The increase in the prevalence of liver diseases and the lack of versatile treatment options for end-stage liver diseases stresses the need for novel approaches and advanced technologies for developing physiologically and pathologically relevant models of the liver. Liver bioengineering through bioprinting has emerged as a pivotal area of research, aiming to address the critical shortage of donor organs, improve drug testing, and improve disease modeling. Through bioprinting the complex structure, composition and geometry of the liver microstructure can be replicated to give rise to liver models that can mimic the healthy or diseased tissue. These capabilities render bioprinted liver models suitable for advancing drug discovery and testing studies, as well as disease modeling. This review paper explores the application of bioprinting technologies in liver tissue engineering, highlighting the progress in the field through exploring materials, cell sources, and new current techniques used in the bioprinting of liver models. Additionally, the article explores spheroid printing and current preclinical models as well as key challenges and future perspectives. This comprehensive overview aims to provide insights into the current state of liver bioengineering through bioprinting and to identify future directions for research and clinical application.

1. Introduction

The liver is the largest gland in the human body and serves several essential functions, including blood protein synthesis, glucose metabolism, and detoxification.¹ Liver diseases, both acute and chronic, result in significant morbidity and mortality worldwide and account for approximately 2 million deaths per year.¹ These diseases exhibit higher occurrence rates, currently accounting for 4% of deaths worldwide.^2,3 Recent studies have shown that liver cirrhosis had a tremendous increase of 8.1% in occurrence since 2017 and was responsible for 1.48 million deaths in 2019.⁴ The risk of infections such as hepatitis B is also a major concern associated with liver diseases that put millions of people globally at high risk of mortality.⁵ Liver transplantations, with the organs obtained from either live or deceased donors, are the gold-standard treatment available for severe liver diseases. However, liver transplantation raises distress that presents with complications and postoperative immune rejections and is limited due to donor scarcity.⁶ As an alternative to surgical treatment and transplantation, drug therapies hold great promise to treat patients with liver disease; however, the development of efficient and targeted therapeutics for liver diseases has major limitations. An important tool in the development of novel therapeutics is cytological studies using two-dimensional (2D) cultures. These cultures, however, lack the crucial cell–extracellular matrix (ECM) and cell–cell interactions and do not exhibit the same pharmacological responses that in vivo models do.⁷ Three-dimensional (3D) models, such as sandwich cultures and organoids, have been used as alternatives where cell–ECM and cell–cell interactions are incorporated, respectively. However, sandwich cultures have cells grown in a plane and organoids may vary in size, leading to variable pharmacodynamic results.^8,9

Recent advances in regenerative medicine have shown that liver bioengineering using bioprinting holds great potential for progress in the treatment of liver disease. 3D bioprinting technology enables precise placement of biomaterials and living cells and control over the shape, structure, and size of the printed materials, allowing for the reconstruction of highly complex tissues and organs.³ Bioprinting offers advantages in terms of high repeatability, controllability, throughput, and positioning of multiple cells simultaneously, allowing for the development of new advances.¹⁰ This new technology enables the mimicry of intricate microenvironments and biological structures to create a reliable platform for the investigation of liver pathologies and study of new therapeutics reliably.⁶

In this review, we provide a comprehensive overview of the rapidly evolving field of liver bioengineering using bioprinting technologies to address liver-related challenges. We explore the anatomical and functional insights of the liver as well as liver pathologies and the promising potential of bioengineering in creating functional liver tissues. We focus our review on the recent key advancements in bioprinting techniques, including novel approaches like spheroid dispersion-based bioprinting. Furthermore, the paper highlights the application of these bioengineering approaches in liver disease modeling, with a particular emphasis on hepatocellular carcinoma and fibrosis models.

2. Fundamentals of liver bioengineering

2.1. Liver anatomy and function

The liver is one of the major organs in the body and it is involved in performing metabolism, detoxification, immunity, digestion, and vitamin storage (Figure 1).^11,12 The liver is a large, reddish-brown organ (Figure 1A) located in the upper right quadrant of the abdomen and is protected by the rib cage.¹³ It is divided into the following two primary lobes: the right lobe and the left lobe. These lobes are separated by a band of connective tissue called the falciform ligament, which also anchors the liver to the abdominal cavity. The functional unit of the liver is the lobule, a hexagonal structure composed of hepatocytes, which is arranged in plates radiating from a central vein (Figure 1B).¹³ Between these plates are sinusoids—small blood vessels that facilitate the exchange of substances between the blood and liver cells. Additionally, bile ducts run between the hepatocytes, collecting bile produced by the liver cells and transporting it to the gallbladder and intestine (Figure 1C).¹⁴ The liver receives blood from following two sources: oxygen-rich blood from the hepatic artery and nutrient-rich blood from the portal vein. The portal vein carries blood, which contains nutrients, medications, and toxins, from the digestive organs. This blood is processed by the liver before being returned to the systemic circulation via the hepatic veins.¹¹ The liver is responsible for numerous essential functions. It plays a key role in fat metabolism by breaking down fats to produce energy and synthesizing important lipids. The liver also produces bile, a yellow-brown liquid crucial for the digestion and absorption of dietary fats. Each day, the liver generates approximately 800 to 1000 mL of bile, which is stored in small ducts before being transported to the main bile duct.¹⁵ This duct carries bile to the duodenum, a section of the small intestine, where it aids in the breakdown and absorption of fats. The liver is also the body’s primary metabolic organ. It processes nutrients from food, stores glucose as glycogen, and regulates blood sugar levels.¹⁵ The organ also helps maintain blood glucose levels by storing excess glucose as glycogen and releasing glucose into the blood when needed. This process ensures a constant supply of energy, particularly between meals and during fasting.¹⁶ The liver also converts other sugars and carbohydrates into glucose to be used as energy.¹⁶ In protein metabolism, the liver modifies amino acids from dietary proteins for energy production or conversion into carbohydrates or fats. This process produces ammonia, a toxic by-product, which the liver converts into urea. Urea is then transported to the kidneys for excretion in urine.¹⁵ The liver is crucial for detoxifying harmful substances, including alcohol, drugs, and metabolic by-products through chemical alteration of these substances to render them harmless or subjecting them to excretion.¹⁵

The liver’s complex anatomy and multifaceted functions (Figure 1D) underscore its importance in maintaining metabolic balance and overall health. From nutrient conversion and storage to detoxification and bile production, the liver is integral to numerous physiological processes.¹³ Understanding its structure and functions is essential for appreciating the liver’s vital role in sustaining life and health.^13,12

2.2. Pathologies of liver

The liver’s complex functions and exposure to numerous toxins make it susceptible to various diseases. Hepatitis is known to be inflammation of the liver, which can be caused by five viruses, alcohol abuse, medications or autoimmune diseases. The most common forms are viral hepatitis, which includes hepatitis A, B, C, D, and E.¹⁷ Autoimmune hepatitis is treated with immunosuppressive drugs. In extreme cases a liver transplant may be necessary.¹⁸ Another common pathology of the liver is cirrhosis, which is the result of long-term liver damage from chronic liver diseases such as hepatitis B and C, alcohol abuse, and non-alcoholic fatty liver disease (NAFLD).¹⁹ Cirrhosis involves the replacement of healthy liver tissue with scar tissue, impairing liver function. This scarring disrupts the liver’s architecture, reducing its ability to regenerate and perform vital functions such as detoxification, nutrient processing, and bile production. As the fibrotic tissue accumulates, it can lead to complications such as portal hypertension and liver failure. NAFLD is characterized by fat accumulation in the liver not caused by alcohol consumption. It is closely associated with obesity, type 2 diabetes, and metabolic syndrome. NAFLD can lead to significant liver damage as fat deposits within hepatocytes (liver cells) trigger a series of cellular stress responses. These responses include inflammation, oxidative stress, and fibrosis, where healthy liver tissue is replaced by scar tissue. This progression can advance to non-alcoholic steatohepatitis (NASH), a more severe form of NAFLD, which is marked by further inflammation, hepatocyte injury, and substantial fibrosis. If untreated, NASH can progress to cirrhosis and eventually liver failure.²⁰

