Q&A Report: The 3D DILI Model of Success – from Biological Relevance to Industry Application

Experts from InSphero answer questions about the application of their 3D human liver microtissues for drug induced liver injury (DILI) experiments.

Part 1 - DILI Screening

How long does a hepatocyte lot last?

Our hepatocyte lots consist of a mixed pool from 10 donors. Each lot last 1-2 years. Before we change the lots we offer our clients bridging studies. In addition, we always offer our clients the reports from our validation studies of the new lots. This reports informs on the CYP450 activity, secretion of albumin, spheroid size and compares the ATP level of a number of reference compounds with the dose response curve and cellular IC50 values obtained from the previous lot. This report aims at reassuring our clients that the new lot is similar to the previous and that the lot change will have minimal impact on the result.

Can the liver models be used for safety assessments of other modalities, for example biologics or oligonucleotides?

We are currently testing both biologics and oligonucleotide-based therapeutic compounds, however, our knowledge working with these types of compounds is smaller compared to our experience with small molecules. Until now, we don’t have any comparative study based on ASOs or biologics, such as the study we showed in the webinar (Proctor 2017), based on small molecules.

In the case of ASOs, no larger comparative study has been performed yet. Nevertheless, we have an ongoing collaborative study together with a few pharmaceutical partners where we aim to evaluate the predictability of our liver models for liver safety assessment following treatment with ASOs. The preliminary results from this study suggest that our model performs very well also with oligonucleotide compounds. In addition, based on previous and ongoing proof of concept studies we know that ASOs are taken up throughout the model, including reaching the inner core of the model, and we could also determine the knockdown efficiency of our tool compound, which was evaluated to be close to 95%.

For testing of biologics we offer a fully immunocompetent model by the addition of the adaptive immune cells through the addition of primary PBMCs. We can measure the cytokine release and intercellular communication between the adaptive and innate immune cells and the hepatocytes. Therefore, we believe that our models is competent and able to inform on liver safety hazard or risk also for biologics.

How well characterized is phase 1 metabolism in your model?

In our validation of each new hepatocyte lot, we test the activity of several CYP450 enzymes in a mass spectrometric assay. In our standard setting we test: CYP1A2, CYP3A4, CYP2C9, CYP2B6, CYP2D6, CYP2C19, CYP2A6. Importantly, we don’t only test the CYP450 enzyme activity but also their inducibility by treating the liver models with inducers of specific CYP450 enzymes.

Does the liver model contain any additional cells in addition to primary hepatocytes and the Kupffer cells?

The primary NPC fraction of our liver model contains Kupffer cells and liver endothelial cells. However, since we are working with the crude NPC fraction our liver models also contain other non-parenchymal cells. For example, the liver models stain positive for OV-6 (hepatic progenitors, giving rise to oval cells), CD3 (T-cells), alpha-SMA (hepatic stellate cells) and CK-19/pan-CK (cholangiocytes). The fact that our models consists of primary hepatocytes co-cultured with the crude NPC fraction is important for us. Thereby we believe that we are obtaining a more physiologically relevant model than if would use purified cell fractions of Kupffer or endothelial cells.

What kind of models are you offering besides the liver DILI model?

InSphero offers several different 3D InSight™ tumor models based on cancer cell lines. We also have primary human 3D InSight™ pancreatic islet models and several 3D liver disease models mimicking different stages of NASH, such as de-novo lipogenesis, steatosis, and fibrosis. In addition, the liver model exists also as rat (Sprague Dawley) liver model, monkey (cynomolgus) liver model and dog (beagle) liver model.

Have you done any transporter functional studies besides CDFDA uptake using this model?

Currently we only offer bile salt export pump inhibition with the use of CLF as a standard mechanistic assay.

How is the batch-batch variability in sensitivity? Are some lots of heps and NPCs are more sensitive than others? Do you have a cut off/sensitivity normalization with DILI positive controls?

To make sure that each production run and each lot delivers the highest quality and consistency we always include a reference compound/positive control for each assay. By looking into our historical data over the last two years we see very little variability of the cellular ATP IC50 value of our reference compound and the data is independent of productions, hepatocyte lot, NPC lot and technical staff preparing the samples. Moreover, before changing from one lot to the next we offer our clients bridging studies and perform a detailed validation of additional parameters of the liver spheroids, such as size and albumin secretion.

Neither ATP loss nor LDH release are cell type selective. How do you know that these endpoints relate to hepatocellular injury?

