Hepatocytes, as the principal functional entities of the liver, comprising a substantial proportion of the liver mass, representing up to 80%. They possess a repertoire of functional characteristics reflective of the liver and can be transplanted into recipients for the restoration of hepatic function, obviating the need for intricate surgical procedures [12,13]. Despite being widely regarded as the gold standard in research, primary hepatocytes in 2D cultures possess drawbacks that significantly restrict their utilization in clinical applications, as their susceptibility to loss of viability, and function during in vitro cultivation or subsequent cryopreservation. Although some scientists have made successful attempts in long-term cultivation of hepatocytes in 3D culture environments [14,15], a consistent cell source remains elusive primarily due to the scarcity of available donor livers. Therefore, it is essential to define and validate novel sources of functional hepatic cells (Fig. 2).

To address the scarcity of hepatocytes, researchers are actively engaged in the pursuit of generating functional hepatocytes from diverse stem cell populations, including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) [16].

MSCs represent a multipotent type of stem cells that can be obtained from a variety of tissues. They express major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules, making them well-suited for allogeneic transplantation without causing rejection [17,18], in addition to their immunomodulatory properties [19–21], making them highly suitable for transplantation purposes. The mechanisms underlying the therapeutic effects mediated by MSCs involves their differentiation into multiple cell lineages, migration towards damaged tissues, immunomodulatory actions, and a wide array of biologically active factors, including cytokines, chemokines, hormones, growth factors, and miRNAs that promote cell survival and proliferation [22,23]. However, the constrained migration and suboptimal viability of transplanted MSCs [24,25] have prompted investigators to delve into alternative stem cell sources for the purpose of liver regeneration.

ESCs originate from the inner cell mass of a blastocyst that possess unique pluripotency and self-renewal capabilities [26]. These cells possess the exceptional capability of undergoing indefinite division in vitro while retaining their potential to differentiate into all types of cells present in an adult body [27,28]. Hence, ESCs hold the potential to offer an inexhaustible supply of transplantable cells to replace or regenerate damaged tissue. Although the clinical application of ESCs for extensive expansion and differentiation into hepatocytes remains a significant challenge owing to lingering uncertainties concerning their acquisition and potential tumorigenic risks, scientists have developed several effective strategies, such as growing ESCs in 3D fibrous matrices or organoids system [29,30]. Another concern is that ESCs are unfortunately mired by ethical controversies.

iPSCs have been demonstrated to exhibit remarkable similarity to ESCs in regard to their transcriptional programs, chromatin modification profiles and overall chromatin configurations [31–33]. As iPSCs can be derived directly from adult tissues, they present the advantage of bypassing the need for embryo destruction and can be personalized for each patient. The advent of iPSCs has thus emerged as a focal point in the realm of stem cell research [34]. Numerous studies have documented the successful differentiation of iPSCs into diverse array of liver cell types, encompassing hepatocytes, endothelial cells, cholangiocytes, and stellate cells [35–37]. Concurrently, the growing fascination with self-assembling or matrix-guided 3D organoids has opened new avenues towards the development of functional bioartificial livers [38]. However, it is crucial to note that while initially believed to eliminate the risk of immune rejection in transplants, accumulating evidence suggests that certain iPSC-derived cells may still provoke immune responses [39,40].

Hepatic progenitor cells (HPCs) represent a class of bipotential cells that are renowned for their exceptional capacity to simultaneously proliferate and differentiate into both hepatocytes and cholangiocytes [41]. HPCs are regarded as a promising cellular reservoir for liver regeneration. Nevertheless, the clinical application of HPCs is impeded by the potential risk of hepatocellular carcinoma development [42,43]. Human-induced hepatocytes (hiHeps) are derived through the direct reprogramming of human fibroblasts. These hiHeps could be easily expanded and maintained hepatocyte-specific functions, including the secretion of plasma proteins and activity of P450 enzymes [44]. The hiHep-based bioartificial liver system demonstrated promising outcomes in both a porcine liver failure model and patients undergoing extended liver resection [45]. Hepatic stellate cells (HSCs) comprise 15% of all hepatocytes and play a pivotal role in the synthesis and secretion of collagen and other extracellular matrix components. HSCs orchestrate liver regeneration by producing a diverse array of growth factors, cytokines, and glycoproteins that are activated by proinflammatory cytokines and transformed into myofibroblasts [46]. Kupffer cells, referred to as hepatic macrophages, play a pivotal role as the liver’s primary immune defense against injurious particles and chemical agents that infiltrate the portal vein. Kupffer cells are responsible for releasing crucial bioactive factors for regulating hepatocytes [47,48].