Liver cancer can be primary, originating in the liver (e.g., hepatocellular carcinoma), or secondary, spreading from other organs.²¹ Risk factors include chronic hepatitis B and C, cirrhosis, and NAFLD. The damage in liver cancer is characterized by the uncontrolled proliferation of abnormal liver cells, which can lead to the formation of tumors that disrupt the normal architecture and function of the liver. As the cancer progresses, these tumors invade healthy liver tissue, causing further damage by impairing the liver’s ability to perform vital functions like detoxification, metabolism, and bile production. Additionally, liver cancer often leads to increased fibrosis and can further exacerbate pre-existing cirrhosis.

2.3. Promise of liver bioengineering

Recent advances in liver tissue engineering offer promising alternatives and complements to traditional liver transplantation, which faces significant challenges due to the scarcity of donor organs. Several non-bioprinting techniques have shown considerable potential. Scaffold-based techniques, employing natural and synthetic polymers, create 3D structures that support cell growth and differentiation. For example, collagen and gelatin scaffolds can provide a favorable environment for hepatocyte function but often suffer from limited mechanical strength and challenges in achieving complex tissue structures.⁶ Decellularization is another prominent method where cells are removed from donor livers, leaving behind an ECM scaffold. This scaffold can then be repopulated with the recipient’s cells, reducing immune rejection risks.²² However, the process can be costly and labor-intensive, and achieving complete decellularization while preserving the matrix’s integrity remains challenging. Organoids and spheroids, which replicate the liver’s microarchitecture and functions, are useful for disease modeling and drug testing. They serve as building blocks for larger tissue constructs but often lack the complexity and vascularization needed for fully functional liver tissues.²³ Scaffold-based tissue engineering involves using synthetic or natural materials to create a scaffold that supports cell growth and tissue formation. These scaffolds can be designed to mimic the liver’s ECM and are often combined with cells to promote tissue regeneration.²⁴ Scaffolds can be customized to support specific cell types and tissue architectures, offering flexibility in designing liver tissue constructs. Materials such as collagen, alginate, and polyglycolic acid (PGA) are commonly used, each providing different benefits in terms of biocompatibility and mechanical properties.²⁵ Scaffolds can sometimes lack the complexity of natural ECM, which may affect the functionality and maturity of the engineered tissue. Additionally, scaffold degradation and integration with the host tissue can be challenging, potentially leading to issues with tissue stability and function.^24,25

Bioprinting represents a significant advancement in liver tissue engineering, offering several advantages over traditional methods. This technique allows for the precise deposition of cells, bioinks, and growth factors in specific spatial patterns, facilitating the creation of complex, multi-layered liver tissues. One of the primary benefits of bioprinting is its ability to generate vascularized structures, crucial for maintaining liver function and viability in vitro and in vivo. Additionally, bioprinting enables customization of tissue constructs to address individual patient needs, advancing personalized medicine.²⁶ Recent studies highlight the potential of bioprinting in liver research. Kang et al.²⁷ have demonstrated the successful bioprinting of multi-scaled hepatic lobules within a highly vascularized construct, mimicking the native liver’s architecture and function. Another study demonstrated a 3D bioprinting system capable of producing human-scale tissue constructs with structural integrity, representing a major advancement in creating functional liver tissues.²⁶ Deng et al.²⁸ demonstrated the potential of 3D-bioprinted livers for clinical applications in liver failure and as an alternative to actual organs used in transplantation. Another study explored the use of polycaprolactone (PCL)-based scaffolds for tissue regeneration, illustrating the capability of bioprinting to fabricate scaffolds that mimic liver architecture and support cell growth.²⁹ These studies underscore the promise of bioprinting in developing accurate models for drug testing and regenerative medicine, paving the way for future innovations in liver tissue engineering.^26,29,30

3. Bioprinting for liver bioengineering

3.1. Bioinks

3.1.1 Natural bioinks

Bioprinting, an advanced technology in regenerative medicine, holds significant potential for addressing the challenges of creating functional liver tissue constructs, offering hope as an alternative to traditional treatments like organ transplantation. Central to this technology is the bioink, a carefully engineered composition of cells, biomaterials, and biological cues designed to mimic the intricate ECM microenvironment of the liver. Biomaterials form the structural backbone of bioinks, providing the necessary mechanical support and biochemical cues for cell–matrix interactions. Natural biopolymers, owing to their inherent biocompatibility and structural similarity to native ECM components, have emerged as the preferred choice for liver tissue engineering. Gelatin methacrylate (GelMA), a chemically modified derivative of gelatin, has garnered significant attention due to its versatility in terms of mechanical property tuning and its ability to support hepatocyte viability and function.³¹ This modification allows GelMA to undergo ultraviolet (UV) crosslinking in the presence of photoinitiator, forming stable hydrogels with tunable properties, and subsequent development of cell-laden constructs via bioprinting. As with most hydrogels with high hydrophilicity, photopolymerized GelMA hydrogels support cell adhesion and proliferation, making them highly suitable for tissue engineering studies.^32,33 Moreover, the incorporation of bioactive molecules, such as growth factors or ECM-derived peptides, within the GelMA matrix has demonstrated enhanced hepatocyte functionality, including albumin secretion and urea synthesis.¹⁹

Alginate, a hydrophilic polysaccharide extracted from seaweed, is another widely employed biomaterial in liver bioprinting. Its excellent printability and gelation properties make it a suitable candidate for creating complex tissue architectures.³⁴ As they are derived from seaweed, they are naturally biocompatible and non-toxic, and considered safe for tissue engineering and other biomedical applications. The mechanical properties of alginate hydrogels can be easily adjusted by varying the concentration of alginate and the type and concentration of crosslinking ions. This tunability allows for the customization of the hydrogel’s stiffness and porosity to match the specific requirements of different tissue types or applications.³⁵ However, the relatively low bioactivity of alginate necessitates the addition of supplementary ECM components or growth factors to optimize hepatocyte performance.³⁶ Despite this limitation, alginate-based bioinks have shown promise in fabricating multi-layered liver tissue constructs.³⁶

Collagen is the most abundant protein in the human body and a major component of the liver ECM. It is widely used in liver tissue engineering due to its natural presence in the liver, promoting cell attachment, proliferation, and differentiation.³⁷ Collagen bioinks closely mimic the native ECM, making them ideal for creating liver tissue constructs. However, collagen is prone to rapid degradation and requires careful handling to maintain its structural integrity, often necessitating crosslinking or blending with other materials to enhance its mechanical properties.^38,39 Collagen scaffolds can be crosslinked using chemical agents (e.g., glutaraldehyde),⁴⁰ physical methods (e.g., UV irradiation),⁴¹ or enzymatic crosslinking (e.g., transglutaminase).⁴² Crosslinking enhances the mechanical properties and stability of the collagen scaffold, ensuring that its structure is maintained during tissue formation and in vivo applications. Fibrin, a fibrous protein involved in blood clotting, is used for its excellent cell compatibility and ability to form a highly porous network, promoting cell migration and vascularization.⁴³ Fibrin-based bioinks are particularly advantageous in liver tissue engineering for creating constructs that require rapid integration with host tissues. However, fibrin degrades quickly in vivo, which can be a limitation unless combined with other materials to prolong its stability.⁴³