If you are looking for a hepatocyte specific leakage marker we offer an ALT assay as an alternative to LDH. Secondly, by comparing data of monoculture hepatocyte liver spheroids with the co-cultured liver spheroid model (hepatocytes + NPCs) it is also possible to decipher whether the observed toxicity comes from the hepatocytes alone or if it is linked to cells of the NPC fraction. However, we believe it is important to assess the role of a compound on drug induced liver injury (DILI) in general and DILI is not an hepatocyte-specific phenomena. For these reason we perform our predictability screenings of compounds with the ATP and LDH assay.

Have you assessed predictive performance using RNA-seq in addition to mechanistic endpoints?

InSphero offers RNA-seq both as low-input bulk RNA and TempO-Seq, and both assays can be performed with as little as one liver spheroid per replicate. For investigative toxicology of compounds with limited in vivo data, we like to use RNA-seq as a hypothesis generator. Changes in toxicologically relevant signaling pathways following a compound treatment can be used to evaluate toxic response signatures and to generate a hypothesis which later can be confirmed with some of our mechanistic causality assays. In addition, InSphero has its own RNA seq analysis platform and a dedicated research associate who can support you with the analysis of the RNA seq data and will supply you with an interactive RNA-seq dashboard report for easy handling of the result.

Have you tried DILI experiments on hepatic organoids?

We have not performed any standard assays on hepatic organoids. Nevertheless, the Akura™ Spheroid Microplates can be used to produce organoids, so if this line of work is interesting for you there is the option to order our plates and perform the studies in your own facilities.

Part 2 - How to integrate a biologically relevant 3D model in an automated and reproducible workflow

Can I buy the presented plates as consumable products?

Our Akura™ Plate family (Akura™ 96 and 384 Spheroid Microplates and the Akura™ Plus Hanging Drop System) can be purchased as consumables in our webshop. There, you can also download the Product Manuals, Technical Specifications and Aggregation Protocols to get you started.

You mentioned that are producing the 3D InSight™ Microtissues in your lab to perform DILI studies as service projects. Is it possible to purchase your models for internal use?

We produce our 3D InSight™ human Liver Microtissues bi-weekly. If you would like to perform the compound testing in house, we can ship the microtissues in Akura™ 96 and 384 plates worldwide to your lab. It takes 2 to 3 days to receive our plates. Upon arrival, you need to remove the sealing membrane, perform medium exchange and then the microtissues are ready to use.

To ensure reliable shipment of the live microtissues to your lab, InSphero developed a shipment solution, which is called the ‘InFloat‘. It consists of a spherical container holding the plates and heating elements to stabilize the temperature during transport. The container floats on water inside a cubical container and can freely rotate. Therefore, we can guarantee that the plates are always transported in an upright position so our customer will have ready-to-use full plates.

Are you deducting the outer wells of your assay due to edge effects?

If you cannot guarantee stable, high relative humidity in your incubator, or there are several users in your lab sharing one incubator, you might experience evaporation in the outer wells. To ensure stable and high relative humidity in your incubator, we use the so-called Incubox™, where we store our plates in.

The Incubox™ is a specially engineered container which creates a high-humidity microclimate inside conventional cell culture incubators and protects your sensitive plates from large humidity drops and fluctuations – the major cause for evaporation effects. By minimizing evaporation-induced reagent gradients across the plate you can use all wells of your plate.

How long does it take to get the plates delivered when ordered? Do you ship worldwide?

It usually takes 2 to 3 days to ship the plates to our customer using our own-developed InFloat system. We regularly ship assay-ready microtissues to Europe, to the East and West Coast of the US, and also to Japan.

Is it possible to generate spheroids inside the Akura™ 96 Spheroid plate or do you need to generate spheroids before?

The Akura™ 96 and 384 Plates are ‘all-in-one’ solutions meaning that you can produce spheroids in the wells, perform long-term experiments due to the stable ultra-low attachment coating and special well design and use the plates for various read-outs, such as bio-chemical endpoints or high-content imaging applications.

Are the Akura™ plates compatible with automated-liquid handling systems? It looks like you need to tilt the tip to ensure no spheroid loss.

The plates have ANSI/SLAS standard and are compatible to automated-liquid handling systems, such as the Integra VIAFLO instrument.

If you perform medium exchange using multi-channel pipettes, we recommend inserting your pipette with a small angle until you reach the contact point of the so-called SureXchange™ ledge. Now you are able to perform a near-complete medium exchange and the spheroids are protected in the lower cavity.

When using semi- or automated-liquid handling devices, you can program your device to insert the pipette tip vertically with a small offset (x/y offsets are given in the Product Manuals) to ensure precise and reliable medium exchange.

Can Akura™ plates be used for organoids culture?

The Akura™ Spheroid Microplates can be used for the production of organoids. As the lower cavity with the flat bottom is only 1 mm in diameter, we recommend to not use spheroids or organoids which are larger than 800 µm in diameter.