Spheroids, which represent dense 3D cell aggregates, serve as optimal building blocks for regenerative medicine. Given their capacity to facilitate tissue formation, spheroids hold potential applications in areas such as wound healing or correcting congenital defects [54]. Approaches to guide spheroid behavior can be categorized as ex vivo or in situ [55]. Ex vivo manipulation of spheroids typically begins during the monolayer culture phase prior to spheroid formation. During this stage, cells undergo priming through exposure to pharmacological agents or other soluble bioactive factors, while the modulation of microenvironmental factors, like cell confluency and localized oxygen levels, can prompt differentiation and enhance cell survival. In situ instruction of spheroids often experience limited viability, swift dedifferentiation, and propensity for migration from the injection site [56]. Biomaterials play a pivotal role in facilitating the localization of cells at the targeted delivery site, while also providing instructive cues to regulate and guide cellular behavior. These biomaterials can be customized to emulate the characteristics of the extracellular matrix (ECM), thereby eliciting stimulation of cell proliferation and enhancement of cell survival. This allows for quicker delivery of therapies and conservation of valuable resources [57].

In preclinical studies using animal models, researchers have explored the prospective capacity of spheroids to amplify and potentiate regenerative processes within the liver. Sun et al. demonstrated that spheroids of human umbilical cord-derived MSCs (hUC-MSCs) exhibited enhanced migratory capabilities towards the injured liver when contrasted with hUC-MSCs cultured in a two-dimensional (2D) manner. Notably, these hUC-MSCs were found to effectively stimulate liver regeneration and facilitate repair in mice afflicted by liver injury [58]. Hepatocyte spheroids of human were effectively engrafted into the livers of mice with viral hepatitis following intrasplenic injection [59]. Collagen fiber-based 3D spheroids comprising adipose-derived stem cells effectively maintained cellular functionality and the capacity for paracrine secretion. Furthermore, transplantation of these spheroids mitigated thioacetamide (TAA)-induced liver cirrhosis in murine models [60]. Spheroids derived from stem cells of human exfoliated deciduous teeth were successfully grafted into the livers of mice afflicted with carbon tetrachloride (CCl4)-induced liver fibrosis. In this transplantation, the spheroids engrafted well, leading to enhanced liver function, and demonstrating antifibrotic efficacy in mice [61]. Innovatively, some researchers have utilized 3D printing to construct hepatocyte spheroids, thereby fabricating functional liver tissue that replicates intricate duct and sinusoid microanatomy [62,63]. Zhang et al. devised a method for fabricating spheroids with precise quantities of distinct cell types using microfluidic flow cytometric printing. These precision spheroids exhibit enhanced variability and functional consistency in comparison to randomly formed spheroids [64].

Organoids represent a condensed and simplified rendition of an organ, often originating from stem cells. They may encompass diverse cell types that spontaneously self-organize in vitro, recapitulating the morphology and intricate biological interplays within a living organism [65]. Here, we elucidate the recent endeavors directed toward constructing next-generation organoids and outline the current potential of utilizing mature organoids in liver regeneration.

ESCs and iPSCs are preferred candidates for liver organoid generation due to their proliferation and differentiation characteristics. For instance, Wang et al. presented a study detailing the generation of hepatic organoids derived from ESCs with expandable attributes, utilizing entirely defined medium devoid of serum and feeder cells. Remarkably, these organoids were capable of expanding for up to 20 passages, facilitating large-scale growth. Furthermore, upon transplantation, they exhibit a notable capacity for repopulating injured livers within Fah^-/-/Rag2^-/-/Il2rg^-/- (FRG) mice, subsequently differentiating into mature hepatocytes in vivo [66]. In 2022, Messina and colleagues successfully developed human iPSC-derived hepatocytes organoids. Specifically, the process involved the initial differentiation of human iPSCs into hepatoblasts through the application of growth factors and cytokines. Subsequently, hepatoblasts were developed into iPSC-derived hepatocytes organoids using hepatic growth factor (HGF), oncostatin M, vitamin K and dexamethasone [67].