Decellularized extracellular matrix (dECM) has emerged as a compelling bioink component due to its intrinsic biological cues that profoundly influence cellular behavior. By preserving the native ECM architecture and composition, dECM provides a more physiologically relevant microenvironment for hepatocytes, leading to enhanced cell function and matrix remodeling.⁴⁴ One recent study by Lee et al.⁴⁵ focused on developing a liver-specific dECM bioink for 3D bioprinting. By encapsulating hepatocytes within the dECM bioink, researchers were able to create liver tissue constructs that exhibited enhanced cell viability, proliferation, and liver-specific functions compared to traditional 2D culture. The dECM scaffold provided a biomimetic environment that supports cell adhesion, proliferation, and differentiation, successfully mimicking the native liver ECM.⁴⁵ However, the complex nature of dECM, including batch-to-batch variability and potential immunogenicity, presents challenges for its widespread application. To recapitulate the heterogeneous architecture and mechanical properties of the native liver, researchers have increasingly explored the use of multicomponent bioinks. Combining different biomaterials, these formulations aim to create a more physiologically relevant microenvironment for hepatocytes. For instance, the integration of GelMA with alginate or dECM has shown synergistic effects on liver tissue construct development, enhancing cell viability, proliferation, and metabolic function.^21,31

3.1.2 Synthetic bioinks

Synthetic bioinks are engineered materials designed to offer controlled mechanical properties, degradation rates, and consistent production quality. Unlike natural bioinks, synthetic bioinks are typically free from biological variability, making them highly reproducible. The most used synthetic bioinks in liver tissue engineering include polyethylene glycol (PEG), polylactic acid (PLA), and PCL.⁴⁶ PEG is a synthetic polymer widely used in various medical, pharmaceutical, and industrial applications due to its unique properties. PEG is frequently used in tissue engineering and regenerative medicine. Its biocompatibility and non-toxic nature make it an ideal candidate for creating hydrogels, which can serve as scaffolds for tissue growth or as delivery systems for cells and bioactive molecules.⁴⁶ PEG can be easily modified with bioactive molecules to enhance cell adhesion and function. In liver tissue engineering, PEG-based bioinks are often combined with natural materials like gelatin or collagen to create hybrid bioinks that offer both mechanical strength and biological activity. By virtue of its non-immunogenic nature, PEG is suitable for clinical applications, though modification is typically required to improve cell interactions.^38,39

PLA is an innovative biomaterial with significant potential in various medical and industrial applications. PLA stands out due to its biodegradability, biocompatibility, and versatility, making it a prominent choice for use in scaffolds, drug delivery systems, tissue engineering, and medical implants. PLA is a thermoplastic polymer derived from renewable resources such as corn starch or sugarcane, which contributes to its sustainability and environmental benefits.⁴⁷ Its biocompatibility and ability to degrade into non-toxic byproducts under physiological conditions are key factors that enhance its appeal in medical applications.⁴⁸ Recent studies showed the use of PLA in liver bioprinting where Mirdamadi et al.⁴⁹ combined HepG2 cells with PLA, which was 3D-printed and coated with gelatin to study the effect of different geometries on liver tissue function. Lee et al.⁵⁰ used a 3D-printed PLA platform, which was populated with 3D-bioprinted HepG2 cells in collagen/alginate bioink. This system was integrated with a glucose sensor made of carbon black PLA for glucose biosensing. The resulting liver-on-chip device showed high cell viability and physiologically relevant drug response.

PCL is a synthetic polymer widely recognized for its applications in tissue engineering and regenerative medicine. Characterized by its biodegradable nature and low melting point, PCL is particularly well-suited for use in 3D bioprinting. Its ability to be processed into complex structures makes it valuable for fabricating scaffolds that support tissue regeneration.⁵¹ PCL’s slow degradation rate compared to other biopolymers is advantageous for applications requiring extended support, such as in scaffold fabrication for pulp-dentin regeneration. Its mechanical properties and biocompatibility enable the creation of scaffolds that provide structural integrity and promote effective tissue repair.⁵² The polymer’s compatibility with 3D bioprinting technologies allows for the development of customized scaffolds that closely mimic natural tissue architecture, facilitating the growth and differentiation of cells essential for tissue regeneration.^51,52

While significant progress has been made in bioink development for liver tissue engineering, several challenges persist. Optimizing bioink printability, mechanical properties, and long-term stability while maintaining hepatocyte function remains a critical area of research. Additionally, the development of standardized evaluation methods to assess bioink performance and liver tissue construct quality is essential for clinical translation. Furthermore, there is a growing need to investigate the interplay between bioink composition, bioprinting parameters, and the resulting tissue microenvironment to achieve optimal outcomes.

3.2. Cell sources

The complex functions of the liver are a result of interactions between different liver cells, mainly described as hepatocytes and non-parenchymal cells. Different cell types, each with their unique advantages, can be used to represent hepatocyte function as shown in Table 1. Hepatocytes are responsible for the metabolism of endogenous and exogenous substances. In the development of liver models, primary human hepatocytes (PHHs) are frequently utilized due to their capability to execute a broad range of liver functions. These functions include protein synthesis, bile production, glucose and fatty acid metabolism, and the detoxification of both endogenous and exogenous substances.⁵³ Nguyen et al.⁵⁴ created an innovative bioprinted liver model utilizing inkjet printing technology. This model involved co-culturing primary hepatocytes, hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells to assess the toxicity of various clinical drugs. Histological examinations revealed a range of cell-to-cell interactions among hepatocytes, desmin-positive staining, networks of CD31+ endothelial cells, and quiescent stellate cells that did not express smooth muscle actin, effectively mimicking the dynamic drug metabolism at the organ level.

Table 1. Advantages and limitations of hepatocyte sources for liver bioengineering

Although PHHs showgreater resemblance to in vivo functionality of the liver, their long-term viability is low, limiting their applications. Cell lines such as HepG2s, Huh7, and HepaRG overcome this limitation. One study showed HepG2 cells when cultured with artificial cells, designed to mimic liver-like functions, can effectively enhance CYP1A2 enzyme activity within 3D-bioprinted structures. The successful proliferation of HepG2 cells within these constructs and the sustained enzyme activity highlight their role in advancing bottom-up tissue engineering and synthetic biology.⁵⁵ Huh7 cells were used in developing 3D liver constructs through bioprinting with gelatin bioink in different strut angles.⁵⁶ When the 90° and 60° angles were compared for construct printing, higher expression of CYP450 enzymes along with higher albumin secretion was observed with 60° angle constructs, showing importance of scaffold geometry. HepaRG cells, which can be differentiated to hepatocytes or bile duct-like cells through dimethyl sulfoxide (DMSO) induction, are also used widely in liver bioengineering as they show cellular polarity and high expression of drug transporters.⁵⁷ Yang et al.⁵⁸ demonstrated that HepaRG cells printed with alginate gained spheroid-like morphology and showed vascularization upon transplantation to mice model with liver injury, prolonging their survival.

