Organoids represent a novel approach in the frame of liver regenerative medicine that could be utilized as transplantable units, disease models, toxicological studies or tools for drug discovery. Organoids, which are 3D self-organized scaffolds based on cellular structures that are derived from differentiated ESCs, iPSCs or adult stem cells (ASCs), are grown in a serum-free condition and display the full spectrum of cellular types in a tissue [20]. More specifically, a liver organoid is a 3D multicellular spherical structure made of one or more liver cell types [76]. Brassard et al. developed some parameters in order to control the process of building a 3D organoid [77]. Importantly, it has been suggested that the choice and control of the number of cells and the density of the dissociation–aggregation phase is mandatory, including the best culture conditions and the combination of growth factors to specifically guide the differentiation process. In liver organoids, the molecular signaling pathways that regulate liver embryonic development, such as HGF, FGF, BMP, Wnt and TGF, could guide the growth factor supplementation, which promotes hepatic progenitor migration, development and survival [78]. Furthermore, in order to build a 3D organoid, it is required to supply (if necessary) some pre-defined extrinsic forces to improve cell-to-cell and cell-to-extracellular-matrix (ECM) interactions [77]. Because the ECM plays a crucial role in supporting cell proliferation, improving cell adherence and the dispersal of nutrients and growth factors, stem/progenitor cells must be in a strict contact with its components, such as collagen, laminin and fibronectin [16]. Finally, micropatterning, microwells and microfluid dynamics are some of the designed geometries that can be utilized to support cell and organoid growth [77].

Liver organoid technology simulates the morphological and physiological properties and tissue-specific functions of the liver in a dish through the self-organization of cell populations that mimic the liver development process [79]. Liver organoids could be derived either from the culture of a single cell type, such as adult stem cells, LPCs or iPSCs, or from a multi-type cell co-culture, such as:

• A combination of iPSCs with human umbilical vein endothelial cells (HUVECs) and MSCs;
• Hepatocytes and stromal cells;
• Primary liver tumor cells or damaged liver cells [80].

The single type of cell culture ensures the proliferation and self-organization of a homogenous cell population and is easier to form, while co-culture of multiple cells types can better mimic the liver organ structure [34,38].

The first single cell-type culture of a human ASC-derived organoid was developed by culturing LGR5⁺ biliary cells derived from a liver injury by adding Rspo1, EGF, FGF10, HGF, nicotinamide, cAMP agonist and TGFβ receptor inhibitor to the differentiation medium [81]. Since then, other large-scale culturing methods for ASC-derived liver organoids have been established [82]. A number of studies have already proposed the development of single-cell culture-derived liver organoids by the use of liver progenitor cells. First, Hu et al. embedded human mature primary hepatocytes in Matrigel to form liver organoids using molecule inducers, such as Rspo1, EGF, FGF7, FGF10, HGF and TGFβ inhibitor [83]. As a result, they developed human fetal hepatocyte organoids (Hep-Orgs), which were cultured for more than 11 months [83]. After that, in 2020, Hendriks et al. established a protocol with similar culture conditions that facilitate the long-term expansion of human fetal hepatocytes as organoids. [84].

Furthermore, the use of iPSC-derived liver organoids has already been proposed for the treatment of liver diseases. Because liver progenitors arise from the endoderm, iPSCs must first be differentiated into definitive endoderm to mimic liver development [85] and further exhibit hepatic maturation in alternate normoxia and hypoxia conditions [86]. Following that, liver organoids will be formed over 2D monolayers of mature hepatocytes that will then be collected and embedded on Matrigel [86]. In 2022, Messina et al. generated human iPSC-derived hepatocytes (iHeps) that self-assembled as organoids (iHep-Orgs) [86]. In particular, human iPSCs were first differentiated into hepatoblasts (iHBs) using growth factors and cytokines according to other already published protocols [86,87]. Then, iHBs were developed into iHep-Orgs using HGF, vitamin K, OSM and Dex, and their culture extended until day 38, as they were still viable, with the absence of necrotic cores [86]. Their successful development of those iHep-Orgs was then confirmed by several studies, including gene expression for EPCAM and CXCR4, the absence of pluripotent markers and the presence of hepatoblast markers as well as by the well-defined morphology [20,80,84,88,89,90,91].