There are some parameters that are crucial to control during the construction of 3D organoids. Firstly, liver organoids require exposure to specific growth factors, including Wnt, fibroblast growth factor, HGF, and bone morphogenetic protein, at precise timings to effectively promote hepatic progenitor survival and their differentiation [68]. The ECM also plays a critical role in the formation of organoids. While Matrigel has been widely employed for organoid culture, its application is limited by factors like complexity, batch-to-batch variability, and its origin from mouse tumor cells. Consequently, various alternatives are under exploration, such as decellularized liver ECM, diverse synthetic hydrogels, as well as peptide and recombinant protein matrices [69]. Lastly, designed geometries like micropatterning or microfluid dynamics represent strategies that can be harnessed to facilitate the growth of cells and organoids [70].

Employing a single cell type in organoids culture ensures the proliferation and self-organization of a homogenous cellular population, thereby simplifying the formation process. However, co-culturing multiple cell types, including endothelial cells, HSCs and MSCs can significantly enhance the functional maturity and complexity of the organoids, leading to a better mimicry of the liver organ structure [71,72]. In 2013, Takebe et al. made the pioneering report of generating a three-dimensional vascularized and functionally active human liver using human iPSCs. Specifically, they demonstrated that iPSCs, co-cultured alongside MSCs and human umbilical vein endothelial cells (HUVECs), could spontaneously self-organize into three-dimensional iPSC-liver buds (organoids) which exhibited an endothelial network and expressed hepatic-specific marker genes [71]. In 2019, Pettinato et al. interlaced iPSCs with human adipose microvascular endothelial cells, resulting in an increased differentiation yield and significant enhancements across a diverse spectrum of hepatic functions [73]. In 2021, Tanimizu et al. generated a functional hepatobiliary tubular organoid through the co-cultivation of mouse hepatocyte progenitors and cholangiocytes. Remarkably, this organoid exhibited the acquisition and sustenance of metabolic functions, including the secretion of albumin and activities of cytochrome P450, persisting over an extended duration [74] (Fig. 3).

Principally, organoids serve as valuable exploration tools, acting as platforms for drug screening and providing insights into the intricate processes underlying human development and diseases [75,76]. For regenerative medicine, the ultimate objective is to achieve the transplantation of tissue-specific organoids, which will facilitate the recovery or enhancement of tissue function. Despite the extensive exploration of regenerative medicine for liver diseases over numerous decades, several challenges persist. Transplanted organoids must exhibit long-term survival, compatibility with extensive expansion, and genetic stability [77].

Several studies have demonstrated that organoids transplantation is a promising strategy for cell-based therapy for liver regeneration (Table 1). Huch et al. established a durable human liver organoid culture, and the cells exhibit the capability to be readily transformed into functional hepatocytes both in vitro and upon transplantation in vivo [78]. Tsuchida et al. verified the safe transplantation of human iPSC-derived liver organoids via the portal vein, with concurrent ligation of the ductus venosus [52]. Sampaziotis et al. delivered remarkable evidence, showcasing the potential of cholangiocyte organoids to repair human biliary epithelium. This groundbreaking work introduces a therapeutic 3D stem cell-based approach for repairing ischemic bile ducts, potentially providing the potential for organoid-based therapies targeting various cholangiopathies [79]. Furthermore, organoids can be derived from patients with genetic diseases, and subsequently, genetically modified organoids hold the potential for transplantation into patients to alleviate disease phenotypes [80]. An illustrative instance of this strategy involves the application of Retinitis Pigmentosa GTPase Regulator (RPGR) gene-edited organoids in tackling retinitis pigmentosa. This strategy has demonstrated the restoration of defects characteristic of the disease, such as photoreceptor reduction [81]. Here, we summarized the typical studies that performing human liver organoid transplantation in the past decade (Table 1).

