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Engineering the liver : Creating reliable models to understand the hepatic physiopathology



The liver is a multi-lobular organ located in the upper abdomen which is critical for many physiological processes such as detoxification of the organism, lipid, and cholesterol homeostasis, and the synthesis of proteins and biochemicals necessary for digestion and growth. (1)

Liver failure is a life-threatening global health problem, and a liver transplant might be the only option when irreversible.


Although significant progress has been made in liver transplantation over the past few years, donor organ scarcity remains a critical limitation and accounts for a large proportion of waitlist mortality.

Thus, other alternatives are being investigated. 3D bioprinting is an emerging technology that uses additive manufacturing technologies to assemble multiple cell types with other biomaterials to create functional 3D constructs layer-by-layer. With bioprinting's arrival, several bioartificial livers models have been developed from functional liver tissue to living mini-organs, playing an essential role in the mechanistic understanding of liver biology interactions in healthy and pathological states and providing therapeutic development for liver diseases. (2)

We illustrate some advances in tissue engineering to understand liver physiopathology. In the future, those advances might help to compensate for the lack of alternatives in treating liver diseases and the need for transplantation.


3D-printed liver tissues

In a study published by H. Kizawa and colleagues, the researchers used cryopreserved hepatocytes to form spheroid culture. Then they cultivated those spheroids in a perfusion chamber until they fused and formed hepatic tissue. They removed the obtained tissues, cultivated them separately, and analyzed functional markers (3). They demonstrated that the bio-printed liver tissue could maintain the active metabolism of drugs, glucose, lipids, and bile acid for several weeks (fig.1).



Fig. 1. 3D bio-printed liver tissue maintained sugar and lipid metabolic functions for long periods. (A) Bio-printed human liver tissue glucose production was stimulated with 500 nM dexamethasone, and 100 μM 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (Dex/8CPT); the increase was reduced with the addition of 10 μM insulin (day 77). Error bars represent SEM (n=3). (B) Time-dependent bile acid accumulation, measured in the medium of bio-printed human liver tissue (day 24–27). Error bars represent SEM (n=4). © Hematoxylin and eosin stained bio-printed liver tissue derived from a Zucker fatty rat (male, age 11 weeks) after 23 days of culture. (D) Oil-red O-stained bio-printed liver tissue derived from a Zucker fatty rat shows lipid deposits after 23 days of culture.


Liver-On-Chip

Microfluidic devices hold significant promise for liver failure. For instance, M. S. Freag and colleagues could mimic human Non-Alcoholic Steatohepatitis (NASH) (4), a metabolic-associated fatty liver disease affecting more than 25% of the world's population. To do so, they co-cultured the four significant types of human primary liver cells (hepatocytes, hepatic stellate cells, Kupfer cells, and liver sinusoidal cells) in a triplet microchannel chip platform. (fig.2)




Fig. 2. Construction of liver-on-a-chip. (A) Recapitulation of the liver cellular microenvironment on a chip. (Ai) LSECs form a continuous monolayer in the lateral channel. (Aii) Collagen compartment containing KCs (blue), HSCs (green), and HCs (red); scale bars are 25 µm. (Aiii) Merged image showing both endothelialized channel and collagen compartments. (B) Representative brightfield microscope image showing the orientation of HCs (red), HSCs (green), and KCs (blue) in the collagen matrix (gray). (C) Representative confocal microscope image of the 3D arrangement of HCs (red), HSCs (green), and KCs (blue) in a collagen matrix. (D) Live/dead assay of liver cells after 14 days of initial culture.


They mimicked NASH exposing the liver cells to fatty acid chains with or without lipopolysaccharide (LPS) - that induces inflammation - and observed that the model could recapitulate the morphological and biochemical hallmarks of the human disease. To get in more details, they collected media effluents from liver tissue cultured on chips five days and ten days after exposure to lipotoxic conditions to analyze inflammatory markers that are known to raise during NASH development, such as monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1α (MIP1α), tumor necrosis factor α (TNF-α). They also measured the expression of TGF-𝛃, a regulator of fibrosis, and observed a NASH-like behavior in the NASH chips.


Liver organoid

The construction of liver organoids as a model system is an appealing experimental approach to exploit liver tumor mechanisms. A study by L Broutier and colleagues created liver tumor-derived organoids that were successfully obtained from patients with primary liver cancer, the second cancer more lethal worldwide. They demonstrated in their study that PLC-derived organoids recapitulate the histological architecture (fig.3) and expression profiles of the corresponding parent tumor (5). They performed drug-sensitivity tests and observed a patient-specific response, showing great potential for biomarker discovery and drug testing.


Fig 3. Patient-derived primary liver cancer organoid cultures expand long-term in vitro while preserving the histological architecture of the tumor subtype they derived from. (a) Experimental design. Healthy (donor-derived) liver tissues, moderate/well-differentiated hepatocellular carcinoma (HCC), combined hepatocellular-cholangiocarcinoma (CHC) and cholangiocarcinoma samples (CC) were obtained from patients undergoing surgery (b) Representative H&E staining of healthy liver tissue and primary tumors (top row), and corresponding brightfield microscopy images (middle row) and H&E histological analysis of the organoid lines derived from these (bottom row). (5)


The liver plays a central role in metabolism. Tissue engineering for modeling the liver is very promissory and can allow new advances in many fields, such as drug testing and cancer research.


At Tissuelabs, we believe in more reliable research through pertinent models. That is why we offer a range of products allowing the creation of all kinds of 3D structures for your research, from tissue-specific hydrogels to 3D bioprinters. Our hydrogels are able in 15 tissues, including the liver!



References:

(1)Trefts E, Gannon M, Wasserman DH. The liver. Curr Biol. 2017 Nov 6;27(21):R1147-R1151. doi: 10.1016/j.cub.2017.09.019. PMID: 29112863; PMCID: PMC5897118.

(2) Deng J, Wei W, Chen Z, Lin B, Zhao W, Luo Y, Zhang X. Engineered Liver-on-a-Chip Platform to Mimic Liver Functions and Its Biomedical Applications: A Review. Micromachines (Basel). 2019 Oct 7;10(10):676. doi: 10.3390/mi10100676. PMID: 31591365; PMCID: PMC6843249.

(3) Kizawa, H., Nagao, E., Shimamura, M., Zhang, G., & Torii, H. (2017). Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery. Biochemistry and Biophysics Reports, 10, 186-191. https://doi.org/10.1016/j.bbrep.2017.04.004

(4)Freag, M. S., Namgung, B., Reyna Fernandez, M. E., Gherardi, E., Sengupta, S., & Jang, H. L. (2021). Human Nonalcoholic Steatohepatitis on a Chip. Hepatology Communications, 5(2), 217-233. https://doi.org/10.1002/hep4.1647


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