Induced pluripotent stem cells (iPSCs) are another widely used cell source for liver bioengineering as they can give rise to patient-specific liver cells. Human iPSC-derived hepatic progenitor cells were bioprinted into hexagonal structures and tri-cultured with human umbilical cord vein endothelial cells (HUVECs) and adipose-derived stem cells where the 3D constructs showed improved morphological organization, higher liver-specific gene expression levels, as well as enhanced cytochrome P450 activity, demonstrating the suitability of iPSC-derived cells for liver bioengineering research.⁵⁹ Another study addressed the challenges of obtaining liver sinusoidal endothelial cells (LSECs) in sufficient quantities, which are crucial for liver development, regeneration, and disease processes. By using human iPSCs to generate LSEC-like cells, the research provides a valuable model for investigating the differentiation process and tissue-specific phenotype of LSECs.⁶⁰ Similar to LSECs, HSCs and Kupffer cells also play crucial roles in liver physiology and pathology. Incorporation of activated or inactive stellate cells or Kupffer cells improves the biomimicry of the developed bioprinted tissue models allowing study of liver diseases such as fibrosis and cirrhosis.³

3.3. Single-cell dispersion-based bioprinting

3.3.1. Extrusion-based bioprinting

Extrusion-based bioprinting is a revolutionary technique that leverages the precise deposition of biomaterials and cells to create complex 3D structures. This method involves forcing a bioink, consisting of a biomaterial and embedded cells, through a nozzle to form layers of a desired shape. The resulting structures have the potential to mimic the natural architecture and function of tissues and organs.⁶¹ Extrusion-based bioprinting is an accessible and cost-effective technology that avoids harmful energy sources, making it suitable for tissue and organ fabrication. However, it faces challenges regarding resolution and accuracy, which are critical for creating microstructures, as well as potential issues with cell viability due to shear stress during the extrusion process.⁶² This bioprinting technology can be classified into pneumatic (Figure 2A), piston-based (Figure 2B), and screw-based (Figure 2C) printing which are distinguished by their distinct mechanical components that facilitate extrusion.⁶³ The piston-based and screw-driven variations use a piston and rotating screw mechanisms, respectively, while pneumatic relies on the force from compressed air (Figure 2).

Cell-laden bioprinting represents a promising biofabrication strategy for creating bioactive transplants to alleviate organ donor shortages. Despite its potential, achieving transplantable artificial organs that incorporate multiple cell types and physiologically relevant architectures has been challenging. A recent study by Jiang et al.⁶⁴ introduces an omnidirectional printing embedded network (OPEN) as an advanced support medium for embedded 3D printing, characterized by its straightforward preparation, rapid removal, and compatibility with various inks. Using this innovative medium, researchers successfully printed hepatosphere-encapsulated artificial livers (HEALs) using primary mouse hepatocytes (PMHs) and endothelial cells. The PMHs self-organized into functional spheroids within the matrix, maintaining structural integrity and hepatic functions for over 10 days in culture. Notably, HEALs with endothelial structures facilitated endogenous neovascularization in vivo, demonstrating enhanced functionality compared to traditional 2D and 3D culture methods. This platform not only enhances the potential for bioactive tissue manufacturing but also holds significant implications for liver function replacement in clinical applications, highlighting the importance of sophisticated bioengineering techniques in disease modeling and regenerative medicine.⁶⁴

The functional performance of bioprinted constructs is often hampered by insufficient cell–cell interactions, necessitating the fabrication of tissue constructs with higher cell densities. A recent study introduced a novel photocrosslinkable GelMA-based bioink formulation designed to overcome challenges related to excess free radicals generated during the photoinitiation process, particularly in large constructs with high cell densities. The bioink combines GelMA with an antioxidant cocktail containing vitamin C (L-ascorbic acid) and vitamin E (α-tocopherol), effectively scavenging intracellular reactive oxygen species produced during photocrosslinking. In vitro and in vivo assessments confirmed the bioink’s biocompatibility and favorable rheological properties. Notably, constructs composed of high-density primary rat hepatocytes exhibited significantly enhanced cell–cell interactions and liver-specific functions, achieving 5-fold and 2.5-fold increases in albumin and urea secretion, respectively, compared to lower-density constructs. These findings highlight the potential of using antioxidant-enhanced bioinks in 3D liver bioprinting to improve the functional performance of liver models for biomedical applications.⁶⁶ Another very recent study presented a novel bioink formulation composed of gelatin and alginate blends (GA) designed for 3D-bioprinting hepatocyte-laden scaffolds, which serve as more physiologically relevant in vitro liver models for drug toxicity assays. In vitro liver models are essential for early drug development, particularly in assessing drug toxicity, with static cultures of liver-derived cell spheroids being the most common. However, these models often fail to replicate the dynamic conditions of the native liver, such as blood flow and glucose gradients, which are critical for studying drug-induced liver injury. The printability of various GA compositions was evaluated using a rotary rheometer, leading to the selection of suitable blends containing human liver carcinoma cells (HepG2) for 3D bioprinting. The scaffolds were stabilized through crosslinking, utilizing calcium chloride for alginate and microbial transglutaminase for gelatin. Cell viability and proliferation of HepG2 cells within the bioprinted scaffolds were assessed over a 28-day period. This innovative approach underscores the potential of dynamic in vitro liver models in drug development and toxicity assessment.⁶⁷

In order to improve the overall biomimicry of the tissue models, the use of decellularized matrix has gained attention in liver bioprinting as well. Deng et al.²⁸ fabricated a mechanically robust and biologically compatible scaffold using a bioink composed of liver decellularized matrix, gelatin, and sodium alginate through extrusion bioprinting. The bioprinted liver tissues exhibited substantial hepatic functionality, including glycogen storage, albumin production, drug metabolism, and CYP450 enzyme activity, outperforming conventional culture methods. When transplanted into tyrosinemia or hepatectomy-induced liver failure mice, the 3D-bioprinted livers restored critical liver functions. Transplanted tissues integrated with host blood vessels, facilitating molecule exchange and vascularization. The 3D-bioprinted livers significantly extended the lifespan of mice suffering from liver injuries, accompanied by enhanced serum markers and reduced native liver damage. The incorporation of artificial blood vessels within the 3D-bioprinted livers enabled efficient nutrient and waste diffusion, addressing challenges associated with vascular integration in tissue engineering.²⁸

An essential challenge in liver bioengineering is achieving effective vascularization. Using extrusion bioprinting, Massa et al.⁶⁸ employed a sacrificial bioprinting technique to create hollow microchannels within a 3D liver construct composed of HepG2/C3A cells encapsulated in GelMA hydrogel. HUVECs were seeded into the microchannels using a bioreactor, forming a vascularized construct with a uniformly coated endothelial layer. The endothelial layer delayed the permeability of biomolecules, offering a more controlled environment for studying drug transport and metabolism. In addition, the vascularized model showed increased cell viability compared to non-vascularized constructs, highlighting the protective role of the HUVEC layer.⁶⁸ In another study, Kang et al.²⁷ fabricated multiscale hepatic lobules within a highly vascularized construct. By using preset extrusion bioprinting, they engineered hepatic lobules, which are approximately 1 mm in size and include spatially organized hepatocytes (HepG2/C3A) and endothelial cells (EA. hy926). The lobules feature a central lumen with a diameter of 150 μm, surrounded by hepatocytes and endothelial cells arranged to mimic the native liver’s architecture. The endothelial cells formed an outer layer around the constructs and interconnected between the central lumen and the exterior, promoting vascularization. This spatial organization preserved structural integrity and improved cellular function, including enhanced albumin secretion, urea production, and cytochrome P450 enzyme activity.²⁷ These studies demonstrate the feasibility of creating complex, vascularized liver models for applications in tissue engineering, drug testing, and regenerative medicine. Yang et al.⁵⁸ bioprinted hepatorganoids, composed of HepaRG cells, with gelatin and alginate composite bioink using extrusion bioprinting. The printed constructs were then transplanted into the abdominal cavities of Fah-deficient Rag2-deficient (F/R) mice. The transplanted constructs showed human albumin production and drug metabolism and formed functional vascular networks. Importantly, a 14-day-long transplantation alleviated liver injury and significantly prolonged the survival of the mice, demonstrating the therapeutic potential of bioprinted liver tissues for regenerative medicine.⁵⁸