Bioscaffolds can mimic the ECM environment of the organ and can effectively induce cell growth and differentiation. As a result, these bioscaffolds possess the capacity to adequately support the colonization of host cells, meeting up the requirements of regeneration and repair. In general, bioscaffolds can be classified into two main groups: natural and synthetic. Natural bioscaffolds, such as collagen, chitosan, silk fibroin, or Polyhydroxyalkanoates (PHAs) are derived from biological sources and demonstrate exceptional biocompatibility and bioactivity. On the other hand, synthetic bioscaffolds, which include polymers like polylactic acid (PLA), polyglycolic acid (PGA), or polycaprolactone (PCL), are artificially produced, allowing for precise manipulation of their physical and chemical characteristics [99–101]. Here, we delve into a discussion of two representative bioscaffolds used in the field of liver regeneration.

3D encapsulation of cells in a collagen hydrogel has shown potential for enhancing maturation compared to 2D culture. Hussein et al. demonstrated that the combining of 3D techniques, namely guided aggregation and microencapsulation, with liver differentiation protocols, constitutes a robust strategy for producing fully mature and functional hepatocytes. This innovative approach presents a sustainable and limitless source of hepatocytes [102]. Song et al. successfully engrafted iPSC-derived hepatocyte-like cells (iPS-H) with stromal cells into immunocompetent mice. This accomplishment was realized through the encapsulation of the cells within biocompatible hydrogel capsules. The secretion of human albumin and α1-antitrypsin by encapsulated iPS-H/stromal cell aggregates attained a level comparable to the primary human hepatocyte/stromal cell control [83].

However, achieving a complete recapitulation of the entire biochemical and architectural intricacies of the natural microenvironment remains a challenge. Decellularized scaffolds from xenogeneic animals and discarded human livers offers a promising approach to tackle this issue. Decellularized liver scaffolds can retain the functional attributes of the native microvascular and bile drainage networks within the liver, along with the essential growth factors required for both angiogenesis and liver regeneration [103]. The techniques for producing decellularized liver scaffolds encompass physical, chemical, and enzymatic treatments, often employed in combination. Currently, substantial strides have been taken towards ensuring safety and clinical applicability. Detailed reviews are available elsewhere [104,105]. The next critical steps are the recellularization of the parenchyma and reendothelialization of the vascular lumen to generate transplantable and functional liver grafts. In the past decade, various proof-of-concept studies have been conducted to assess the viability of repopulation of liver grafts in both small and large animal models (Table 2).

3D bioprinting, a technique that employ computer-aided processes to precisely assemble cells and biomaterials into precise 3D structures, has emerged as a promising avenue for replicating hepatic tissue and facilitating translational endeavors. Bioprinting can be broadly classified as extrusion-based, inkjet-based, or light-assisted bioprinting. Notably, 3D-bioprinted liver models are commonly classified as either static scaffold-based models or dynamic liver-on-chip models [116]. Diverse forms of scaffolds featuring intricate structures have been designed for the exploration of liver regeneration. Kang et al. utilized a preset extrusion bioprinting technique to generate multicellular and multi-material structures comprising hepatic cells, endothelial cells, along with a lumen. This construct, comprising hepatic lobules within a densely vascularized framework, facilitated the fabrication of tissue at both micro-and macro-scales (Fig. 4). Such an approach can be pivotal in developing expansive 3D tissue constructs for multiscale tissue engineering purposes [117]. In a recent study, 3D-bioprinting was employed to construct hepatorganoids using HepaRG cells and bioink. Following transplantation into Fah-deficient mice, these hepatic organoids exhibited the formation of functional vascular systems, which consequently enhanced material transport and liver function. Consequently, the survival rate of the mouse model afflicted with liver injury exhibited significant improvement [118].

