Scaffold-mediated liver regeneration: A comprehensive exploration of current advances

Abstract

The liver coordinates over 500 biochemical processes crucial for maintaining homeostasis, detoxification, and metabolism. Its specialized cells, arranged in hexagonal lobules, enable it to function as a highly efficient metabolic engine. However, diseases such as cirrhosis, fatty liver disease, and hepatitis present significant global health challenges. Traditional drug development is expensive and often ineffective at predicting human responses, driving interest in advanced in vitro liver models utilizing 3D bioprinting and microfluidics. These models strive to mimic the liver's complex microenvironment, improving drug screening and disease research. Despite its resilience, the liver is vulnerable to chronic illnesses, injuries, and cancers, leading to millions of deaths annually. Organ shortages hinder liver transplantation, highlighting the need for alternative treatments. Tissue engineering, employing polymer-based scaffolds and 3D bioprinting, shows promise. This review examines these innovative strategies, including liver organoids and liver tissue-on-chip technologies, to address the challenges of liver diseases.

Keywords: 3D bioprinting; 3D scaffolds; bioink; hepatic organoids; hydrogels.

Graphical abstract

Figure 1.

Anatomy of the liver. This figure illustrates the basic anatomy of the liver, highlighting different cell types of liver such as the Hepatocytes, Endothelial cells, Kupffer cells and stellate cells.

Figure 2.

Treatment strategies for liver disorders. This figure summarizes various treatment strategies for liver disorders including liver transplantation, tissue engineering, 3D bioprinting and emerging therapies.

Figure 3.

Number of publications from 2017 to 2024 derived from Scopus-advanced literature search (https://www.scopus.com) with keywords “Liver Tissue Engineering,” “Scaffolds for Liver Tissue Engineering,” “Liver Scaffolds,” “Scaffold Fabrication” AND “Liver Organoids” were used as keywords.

Figure 4.

Decellularized extracellular matrix (dECM) based scaffolds. Created by Joao Vieira and Daid Ahmad Khan by biorender.com. This figure showcases different types of dECM scaffolds used in hepatic tissue engineering. dECM scaffolds retain the native extracellular matrix structure and are utilized to support cell attachment and growth. Various methods of dECM preparation and their fabrication techniques in liver tissue regeneration are illustrated.

Figure 5.

Types of 3D scaffolds. This figure presents various types of 3D scaffolds used in liver tissue engineering, including porous scaffolds, hydrogels (synthetic and natural polymers), 3D scaffolds, 3D nanofibrous scaffolds and microspheres.

Figure 6.

Process of 3D bioprinting. This figure outlines the step-by-step process of 3D bioprinting used in hepatic tissue engineering. The process includes bioink preparation, scaffold design, printing, and post-printing maturation.

Figure 7.

(a) micro physiological structure of Liver tissue. (b) Schematic diagram of Liver-On-A-Chip. (Reproduced from Deng et al. 2020, Biomicrofluidics 2020, under creative common attribution license CC-BY).

Figure 8.

iPSCs-derived liver organoid generation. (a) utilizing hepatoblasts generated from iPSCs, mesenchymal cells, and endothelial cells in a co-culture system. After the initial aggregation, different cytokines are added to the culture media to aid in the creation of organoids. (b) developing organoids with only cells generated from iPSCs. Hepatoblast aggregates obtained from induced pluripotent stem cells (iPSCs) are distributed, cultivated in Matrigel with various cytokines, and differentiate into liver organoids that comprise cholangiocytes and hepatocytes. (c) using “Liver-on-a-chip” methods, which include growing primary liver cells or embryoid bodies generated from iPSCs on a chip in matrix-dependent or matrix-independent environments to encourage organoid aggregation. (d) Using primary hepatic cells and gelatin-methacryloyl hydrogels as ink, a three-dimensional (3D) printing technique is applied. These cells are then printed onto trans wells or perfused microwells to make liver organoids. (Reproduced from Cristina et al. 2020, Int. Journal of Molecular Sciences, under creative common attribution license CC-BY).

Figure 9.

Human multi-lineage Hepatic Organoid model forms complex structures that resemble those in human liver. (Reproduced with permission from Guan et al. 2021, Nat Commun,) (a) A schematic representation of the in vitro culture system that directs IPSC to differentiate into HOs. The following structures are indicated in the images: cv, central vein; pv, portal vein; and bd, bile duct; bc, bile canaliculus; a, artery. (b) A low-power, bright field view of HOs obtained after 21 days of differentiation. Scale bar is 500 μm. (c) Calcein AM staining indicates that cells within an organoid are viable, the Scale bar is 500 μm. (d) A high-power bright field image of the region indicated in (c) shows the polygonal hepatocyte morphology of the cells within an HO. These cells also have lipid vesicles, which appear as bright areas. (e) Immunostaining shows Albumin+ hepatocytes and CK19+ cholangiocytes within the HOs. The dotted circles indicate bile ducts. Scale bar is 50 μm. (f) Left: Trichrome staining shows that some of the structures present in normal liver (top) are also present in HOs (bottom). Right: Immunostaining shows that collagen is present in peri-ductal and vascular areas. The yellow dotted line delineates an area with hepatocytes (Hep+) in normal liver. Scale bars are 50 μm. (g) HOs were immunostained with antibodies to endothelial cell (CD31), and hepato-biliary (HNF4A, CK8) markers. Structures resembling bile ducts (bd), portal vein (pv), and venules (v) are present in HOs. Scale bars are 50 μm. (h) A primary cilium in a day 21 HO was visualized with an ARL13B-GFP fusion protein (GFP), and by immunostaining with acetylated tubulin (ac-T). Scale bar is 5 μm. (i) scRNA-seq data indicates that HOs express multi-lineage markers, which include CK19 (Cholangiocyte), PDGFRB (hepatic stellate cells), and ICAM1 (endothelial cells). (j) Left: A schematic diagram of SHG and CARS microscopic imaging of a HO with collagen fibers (cyan).

Figure 10.

Evaluation of Drug toxicity. analyzing the toxicity of the medication after nine days of exposure on steatotic HepaRG cells and HML organoids. On day 5, HepaRG cells and HML organoids were exposed to a fatty acid combination. The medium was then supplemented with 5-fluorouracil (5-FU), paracetamol, valproate, voriconazole, or troglitazone from Day 7 to Day 14. Intracellular ATP levels were measured to evaluate the levels of toxicity. (Reproduced from Bronsard et al. 2024, Toxicology Invitro under creative common attribution license CC-BY-NC).