To ensure cell-to-cell contacts during liver development, multi-cell co-cultures are able to generate a complex vascularized 3D structure in dishes [92]. In 2013, Takebe et al. generated, for the first time, a vascularized and functional human liver from human iPSCs by the transplantation of liver buds that were created in vitro [93]. More specifically, iPSCs were co-cultured with HUVECs and MSCs and spontaneously self-organized into macroscopically visible 3D cell aggregates showing endothelial network and the expression of hepatic-specific marker genes [93]. After that, the whole liver buds were connected to the recipient’s vasculature to generate functional vascular networks, promoting liver bud development [93]. Since MSCs can provide a number of signals promoting hepatocyte growth and development, co-culture of BM-MSCs and hepatocytes exhibited higher rates of hepatocyte-specific functions, maintained hepatocyte metabolism and generated higher quality hepatocyte organoids [94]. Although some studies suggested that MSC and HUVEC paracrine signals can both drive hepatocyte differentiation, both must co-exist to allow for cell-to-cell interaction and organization into a 3D liver organoid [95]. In 2018, Nie et al., successfully generated human liver organoids derived from multiple cells from a single donor, and these organoids were finally able to rescue a mouse model of ALF [96]. In more detail, umbilical cord endothelial cells (UC-ECs) were able to generate donor-derived human iPSCs, which then were able to efficiently differentiate into pure definitive endoderm and further into the hepatic lineage, and with a co-culture of these three cell types (UC-ECs, iPSCs and MSCs), the investigators successfully generated a single-donor cell-derived liver organoid [96]. In 2021, Qiu et al. generated a functional 3D sheet-like human hepatocellular carcinoma (HCC) organoid in vitro by co-culturing luciferase-expressing Huf7 cells, hiPSCs-derived endothelial cells and hiPSC-derived MSCs [97]. According to their results, once they added iPSC-MSCs, the organization of the 3D HCC organoid was easier, suggesting that iPSC-MSCs play a crucial role in promoting HCC organoid growth [97].

Although iPSCs appear to be beneficial in 3D liver organoid development, some studies tested a co-culture of MSCs and HUVECS with other cell types, such as amniotic stem cells (ASCs), resulting in the generation of a 3D structure with polarity and hepatic-like glycogen storage [98]. Co-culturing methods of human hepatocytes, HUVECS and MSCs have also been applied [99]. Moreover, the privilege of co-culturing human hepatocytes, HUVECs and MSCs has been tested in fusing hundreds of liver-bud-like spheroids using a 3D bioprinter [100]. Currently, studies for organoid development focus on co-culturing mesenchymal and endothelial cells, as these cells are known for their ability to regulate liver progenitor cells’ fate and growth [101].

Furthermore, the co-culturing of primary liver tumor [102,103,104] or damaged liver cells [79,105] has also been studied. Patient-derived tumor organoids (PDO) are 3D cell culture models that closely mimic the form and function of tumor tissue, illustrate cell-to-cell and cell-to-matrix interactions and have similar pathophysiological characteristics to differentiated tumor tissue in vitro [92]. Primary liver tumor organoids can be derived from liver tumor specimens, preserving the histological architecture, gene expression and genomic landscape of the original tumor, providing a tool for biomarker identification and drug screening [103]. Liver organoids can also be derived from patient biopsies with alpha-1-antitrypsin (ATT) syndrome, which is a useful tool for modeling disease pathology [80]. In addition, in 2021, McCarron et al. received wedge biopsies from patients with nonalcoholic steatohepatitis (NASH) and developed hepatic organoids, suggesting that these organoids can be used for personalized disease modeling and drug development [105]. Consequently, liver organoids can be derived from either healthy or injured patient tissues. Genetic modifications of these organoids enable disease modeling, the clarification of molecular pathogenesis, patient-specific drug testing, personalized medicine and biomarker discovery, causing them to be an alternative model for biomedical research [106,107].