3.3.2. Inkjet bioprinting

Inkjet bioprinting is a sophisticated 3D printing technique that employs inkjet technology to deposit bioinks containing living cells in a controlled manner. It includes two main types: continuous and drop-on-demand inkjet printing, which enables the generation of picoliter-sized droplets for high-resolution and precise cell placement.¹ This method was pioneered by Prof. Thomas Boland in 2003, who demonstrated live cell printing using a PBS solution with Chinese hamster ovary cells and mouse embryonic motor neurons. Inkjet bioprinting has since been used to create complex biological structures, such as 3D culture systems for studying liver functions and integrating electrochemical sensors in microfluidic devices. Its non-contact and rapid droplet firing capabilities make it a valuable tool for applications in tissue engineering, drug development, and regenerative medicine.⁶⁹

Inkjet bioprinting offers several advantages, notably its low cost and ease of operation, making it accessible for many research laboratories. The technology’s ability to generate precise and high-resolution structures quickly through non-contact droplet deposition further enhances its appeal in biomedical applications. However, it also has notable disadvantages, primarily its limitation to low-viscosity bioinks, which restricts the types of materials that can be used. Additionally, the technique is typically only applicable to low-cell-density constructs, potentially compromising cell–cell interactions and functionality in more complex tissue engineering applications. This trade-off necessitates careful consideration of the desired outcomes when selecting inkjet bioprinting for specific bioprinting projects.⁷⁰ Further classified into thermal (Figure 3A) and piezoelectric (Figure 3B) processes,⁶³ the thermal or ``bubble’’ method works by rapidly heating a thin film resistor inside the printer. This creates a vapor bubble that expands and ejects a small droplet of bioink from a nozzle. The process is precise and non-contact, enabling controlled deposition of cells, biomaterials, or other bioactive components.⁷¹ With piezoelectric inkjet bioprinting, electrical signals cause piezoelectric materials in the printhead to deform mechanically, leading to volumetric changes in the ink chamber and the ejection of controlled droplets. The process is particularly valuable in tissue engineering and regenerative medicine due to its precision, scalability, and compatibility with living cells.^72,73

The advancements in 3D bioprinting techniques present a transformative opportunity for liver bioengineering and disease modeling by enabling the fabrication of functional tissues and organs. A recent study highlights a critical challenge in this field: cell sedimentation-induced aggregation during inkjet-based bioprinting. The investigation revealed that as printing time increased from 0 to 60 min, the percentage of cells forming aggregates at the bottom of the bioink reservoir rose significantly, from 3.6% to 54.5%. Notably, within just 15 min of printing, over 80% of the cells in the nozzle had aggregated, underscoring the urgency of addressing this issue for effective bioprinting. The study found that both individual cells and aggregates migrate towards the centerline of the nozzle, primarily due to the bioink’s weak shear-thinning properties. Post-printing analysis indicated a rise in the mean cell number per microsphere from 0.38 to 1.05 as printing time increased, with a substantial proportion of microspheres containing aggregates. These findings emphasize the necessity of optimizing bioink formulations and printing parameters to minimize cell aggregation, which is essential for enhancing the viability and functionality of bioprinted liver tissues. By addressing these challenges, the study contributes valuable insights that can facilitate the development of more effective in vitro liver models for drug testing and disease modeling.⁷⁵

The use of bioprinted tissues derived from human iPSCs has significant implications for understanding disease mechanisms and assessing toxicity, particularly in the context of liver bioengineering and disease modeling. Recent findings have highlighted concerns regarding the hepatotoxicity of microplastics found in liver tissues of cirrhotic patients, as these particles can absorb harmful endocrine disruptors like tetrabromobisphenol A (TBBPA). To explore the toxic mechanisms associated with these pollutants, researchers employed electro-assisted inkjet printing technology to create Disse space organoids that accurately mimic the cellular composition and transcriptional features of the liver’s microenvironment. The study revealed that microplastics accumulated in the organoids, with TBBPA exacerbating this effect. Importantly, while neither microplastics nor TBBPA alone impacted liver function in healthy donor-derived Disse space organoids, co-exposure notably altered the transcriptional and biochemical profiles of organoids derived from patients with alcoholic liver disease. These findings suggest that both genetic predispositions and environmental pollutants can influence susceptibility to liver toxicity, underscoring the value of human iPSC-derived organoids in environmental toxicology. This innovative approach paves the way for advancing personalized medicine and risk assessment in liver disease, offering a robust platform for investigating the interplay between genetic factors and environmental exposures.⁷⁷

3.3.3. Stereolithography and digital light processing

In 3D bioprinting and additive manufacturing applications, common stereolithographic techniques typically produce structures from photocuring and photopolymerization using a light source. In particular, 3D constructs formed from stereolithography are developed in layers as a result of photosensitive materials being exposed to UV irradiation from a laser.⁷⁶ This form of 3D printing has been devised and actualized by Charles W. Hull, who patented the first stereolithographic 3D printing technology.⁷⁸ In Figure 4, Hull’s system features a laser source (scanner) positioned above a vessel containing liquid resin or other curable polymer, an adjustable stage which can be adjusted along the z-axis, and a computer to control the configuration of the stage (z-axis moving) and laser scanner. Additionally, the process of emerging solid structures from a vat of liquid is traced to the work of Tumbleston et al.,⁷⁹ where a more time-efficient process for 3D model preparation in a liquid interface is proposed. In the field of bioprinting, this process can be applied and is commonly referred to as stereolithography apparatus (SLA)-based bioprinting. This technique is highly effective and superior in creating complex, high-resolution, and architecturally sophisticated biostructures. The spot size of the laser in SLA can be reduced, resulting in the formation of higher-resolution constructs, generally up to 10 µm.⁸⁰ SLA bioprinting relies on photosensitive bioinks, typically composed of cells encapsulated in a hydrogel matrix that can be crosslinked upon exposure to light.⁴⁰ Following bioink preparation, a digital 3D model of the desired tissue structure can be created using computer-assisted design (CAD) software. This model can then be sliced into thin layers (Figure 5B), which would be sequentially cured during the printing process⁸¹ (Figure 5D).

