear stress as they are separated from the blood by the fenestrated (i.e. has holes) endothelium. However, as the endothelium is fenestrated, there is substantial mass transport, and this is influenced by the flow rate in the sinusoid. Our PhysioMimix OOC Systems provide sufficient flow to ensure good mass transport with low shear stresses of a similar order of magnitude to those observed in the liver sinusoid/capillary. To learn more about the flow in our system please read Domansky et al, 2010 and  Ebrahimkhani et al, 2014. 

We have stained the liver microtissue for markers that are known to be trafficked into the canaliculi space. We have also labelled it with antibodies for the expression of markers on the canaliculi. When looking at scanning electron microscopy (SEM) image of the actual liver-on-a-chip tissues, we observe lumens, with microvilli formation, developing for the bile. (Please refer to the image in the “Dynamic 3D microenvironment” section above). 

When developing a co-culture model, confirming the presence of the required cells is critical. Depending on the organ that you are trying to recapitulate and its composite cell types, specific biomarkers can be used to confirm the presence of the different cell types.

In our duo-, and tri-culture liver models, Kupffer cells are incorporated to represent the liver’s innate immune system. Their presence is confirmed by measuring inflammation levels, via inflammation biomarkers (such as interleukins or cytokines) and compared to the control – primary human hepatocytes monocultures (PHH).

In our triculture model of Non-alcoholic steatohepatitis (NASH), stellate cells are added, and their presence is confirmed by comparing fibrosis levels to PHH monocultures in a fat media (Kostrzewski et al 2021). Transcriptomic analysis to detect the RNA expression of cell-specific markers provides an alternative or additional approach to confirm cell types in the culture. 

Simpler in vitro systems successfully flag “yes/no” drug responses. These are perfectly complemented with the use of more advanced perfused Organ-on-a-chip (OOC) models to provide detailed data. To achieve data richness and high assay sensitivity, we require large cell culture volumes. In the Liver-12 plate, approximately half a million primary cells, perfused by large volumes of media (>1 mL), generate enough horsepower to reproduce phase 1 and 2 metabolism (particularly important because one of the reasons standard animal and in vitro models have proven to be poor predictors is due to their non-human metabolic profiles) and to increase assay sensitivity so more sophisticated clinical chemistry outputs of liver function, such as ALT/AST concentrations, can also be detected. 

Samples taken over time in longitudinal studies provide biochemical assessments that build up a picture over time. This (relatively) large amount of tissue recovered at the end of the experiment can also be examined in additional proteomics or genomics studies, to investigate gene up- and down-regulation to generate a more complete mechanistic profile of drug action.

Hepatocyte performance in PhysioMimix OOC Systems is significantly greater compared to other standard approaches such as collagen-sandwich cultures and spheroids. CYP activity can be detected throughout the culture period, as can the expression of transporters.

For an independently generated comparison please see our peer reviewed co-publication with the US FDA

For more information read the following Blog : De-risking drug-induced liver injury (DILI) through the predictive power of organ-on-a-chip.

Accounting for door-to-donor variation has always been a challenging in in vitro experiments. There are several things to consider when donor-donor variability is considered: 

• What is the adequate number of donors to include in a study, without drastically increasing the cost and timeline of the study? 
• What genetic, gender, or ethnic background should we look for in the different donors? 
• Can we access a donors that allow for diversity and also perform well under my experimental conditions? 

When validating donors, we always try to find the right balance between the requirement for the different donors and the quality of donors. It is important to source cells from more than one supplier as access to the number of different donors greatly depends on the cell suppliers’ availability.

As previously mentioned, the number of donors that you evaluate within a study affects the cost of the study. So, the rule is simple, the more donors you incorporate, the more expensive the study will be. One way to minimize cost is to use pooled donors, which is a good compromise for rapid assessments. However, in our experience, pre-mixed pooled donors purchased off-the-shelf generate poor quality microtissues in OOC culture.

If you want to run a study using pooled donors, we recommend identifying three to five donors that have passed the pre-validation tests individually (to test for liver microtissue formation and function) before pooling them together (Tsamandouras et al, 2017).

For more information, read our Blog: A guide to pre-validating primary cells for use in organ-on-a-chip assays.

For more info about 3D validating cells, or to find out how to circumvent this step you can source 3D validated cells from us directly. 

As practical as pooled donors may be, they do have limitations. For example, one donor may be more represented than the others in the microtissue, as we cannot control which donor cells attach best to form the microtissues in the OOC plates.

Using a higher throughput OOC system can help reduce the financial and resource burden for such experiments, as more conditions can be combined and studied on each Multi-chip Liver plate. For example, our PhysioMimix Single-organ HT System and its Liver-48 plate enables four times (n= 16 conditions in triplicate) the experimental capacity of our standard PhysioMimix Liver-12 plate. 

This poster from the US FDA: Characterization of the effect of primary human hepatocyte (PHH) lots on function in CN Bio Innovations Liver-Chip using acetaminophen, provides some useful insights into the effect of donor-to-donor variability in OOC assays.