The bioprinted liver

Bioprinted liver tissue containing both parenchymal and non-parenchymal cells in spatially controlled, user-defined geometries that reproduce compositional and architectural features of native tissue are making it possible to assess drug effects over timeframes much longer than those offered by 2D liver cell culture systems.

The liver is responsible for many things: filtering the blood, metabolising and transporting drugs, and producing a myriad of proteins that are critical to homeostasis (albumin, clotting factors, enzymes involved in protein metabolism). It is also central to the pathogenesis of several infectious diseases, including hepatitis, and it can also be seriously and irreversibly injured by chronic exposure to alcohol. Many genetic disorders are linked to reduction or absence of proteins that would normally be produced by the liver. No wonder that a lot of research centres on liver function and biochemistry.

Most liver functions are dependent, in part, on architecture. Hepatocytes inside the body have a nearly unlimited capacity for replication. When as much as two-thirds of a whole healthy liver is surgically removed, the hepatocytes within the liver remnant undergo rapid and extensive proliferation to restore liver mass completely. Inside the body, hepatocytes are polarised along a border of endothelial cells, with formation of canaliculi along their apical surface and tight junctions between neighbouring cells.

However, once removed from the body, hepatocytes replicate poorly and rapidly lose critical liver-specific functions. Loss of polarisation - as occurs when hepatocytes are cultured in simple monolayers on standard tissue culture-treated plastic - leads to loss of function and an inability of the hepatocyte to maintain the intracellular architecture that enables absorption, transport and bile production.

While liver cells, in particular the parenchymal hepatocytes, have been widely used in the laboratory to assess the potential toxicity or efficacy of drugs, the loss of function has limited their usefulness. What is needed is a method whereby hepatocytes can be maintained in culture environments that support polarisation and three-dimensionality and so retain critical functions for longer periods outside the body.

Bioprinted liver tissue model

Organovo’s NovoGen Bioprinting platform has been used to generate bioprinted liver tissue prototypes that contain both parenchymal and non-parenchymal cells in spatially controlled, user-defined geometries that reproduce compositional and architectural features of native tissue.

One advantage of the automated bioprinting platform is that it enables fabrication and comparative testing of multiple compositions and geometries so that winning combinations can be identified systematically based on histological and functional outcomes.

Cross-section of multicellular bioprinted human liver tissue, stained with hematoxylin & eosin (H&E).

Beginning with hepatocytes (the predominant parenchymal cells of the liver), designs were created based on shapes and cellular interfaces found in native liver tissue. Non-parenchymal cells, including endothelial cells and hepatic stellate cells, were positioned in defined locations relative to hepatocytes, creating a compartmentalised architecture that was established at the time of fabrication and substantially maintained over time in culture.

Cross-section of bioprinted human liver tissue demonstrating compartmentalisation between the hepatocytes (shown as blue nuclei), endothelial cells (red) and hepatic stellate cells (green).

In addition to the cell type-specific compartmentalisation, two histomorphological features can be appreciated in these bioprinted liver tissues: 1. The development of microvascular networks within the tissue; and 2. the formation of tight intercellular junctions among the hepatocytes.

The image above shows bioprinted human liver with CD31+ microvessels (green) forming within the tissue.

The image above shows formation of intercellular junctions between hepatocytes in bioprinted liver tissue, highlighted by E-Cadherin immunochemistry (green).

Importantly, these multicellular, 3D liver tissues possess critical attributes central to liver function, including production of liver-specific proteins such as albumin and transferrin, biosynthesis of cholesterol and inducible cytochrome P450 activities, including CYP1A2 and CYP3A4. Production of the liver-specific protein, albumin, was 5 to 9 times greater on a per-cell basis when compared to matched 2D controls. These functional data, combined with the unique histological features of the tissues, suggest they may be a compelling alternative to traditional 2D hepatocyte cultures for predictive studies, especially those involving longer-term tissue toxicity assessments or studies of disease development and progression where results need to be interpreted in the context of cell-cell interactions.

CYP1A2 and CYP3A4 were measured with Pro-Glo CYP450 assays (Promega), after induction with verapamil or dexamethasone, respectively. Measurements were taken at 135 hours after the 3D liver tissues were bioprinted and reported as fold induction over matched, non-induced controls.

The overall goal of studies like these is to develop living, multicellular human tissues that can be maintained in the laboratory environment for extended periods of time and sampled serially for both functional and histological changes in response to injury, pathogens or treatments.

3D bioprinted human liver tissue now available

Using 3D bioprinting technology, Organovo Holdings has released its exVive3D Human Liver Tissue for preclinical drug discovery testing. This model is intended to provide human-specific data to aid in the prediction of liver tissue toxicity or ADME outcomes in later-stage preclinical drug discovery programs.

Organovo’s exVive3D Liver Models are bioprinted, living 3D human liver tissues consisting of primary human hepatocytes, stellate and endothelial cell types, which are found in native human liver. The exVive3D Liver Models are created using Organovo’s proprietary 3D bioprinting technology that builds functional living tissues containing precise and reproducible architecture. The tissues are functional and stable for at least 42 days, which enables assessment of drug effects over study durations well beyond those offered by industry-standard 2D liver cell culture systems.

Organovo has previously shown that exVive3D Liver Models produce important liver proteins including albumin, fibrinogen and transferrin, synthesise cholesterol and possess inducible cytochrome P450 enzymatic activities, including CYP 1A2 and CYP 3A4. The exVive 3D Liver has successfully differentiated between structurally related compounds with known toxic and non-toxic profiles in human beings and the model has also been employed successfully in the detection of metabolites at extended time points in vitro. Importantly, the configuration of the bioprinted liver tissues enables both biochemical and histologic data to be collected so that a customer can investigate compound responses at multiple levels.

The durability and functionality of the 3D liver product enable the assessment of the effects of low dose or repeated dosing regimens across a spectrum of biochemical, molecular and histologic end points. Initially, users will be able to access the technology through Organovo’s contract research services program. All testing will be performed at Organovo’s facility by the company’s laboratory services tissue experts.

Organovo Holdings