With reference to the Hull model (Figure 4), the SLA bioprinter also utilizes a light source, often a UV laser, to selectively trace and cure each layer of the bioink. The light solidifies the bioink in predefined areas according to the sliced model layer images.⁷⁹ After undergoing initial photopolymerization, the printed structure may require additional crosslinking to enhance its mechanical properties and stability. This may involve further exposure to light or chemical crosslinkers.⁸² The bioprinted structure is incubated under controlled conditions to allow cell growth, differentiation, and tissue maturation (typically growth medium and appropriate supplementary factors). Functional testing, including histological, biochemical, and mechanical assessments, is performed to evaluate the quality and functionality of the bioprinted tissue.⁸³ Highly comparable to SLA bioprinting techniques, digital light processing (DLP)-based bioprinting in stereolithography expands on the foundation of SLA by introducing a projection of cross-sectional layers (Figure 5A). DLP bioprinters typically involve a digital micromirror device (DMD), a device containing thousands of microscale mirrors which function to project light to form the image of each layer (Figure 5D). The main advantage offered by DLP technology over traditional SLA printing is the speed at which layers are cured, completing entire layers simultaneously. Additionally, due to the fine resolution of the projected images, DLP also produces highly detailed parts with smooth surfaces.⁸⁰

DLP technology is particularly suitable for bioprinting due to its precision and ability to work with photosensitive hydrogels. Like SLA, DLP technology can be employed to create complex tissue scaffolds, microfluidic devices, and detailed anatomical models.⁸⁴ SLA technology has proven to be a promising tool in biomedical research and tissue fabrication applications. Sphabmixay et al.⁸⁵ developed a mesoscale microfluidic physiological system (MePS) using high-resolution stereolithography techniques for liver disease modeling. The engineered tissue was fabricated from a composite photopolymerizable resin scaffold and punched into a perfusion bioreactor for hepatocyte cell culture. Scaffolds supported cell function for 7 days. The resulting system achieved biomimicry of native tissue, characterized by albumin and urea secretions, and increased CYP3A4 enzyme activity. In comparison to polystyrene scaffolds, these scaffolds experience a huge reduction in inflammatory cytokine production rates, which were quantified under the MePS condition. Likewise, Anandakrishnan et al.⁸⁶ utilized a fast hydrogel stereolithography printing method (FLOAT) to create solid perfused (embedded vessel networks) or non-perfused hydrogels for HepG2 cell-laden culture. Photopolymerization conditions and hydrogel flow velocity parameters were fine-tuned to optimize structural integrity. Culture medium perfusion was maintained for up to 6 days, and perfused models showed an approximately 80% cell viability. Based on the ELISA analysis, 5.2 µg albumin per million cells were quantified in perfused models, almost three times the secretion in non-perfused models.

3.4. Spheroid dispersion-based bioprinting

Spheroids are 3D aggregates of cells which self-assemble under non-adherent conditions. They are commonly produced by hanging drop (Figure 6A) and liquid overlay (Figure 6B)^87,88 fabrication techniques. Their self-assembling properties capacitate the advanced technique of spheroid-based bioprinting to be employed in tissue engineering studies. Prepared spheroids are suspended in a bioink, often a biocompatible material (hydrogel-based) which can support cell proliferation, adhesion, and functionality. The bioink acts as a medium, delivering and positioning spheroids with controlled accuracy to create coherent tissue structures.⁸⁹ With spheroid dispersion-based bioprinting, the bioink containing spheroids needs to be dispensed through a bioprinter to create the desired 3D structure. This process may involve either direct deposition or encapsulation of spheroids within a supportive matrix.⁹⁰ After printing, the spheroids undergo a fusion process where they merge to form a continuous tissue structure. This process can be facilitated by placing the construct in a bioreactor, which has been shown to provide optimal conditions for tissue maturation, including appropriate mechanical, electrical, and biochemical stimuli.^91,92 Spheroid dispersion-based bioprinting offers several advantages over traditional tissue engineering and bioprinting methods, primarily due to the use of multicellular spheroids as building blocks. Benefits include enhanced cell–cell and cell–matrix interactions,⁸⁸ improved vascularization via spheroid prevascularization,⁹³ and high cell density necessary for certain types of tissues (e.g., liver).⁹⁴ The utility of the spheroid dispersion technique makes it a promising tool for the development of a wide variety of tissues.

3.4.1. Scaffold-based bioprinting with spheroids

As a result of their 3D nature, spheroid aggregates can mimic the microenvironment of tissues more accurately than 2D cell cultures. With the inclusion of scaffolds, the structural support enables and guides the organization and growth of cells within bioprinted constructs.^88,95 Gaskell et al.⁹⁶ devised a method to create 3D spheroid liver models using C3A hepatoma cells. Spheroid structures remained stable and viable after 32 days, exhibiting native liver tissue properties. The models were stained with CPS1, a marker of zonation for periportal liver regions due to oxygen and nutrient gradients, and shared temporal and spatial similarities in expression compared to native tissue. Polarization properties were also confirmed from expression of functional MRP2 and Pgp transporters. Spheroids displayed higher sensitivity to hepatotoxins like acetaminophen compared to 2D cultures, demonstrating their potential for improved drug toxicity screening models.

Hydrogels are widely used in spheroid bioprinting due to their high water retention properties, which closely resemble those in natural tissues. They possess tunable mechanical properties, making them both biocompatible and biodegradable constructs. Hydrogels are ubiquitously used for encapsulation of cells and bioactive molecules, promoting cell proliferation and tissue regeneration. Taymour et al.⁹⁷ utilized an alginate-methylcellulose hydrogel bioink (algMC) to develop 3D liver tissue in vitro models via pneumatic extrusion bioprinting. Matrigel, a protein mixture of ECM molecules, was also incorporated to enhance cell proliferation and morphology. A core–shell bioprinting technique was applied and enabled printing multiple cell types in a spatially defined manner. Fibroblasts were co-cultured with hepatocytes in a core–shell structure. The core contained NIH 3T3 fibroblasts, while the shell contained HepG2 cells, encapsulated in algMC bioinks. Cell-laden bioinks were crosslinked with calcium chloride to stabilize the constructs. Monophasic scaffolds were also fabricated for comparison. Hepatocytes showed high viability throughout the culture period, forming larger and more numerous clusters in bioinks supplemented with Matrigel compared to algMC alone. The fibroblasts in the core supported the hepatocytes’ viability and function. Cell proliferation was higher in constructs containing Matrigel. The number of hepatocytes increased significantly in Matrigel-supplemented algMC over time, with larger and more organized cell clusters. HepG2 cells in both core–shell and monophasic scaffolds secreted albumin, indicating liver-like functionality. The presence of fibroblasts in the core-enhanced albumin secretion, where low secretion was shown in monoculture scaffolds. Furthermore, the core– shell structure allowed indirect communication between the two cell types through diffusion of signaling molecules, resulting in highly enhanced hepatocyte function and biomarker expression. Fibroblasts encapsulated in a fibrin or plasma-supplemented core formed interconnected networks, which positively influenced the neighboring hepatocytes’ functionality.

