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International Journal of Bioprinting New challenges in liver tissue engineering

survival. Under normal conditions, liver ECM comprises with several limitations, such as the scarcity of liver tissue
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less than 3% of the relative area and is mainly composed of and the vulnerability of PHHs to freezing and thawing
collagens (types I, III, IV, and V being the most abundant); procedures. 70-72 Researchers have recently focused on
glycoproteins such as fibronectin, laminin, or nestin; and exploring new culture systems (3D cultures, co-culture
glycosaminoglycans such as hyaluronic acid, chondroitin, systems, microfluidic devices) that maintain the cultures
heparin, and dermatan sulfates. 53,54 Liver ECM is composed over longer periods and even improve their functionality.
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by two different structures (basement membrane and Human liver cell lines (HepG2, Huh7, and HepaRG) are
interstitial matrix) that differ in both morphology and alternatives to PHHs, that are widely available and easy to
biochemistry. The interstitial matrix is composed of handle, 73,74 despite their reduced metabolic performance
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collagen (types I and III) and fibronectin. The basement and inability to reflect inter-individual differences.
membrane mainly comprises laminin, heparan sulfate Stem cell research has driven a series of efforts
proteoglycan, and collagen IV and forms a thin acellular leading to the generation of induced pluripotent stem
layer that facilitates diffusion. However, it is not present cells (iPSC), reprogramming of distinct cell types, and
in the sinusoids and parenchyma of healthy livers. Liver thus, their differentiation toward a hepatocyte phenotype
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ECM has a critical role in the regulation of cell behavior, (hepatocyte-like cells, HLCs) by means of a sequential
phenotype, migration, and proliferation. Remodeling protocol that mimics liver development. The generation of
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the liver ECM can help to maintain both the viability HLCs would supply an unlimited cell source for cell-based
and functionality of the tissue and induce diseases. For assays and also as therapy for liver disease. 75-77 One of their
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instance, in liver fibrosis, different chemicals or viruses can major advantages is that these iPSC models could reflect
induce cell death, through which different mediators that inter-individual variability, offering more representative
can trigger HSC activation are liberated. Activated HSCs models, while HLCs could be also a therapeutic
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deposit elastin and collagen, thus increasing stiffness. alternative to organ transplants, although protocols that
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ECM modification is an extremely coordinated permit the differentiation of iPSCs into a more mature
process that involves synthesis, secretion, degradation, form are urgently needed.
and reorganization of the different components. A Monolayer (two-dimensional [2D]) cultures have
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pathological deposition of ECM would occur when there is traditionally been used in hepatology; however, these
an injury or the enzymes involved in modifying the ECM, systems do not accurately reproduce the liver’s spatial
such as the matrix metalloproteinases (MMPs), become organization or the cell–cell or cell–matrix interactions
malfunctioned. 58,59 that improve and stabilize hepatocyte’s functionality. 79,80
Several cell types are implicated in modifying the liver Co-cultures of hepatocytes and other liver cells have
ECM, e.g., the cross-talk between HSCs, portal fibroblasts, also been proposed to improve functionality and better
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LSECs, and KCs has been extensively discussed. In this recapitulate liver (patho)physiology. 81-83
regard, Ramachandran et al. used RNAseq technology to
identify pathogenic subpopulations of endothelial cells, 5. Hydrogels for liver tissue engineering
macrophages (KCs, mononuclear phagocytes, and scar- Tissue engineering approaches are utilized to simulate
associated macrophages), and myofibroblasts in fibrotic the functions of the target organs but not necessarily their
human tissues. architectures. These approaches entail the combination of
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cells, 3D scaffolds, and signals for the correct regeneration
4. In vitro models in hepatology of the tissue/organ or the in vitro simulation of healthy

Animal models offer an indispensable tool for exploring and diseased tissues/organs. As the liver is a relatively
the mechanisms underlying liver disease and deciphering soft organ, hydrogels or soft scaffolds have mostly been
the process of liver regeneration. However, different applied as synthetic ECM components for the cells. Great
human in vitro models are now used to fully recapitulate advances in liver tissue engineering techniques tailoring
human liver disease and develop safer and more efficacious complex multicellular models to better recapitulate the
treatments. PHHs are the gold-standard cells used in liver functions, microarchitecture, and microenvironment
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liver cell-based assays 62-68 as they can be isolated and have been achieved in recent years.
cultured, but the functionality and quality of the cells Hydrogels are polymeric hydrophilic networks that can
may significantly decline over long periods of culture absorb large amounts of water without dissolving. They
in monolayer configuration. 4,62-64,68 One of the major maintain their integrity in the presence of an aqueous
advantages of cultured PHHs is that they can reflect inter- environment due to the crosslinking points between the
individual variability, although this approach is fraught polymeric chains. These crosslinks can be chemical,
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Volume 10 Issue 3 (2024) 120 doi: 10.36922/ijb.2706