3D Human Liver Tissue Model

ORIGINAL: Organovo

OVERVIEW

Liver cells, in particular the parenchymal hepatocytes, are widely used in the laboratory to assess the potential toxicity or efficacy of drugs. 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.1, 2, 3 

However, once removed from the body, hepatocytes replicate poorly and rapidly lose critical liver-specific functions. The liver is responsible for

• filtering the blood, 
• metabolizing and transporting drugs, and 
• producing a myriad of proteins that are critical to homeostasis (albumin, clotting factors, enzymes involved in protein metabolism). 

Many genetic disorders are linked to reduction or absence of proteins that would normally be produced by the liver. Furthermore, the liver is central to the pathogenesis of several infectious diseases, including hepatitis, and it can also be seriously and irreversibly injured by chronic exposure to alcohol.

Most liver functions are dependent, in part, on architecture. Hepatocytes inside the body are polarized along a border of endothelial cells, with formation of canaliculi along their apical surface and tight junctions between neighboring cells. Loss of polarization—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. It is known from the literature that hepatocytes which are maintained in culture environments that support polarization and three-dimensionality retain critical functions for a longer period outside of the body.4 

BIOPRINTED LIVER TISSUE MODEL

Organovo’s NovoGen Bioprinting platform was utilized 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 our 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 multi-cellular 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 compartmentalized architecture that was established at the time of fabrication and substantially maintained over time in culture.

This image is a cross-section of bioprinted human liver tissue demonstrating compartmentalization between the hepatocytes (shown as blue nuclei), endothelial cells (red), and hepatic stellate cells (green).

In addition to the cell type-specific compartmentalization, 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 multi-cellular, 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, multi-cellular 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.

REFERENCES

1. Nagasue N, Yukaya H, Ogawa Y, Kohno H, Nakamura T. Ann Surg. 1987 Jul; 206(1):30-9.
2. Marcos A, Fisher RA, Ham JM, Shiffman ML, Sanyal AJ, Luketic VA, Sterling RK, Fulcher AS, Posner MP. Transplantation. 2000 Apr 15; 69(7):1375-9.
3. Yamanaka N, Okamoto E, Kawamura E, Kato T, Oriyama T, Fujimoto J, Furukawa K, Tanaka T, Tomoda F, Tanaka W. Hepatology. 1993 Jul; 18(1):79-85.
4. J Pharm Sci. <http://www.ncbi.nlm.nih.gov/pubmed/20533556#> 2011 Jan; 100(1):59-74. doi: 10.1002/jps.22257. Epub 2010 Jun 8.