Through the digestion of decellularized porcine liver (dECM), Kim et al.⁹⁸ presented a method that enabled the formation of hepatocyte spheroids using dECM-based bioink. The process involved printing a PMHs-laden bioink (with or without digested dECM) into a hydrogel matrix ink consisting of alginate, hyaluronic acid (HA), and gelatin, which created circular cavities for development. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed 4.3- and 2.5-times greater albumin and urea secretion, respectively, and double cytochrome P450 (CYP) enzyme activity. Models with dECM-incorporated hepatocyte spheroids also showed up to 1.8 times higher responsiveness to hepatotoxic drugs, revealing its potential for an effective drug toxicity assessment model. Furthermore, a recent study from Wu et al.⁹⁹ featured a composite hydrogel bioink containing GelMA (5%), alginate (1%), and 3% cellulose nanocrystal (135ACG), and standalone GelMA was used to fabricate different variations of biomimetic liver lobule honeycomb lattices for structural ECM. Bioprinted variations (S1–S4) included an acellular/cell-laden approach of either the honeycomb or middle lattice, or a combination of them, with HepG2 or NIH/3T3 encapsulation. Fibroblasts were shown to follow durotaxis, aligning themselves along 135ACG-GelMA boundaries, whereas HepG2 cells developed into spheroids solely in the softer standalone GelMA matrix. In both direct contact and non-contact NIH/3T3 + HepG2 cocultures, increased albumin secretion was shown compared to HepG2-only cultures. However, 3D HepG2 cultures exhibited increased albumin production compared to 2D cultures. The true- to-scale variations of the liver lobule-mimetic lattices presented in this study demonstrate the potential of bicellular bioprinted constructs in tissue engineering and biomedical research.

Goulart et al.¹⁰⁰ explored the effect of bioprinting single cell dispersions versus spheroids of human iPSC-derived hepatocyte-like cells on liver functionality. They printed 3D structures shaped like donuts using alginate along with differentiated hepatocyte-like cells either as single cells or in spheroids form. In both cases, the cells or spheroids were accompanied by endothelial cells and mesenchymal cells to improve biomimicry and overall functionality of the constructs. They observed that after 18 days of culture, the constructs with bioprinted spheroids showed significantly higher cell viability, albumin and urea secretion, and CYP P450 enzyme activity. In addition, metabolomics analysis revealed reduced hepatic metabolic activity in single-cell-printed models, which was supported by the increased fibrosis markers such as type IV collagen in single-cell-printed models, pointing out to poor long-term functionality of these constructs.

3.4.2. Scaffold-free bioprinting with spheroids

Without the dependence on scaffold biomaterials, scaffold-free bioprinting is an advanced technique in tissue engineering which instead relies on cellular properties to form functional, 3D structures. In other studies, this technique is not extensively reviewed. In some cases, certain scaffolds may exhibit cytotoxic properties or elicit immune responses and reduce cell–cell interaction.¹⁰¹ Alternatively, this approach can be particularly useful for developing tissues which closely mimic the natural microenvironment of cells, promoting increased functionality and integration within host tissue. Furthermore, spheroids without the scaffold integration can produce their own ECM to provide structural support. In scaffold-free bioprinting, most bioinks are composed of solely spheroids rather than having the guiding support of hydrogels. In the Kenzan method, bioinks are printed in a layer-by-layer fashion onto an array of needles¹⁰² to expand the desired structure (Figure 7),¹⁰³ allowing for the creation of functional tissues without the need for scaffolding materials. In the domain of bioprinting, this novel method is typically overlooked in literature review. Yet, of all possible biofabrication processes, it is known to produce 3D tissue constructs with the highest resolution. As the machine distributes the spheroids, they are impaled by the needle array. After the tissue has matured and sufficient ECM has been produced to maintain the structure, the needle array is carefully removed, leaving the high-resolution construct intact.^104,105 This process takes place in a sterile environment.

4. Bioprinting for liver disease modeling: preclinical models

4.1. Hepatocellular carcinoma models

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the fifth most common cancer worldwide.¹⁰⁶ HCC has been shown to be resistive to systemic chemotherapy¹⁰⁷ and surgical resection is only suitable for 10–15% of the cases.¹⁰⁸ As an extremely heterogeneous tumor, understanding the complex tumor microenvironment (TME) of HCC remains one of the main challenges. Thus, recapitulating the TME is a crucial aspect of exploring new diagnostic, preventive, and personalized medicine approaches for HCC. Although 2D models have widely been used for studying HCC, the recent studies show the importance of the TME in mimicking the pathology of tumors in vitro. The TME consists of an altered, vascularized ECM as well as non-malignant cell types. Therefore, it is deemed critical to develop models of HCC where the ECM is precisely organized, which can be achieved using the versatile applications of bioprinting.¹⁰⁹

Sun et al.¹¹⁰ used extrusion printing to construct 3D hepatocarcinoma model using HepG2 cells and alginate/gelatin gels as the bioink for antitumor drug testing. The layer-by-layer assembled model was crosslinked with calcium chloride, and the structure was stable through the 10-day culture period. Cells in the printed constructs maintained the viability over 90% and showed a higher growth rate compared to 2D cultures. Transcriptome analysis of the 3D-printed models showed significantly higher expression of liver function-related genes, including albumin and CYP P450s as well as a differential expression of cancer-related genes compared to the 2D cultures. Importantly, the printed models were treated with antitumor drugs cisplatin, sorafenib, or regorafenib, and the IC₅₀ for all the three drugs were found to be higher compared to the 2D models, showing the importance of the 3D microenvironment in tumor models replicating the pharmacodynamic conditions in vivo. In another study, Mao et al.¹¹¹ used 3D printing to develop a tumor model of intrahepatic cholangiocarcinoma where primary tumor cells isolated from a 62-year-old male patient, along with gelatin/alginate/Matrigel composite hydrogel, were used as bioink. The extrusion printed models gave rise to the formation of microspheres within the 7-day-long culture period and showed higher expression of tumor markers such as carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) compared to 2D cultures of the same cells. In addition, the metastatic potential of the cells in 3D constructs was found to be higher compared to 2D cultures as higher expression of matrix metalloproteinase (MMP)-2 and MMP9 were detected. Interestingly, the constructs also showed a higher drug resistance when treated with sorafenib, cisplatin, or 5-fluorouracil. These results outlined the importance of personalized tissue models in finding the correct anti-cancer treatment and the important role of developing a controlled 3D tumor microenvironment. In a similar study, 3D patient-derived hepatocellular carcinoma models were printed using gelatin/alginate and the cells isolated from six patients.¹¹² The models were shown to successfully retain the specific biomarkers of HCC, while maintaining stable genetic alterations and expression profiles similar to those of the original tumors. When the resulting tumors were subcutaneously transplanted to mice after 3 weeks of 3D culture in vitro, tumorigenic potential and histological features were observed to be well retained. The efficacy of targeted drugs sorafenib, lenvatinib, regorafenib, and apatinib was also tested on the printed patient-specific HCC models and showed clear differences among the patients.

In a recent study, the use of GelMA and poly(ethylene oxide) (PEO) composite hydrogels were shown to be printed successfully with SMMC-7721 hepatocarcinoma cell line to develop 3D HCC models.¹¹³ The printed tumor models were crosslinked using UV irradiation and when compared to 2D cultures or 3D cell encapsulation models, a significantly improved cell proliferation and tumor formation-related protein expression was observed. In addition, when transplanted into nude mice, the cells isolated from the printed models showed higher tumorigenicity showing suitability of the bioink for evaluating anti-cancer drug testing platforms. The same group improved on their model in another study by introducing an external hydrogel with stromal cells of the TME that surrounds the printed hepatoma model.¹¹⁴ A GelMA-gelatin hydrogel along with SMMC-7721 cells was used to print the hepatoma model, which was then surrounded with the second GelMA-gelatin hydrogel containing human fibroblasts and HUVECs. The 3D tri-culture model demonstrated superior tumor-related characteristics, such as higher cell proliferation, tumorigenicity, drug resistance, and gene expression compared to the control model that lacks the external TME. The model was used to assess the efficacy of anticancer drugs like doxorubicin and cisplatin, showing higher resistance in the 3D tri-culture model, which better reflects clinical drug resistance.