Unlike conventional static platforms, the integration of bioprinting with dynamic microfluidic liver chips offers the capacity to recreate intricate microenvironments encompassing factors like temperature, pH, cell shear stress, nutrient supply, oxygen gradient, and waste removal. Although the primary focus of research in the field of liver-on-chip technology is directed towards the application of these platforms for drug screening and disease modeling, a recent study introduced a novel microfluidic liver system that integrates hiPSCs-derived hepatocytes-laden microparticles and semipermeable microtubes. This system exhibited high cell viability, functional regeneration, and effective circulation system. Building upon these merits, a liver chip integrated with multiple microfluidic liver systems was employed in a rabbit model of acute liver failure. This intervention not only mitigated inflammation, but also facilitated the production of serum proteins, ultimately leading to improved survival rates [119].

Transitioning from small-scale laboratory experiments to large-scale production of functional liver tissue constructs presents a substantial manufacturing challenge. Consistency in quality, reproducibility, and scalability is essential for guaranteeing the safety and effectiveness of liver constructs on a human scale. For spheroids or organoids, culture conditions that seamlessly integrate with the large-scale manufacturing platforms need to be developed. Indeed, the commonly used Matrigel carries potential risks for tumorigenesis and immune reactions due to its mouse origin, and batch-to-batch variability further complicates its reliable usage. Synthetic hydrogels, like polyethylene glycol (PEG), offer enhanced reliability and safety. However, cells within these hydrogels often exhibit restricted liver functionality [121]. Thus, it is necessary to develop reliable and safety ECM for generating reproducible and functional organoids. Furthermore, ensuring adequate oxygenation and nutrient exchange throughout the entire tissue construct, as well as the complex microenvironment and metabolic demands, are crucial for the viability and functionality of liver constructs.

The choice between allogeneic and autologous cell sources in clinical applications involves careful consideration of factors, such as immune compatibility, treatment timeline, availability, and ethical considerations. In cases of patients with acute liver failure, the feasibility of utilizing autologous cells for readily available regenerative medicine solutions could be limited. Meanwhile, the use of allogeneic cells carries the inherent risk of inciting immune reactions within the host’s physiology, possibly culminating in graft rejection or analogous immune-mediated intricacies [122]. Genetic engineering techniques can be employed to manipulate allogeneic donor cells to diminish immunogenicity, presenting a promising avenue, albeit accompanied by a spectrum of inherent challenges [123].

Hepatocytes or stem cell-derived cells in 3D cultures often struggle to fully mature and maintain their functionality over time. For instance, the presence of unrelated cells or undifferentiated stem cells or within organoids can give rise to the subsequent development of undesirable and potentially hazardous tissue formations characterized by genetic abnormalities [124]. This issue highlights concerns regarding the safety and potential tumorigenicity associated with organoids. To address this challenge, the genetic stability of organoid cells should undergo regular check. The cells utilized for generating organoids should undergo purification by microdissection methods to ensure separation from their surrounding stromal components. Modulating the microenvironment composition, particularly by manipulating the presence of growth factors, can effectively guide the differentiation of organoids towards the desired pathways [125].

Vascularization is critical for the growth and efficient exchange of factors within organoids and liver constructs. Ensuring efficient vascularization within 3D liver tissue constructs remains a significant challenge. Although the vascularization of bioprinted liver scaffolds has been generated successfully in a few models, there remains a need for the development of advanced technologies that enable more precise bioprinting of small-diameter blood vessels. Currently, spheroids and organoids still encounter limitations in establishing proper vascularization upon engraftment. To address this, incorporating endothelial cells or HUVECs as organoid components may presents a promising strategy [71]. Exploring transplantation and cytokine induction methods for in vivo vascularization should receive more attention.

Ethical concerns pertaining to the origin, potential hazards, and long-term consequences of transplanted liver grafts introduce intricate questions. Participants may face risks including tumorigenesis, adverse immunogenic responses, and infections [126]. Therefore, the progression of 3D cell-based liver regeneration therapies from laboratory breakthroughs to clinical implementation demands rigorous safety evaluations and regulatory approval. To attain regulatory approval and ensure patient welfare, it is imperative to substantiate the safety and efficacy of these therapies through well-designed clinical trials and comprehensive preclinical studies. At present, no established clinical or ethical guidelines are in place to evaluate the safety of 3D cell-based liver grafts for clinical trials. Researchers could gain insights from analogous fields like cell therapy, for addressing matters such as cell sourcing, risk evaluation, patient selection criteria, trial design and oversight.