4.2. Fibrosis models

Chronic liver stress and disease, when untreated, leads to accumulation of ECM in the form of scar tissue, resulting in fibrosis.¹¹⁵ Early diagnosis and prevention of liver diseases such as cirrhosis requires an in-depth understanding and therapeutic approaches against liver fibrosis. The lack of availability of robust and representative in vitro models for studying liver fibrosis is one of the most eminent roadblocks in developing efficient therapies.¹¹⁶ Since the altered state of cells and the ECM are the defining characteristics of a fibrotic liver tissue, research has focused on replicating these conditions. Cuvellier et al.¹¹⁷ developed a bioprinted 3D co-culture of HepaRG, LX-2, and HUVECs in GelMA as a fibrotic model. They observed that when treated with transforming growth factor beta 1 (TGF-β1), a promoter of fibroblastic/migratory phenotype, the printed co-culture models had induced COL1A1 gene expression and pro-collagen 1a1 secretion, a major marker of liver fibrosis. In addition, a significant decrease in hepatic function was also observed, mimicking the fibrotic state, confirming the importance of replicating the complex cell–cell and cell–ECM interactions to obtain a relevant pathological liver fibrosis model. In order to recapitulate the complex composition of the native microenvironment of the liver tissue, Ma et al.¹¹⁸ developed 3D-printed liver constructs using decellularized porcine liver ECM mixed with GelMA. They used HepG2 cells in the constructs and modified the mechanical properties to model the different stiffness liver tissue gains through fibrosis and cirrhosis. The stiff gels, matching the stiffness of cirrhotic liver, were found to decrease viability and function of HepG2 cells in in vitro culture over a 7-day period. This study emphasized the crucial role of mimicking not only the cellular components of a tissue, but also the environmental factors when developing a disease model.

Norona et al.¹¹⁹ developed a 3D-printed a triple co-culture liver tissue to model compound-induced fibrogenesis in vitro. They used a model with primary hepatocytes, HSCs, and endothelial cells to mimic the complex cell–cell interactions in liver fibrosis. Through repeated exposure to methotrexate, thioacetamide, and TGF-β1, they successfully induced liver injury and fibrosis, as evidenced by hepatocellular damage, collagen deposition, and cytokine release, which are hallmarks of fibrotic processes. The induction of different cytokines over different time points of their treatment was observed. Proinflammatory cytokines were found to express during early exposure, whereas an increase in chemotactic cytokines, such as MCP-1, was observed at later time points. This approach offers valuable insights into liver fibrosis mechanisms and enhances the potential for drug screening and toxicity testing. Another triple co-culture liver fibrosis model was developed by Lee et al.¹²⁰ A multi-layer “fibrosis-on-a-chip” structure that mimics the native organization of hepatocytes, stellate cells, and endothelial cells was 3D-printed. The researchers achieved this complex organization by printing differentiated HepaRG cells in the decellularized liver matrix at the bottom, and activated LX2 stellate cells in gelatin in the middle layer, and HUVECs in gelatin in the top layer. Before and after the printing of cellular layers, polyethylene vinyl acetate (PEVA) layers were printed to enclose the chip structure. The multilayer construct was incubated at 37 °C to remove gelatin bioinks, giving rise to a structure more closely mimicking the native organization in the liver. They observed that constructs with activated stellate cells had increased expression of fibrosis markers such as type I collagen, vimentin, and desmin, as well as experienced fibrosis-associated functional deterioration marked by a decrease in albumin and urea secretion. The drug response of the fibrosis-on-a-chip model was analyzed, which determined that a combination treatment of retinol and palmitic acid may be a promising therapy, highlighting the importance of mimicking the native structure of the liver for successful drug testing.

5. Challenges and future directions

Advancements in 3D bioprinting in liver bioengineering made the development of more complex and physiologically relevant models of the liver possible for tissue engineering and regenerative medicine, disease modeling, and drug testing applications. Although these models provide a critical platform for advancing our knowledge of the liver pathologies and related treatments, there still remain significant challenges in replicating the liver tissue in vitro. One important challenge is achieving the spatial arrangement and intercellular interactions that are crucial for liver function and mimicking liver pathologies. The precise arrangement of the distinct cell populations of the liver, including hepatocytes, endothelial cells, Kupffer cells, stellate cells, and bile duct cells, is crucial for successful mimicry of liver function. An important consideration that lacks in many studies is achieving the polarization of hepatocytes, and their proper spatial organization with endothelial cells to form functional liver sinusoids. Although more studies involve the culture of two or more cell types in the liver constructs, a comprehensive model that includes all the cell types found in the liver, as well as ones that replicate the interactions with immune cells does not exist.

Another central challenge is the lack of models with sufficient vascularization and perfusion providing physiological resemblance. The viability and proper function of the liver heavily rely on the efficient transport of oxygen and nutrients through both large vessels and capillary-like microvessels. Although direct printing of vascularized models as well as ones that incorporate sacrificial materials have been shown, these models remain limited in size and long-term perfusability. Another limiting factor in the bioprinting technology is the speed and resolution of bioprinters not suitable for printing of delicate microstructure of the liver tissues. The highly specialized structures such as the hepatic lobule and bile canaliculi are essential; however, commonly used techniques, particularly those based on extrusion and inkjet printing, fail to reach the required fine resolution. To enhance the complexity and functionality of liver constructs, innovations in bioprinting technologies, such as multi-material printing and dynamic printing strategies, may improve outcomes.

Although a few studies, as explained above, incorporate two or more cell types in their liver models, lack of all cell types found in the liver and maintaining long-term cell viability and functionality over time poses challenges, particularly for primary cells which often lose their phenotype in long-term culture. For this reason and for their ability to be used in personalized medicine, iPSC-derived hepatic cells have gained attraction in liver bioengineering. Despite the many advantages of iPSCs, the resulting cells require lengthy procedures to mature and yet underperform the essential functions of the liver cells. Through further advances in the development of iPSC-derived hepatic parenchymal and nonparenchymal cells, the lack of patient diversity in drug testing can be addressed.

Integrating microfluidic systems with bioprinting technology is gaining focus, which could lead to better transport of oxygen and nutrients, providing long-term maintenance of the tissues. Another aspect gaining attention in the recent years is the incorporation of the decellularized liver matrix to bioprinting applications. This not only enhances the biomimicry of the liver models, but also helps with improved long-term viability and functionality of the cells. Through a combination of improved innovation and complexity of the models, as well as the use of more versatile and patient-specific cells, 3D-bioprinted liver tissues will become important tools in liver disease research and drug testing.

6. Conclusions

In light of the continuous escalation of liver disease incidence globally, innovative solutions are essential to address the limitations of current therapeutic approaches. Liver bioengineering through bioprinting holds tremendous potential as it offers a promising avenue for creating complex, functional liver tissues that closely mimic the native organ’s architecture and function. The current focus in the use of bioprinted models of the liver is on improving the complexity of the developed tissues through incorporating non-parenchymal cells and native ECM of the liver for studying drug response and metabolism as well as pathologies like hepatocarcinoma and fibrosis. Through future efforts to incorporate patient-specific cells, co-cultures of non-parenchymal liver cells, and targeting complex organization of cellular and acellular components similar to the native tissue architecture, bioprinting-based liver bioengineering will present exciting prospects for personalized in vitro models for drug testing.