Applications of Bioprinting on Pathological Liver Models

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This entry is adapted from the peer-reviewed paper 10.3390/molecules28093683

Significant progress has been made in liver tissue engineering through the use of 3D bioprinting technology. This technology offers the ability to create personalized biological structures with precise geometric design capabilities. The complex and multifaceted nature of liver diseases underscores the need for advanced technologies to accurately mimic the physiological and mechanical characteristics, as well as organ-level functions, of liver tissue in vitro. Bioprinting stands out as a superior option over traditional two-dimensional cell culture models and animal models due to its stronger biomimetic advantages.

3D bioprinting bioink Pathological Liver Models

1. Introduction

The World Health Organization (WHO) reports that liver diseases, including the hepatitis B and C viruses (HBV and HCV), cirrhosis, liver cancer, fatty liver, and drug-induced liver injury, affect 1.5 billion individuals globally ^[1]. Unfortunately, liver disease causes over 2 million deaths annually, which is steadily increasing ^[2]. A recent publication in the International Journal of Epidemiology predicts that without intervention, the incidence and death rate of liver cancer in China will continue to rise until 2030, with the most significant increase seen in steatohepatitis-related liver cancer ^[3].

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 ^[4]. The resulting 3D structures can be customized to meet individual needs. Since patient cells are used, there is a low risk of rejection, making these structures suitable for direct implantation into the human body ^[5]. With the growing popularity of personalized patient treatment and precision medicine, the potential benefits of 3D bioprinting are becoming increasingly apparent ^[6]. Bioprinting offers significant biomimetic advantages in simulating the physiological characteristics and functions of liver tissue within the body ^[7]. This technology can provide new ideas and methods for exploring the biological characteristics and disease mechanisms of liver tissue and for the treatment, prevention, and drug screening of liver diseases.

2. Three-Dimensional Bioprinting Technology

The introduction of 3D bioprinting as a highly innovative manufacturing technique addresses the limitations of conventional manufacturing methods, making it one of the most thrilling and pioneering fields of the 21st century, especially in the field of biomedical research ^[8]. Regenerating and reconstructing damaged tissues and organs through organ and tissue regeneration techniques can serve to replace, restore, maintain, or enhance their functions ^[9]. The success of organ tissue engineering hinges on the ability to recreate three-dimensional structures that closely mimic human organs ^[10]. The utilization of 3D bioprinting technology enables the creation of organ tissue scaffolds with precise spatial geometry, microstructure, and mechanical properties. These characteristics are crucial in the fabrication of functional tissue constructs, making 3D bioprinting a promising tool in tissue engineering and regenerative medicine ^[11]^[12]. Depending on the specific application, various bioprinting technologies can be employed to achieve the desired results. As a complex and large organ, the liver requires a combination of bioprinting methods for its successful reproduction. Extrusion-based 3D bioprinting, for example, is highly flexible and efficient for printing liver lobules. However, for the intricate dual circulatory system of the liver, high-resolution, high-precision light-assisted 3D bioprinting might be preferred. Standard bioprinting methods include extrusion-based 3D bioprinting, inkjet-based 3D bioprinting, and light-assisted 3D bioprinting ^[13]^[14]^[15]. These methods can be used individually or in combination to build organ tissue through additive fabrication.

3. Liver Tissue Printing Materials

3.1. Bioink

Bioink refers to a solution of biomaterials that is loaded with cells, which is essential for the ex vivo development of organ tissues in current 3D bioprinting technology. Bioink not only needs to help fix cells in the right place to form microstructures similar to those in the human body, supporting cell adhesion and proliferation ^[16]^[17], but also needs to support the physical demands of the printing process to withstand mechanical and thermal stresses during printing. Bioinks can be formulated using both natural and synthetic polymers. Natural polymers derived from natural sources are highly desirable, as they best mimic the characteristics of natural tissues. They possess similar properties as the extracellular matrix structure and exhibit good biocompatibility, biodegradability, and low cytotoxicity, and they support stable cell growth and proliferation; therefore, they are extensively utilized in bioprinting applications. Gelatin, alginate, collagen, decellularized liver extracellular matrix, hyaluronic acid, chitosan, and several other materials ^[18] are natural polymers that are widely applied in fields such as organ transplantation and regeneration. Commonly used synthetic polymers in 3D bioprinting include poly(ethylene glycol) diacrylate (PEGDA) ^[19], polyethylene glycol (PEG) ^[20], polycaprolactone (PCL) ^[21], polyvinylpyrrolidone (PVP) ^[22], and polyhydroxybutyrate (PHB) ^[23]. During printing, synthetic polymers can be tailored to provide essential biochemical and rheological properties and mechanical support and performance as softer materials and complex structures by adjusting their physical and chemical properties accordingly. Overall, natural polymers have inferior mechanical properties and cannot sustain structural integrity in the body for prolonged periods, leading to low cell support and a high risk of collapse ^[24]. Synthetic polymers may have higher cytotoxicity, lack natural cell attachment sites, and fail to mimic the properties of natural tissues accurately, which can negatively impact cell survival and proliferation ^[25].

Therefore, a single type of bioink is insufficient to meet the demands of complex structures. Natural and synthetic polymers are often combined during printing to obtain structures with high stability, resolution, fidelity, and biocompatibility ^[20].

3.2. Cell Sources

The complex signaling and metabolic environment in the liver are formed by the interaction of autocrine and paracrine signals among its cells ^[26], which drive the various activities of the liver. The following provides an overview of the different cell types and their functions in the liver.

4. Bioprinted Liver Tissue Models

In recent years, there has been a surge in the creation of liver tissue models using bioprinting technology. This research summarizes the various models that are currently available.

4.1. Liver Tissue Models

4.1.1. Scaffold-Based Models

Significant advances have been made in bioprinting livers ^[27], making it possible to produce functional liver tissues and mini-liver organs for drug research and liver tissue engineering. Yang et al. ^[28] used a 3D bioprinter to create in vitro liver tissue models with high structural integrity and high expression of liver-specific proteins and receptors, such as albumin, MPR2, CYP enzyme family, and glycogen, demonstrating the biological functions that the printed liver tissue should possess. These transplanted liver tissues have been shown to reduce liver injury and increase survival in mice with liver failure. Arai et al. ^[29] used the bionic hydrogel structure, which provides an enclosed 3D microenvironment with improved hepatocyte adhesion. It may be employed efficiently to create liver tissues for the long-term evaluation of cellular responses and drug analysis.

The absence of a circulatory system has hindered the accurate replication of the liver in traditional tissue models. To address this challenge, creating a complex, perfusable vascular network is crucial for building realistic liver organ structures. Kang et al. ^[30] utilized low concentrations of GelMA and fibrin as bioink to print vascularized tissues with high cell viability and excellent resolution. The resulting macroscopic channels, endothelialized for bioprinting, allow for the creation of multiscale, vascularized liver tissues that can meet the centimeter-scale nutritional and oxygen demands of the liver. Rania et al. ^[31] aimed to create an in vitro vascularized liver tissue that mimics the hepatic sinusoidal-like structure, using a coaxial extrusion-based bioprinting approach. This approach allowed for the study of cellular interactions between hepatocyte clusters and tubule-forming endothelial cells, leading to the development of a functional hepatic sinusoidal-like model.

4.1.2. Scaffold-Free Models

Scaffold-free 3D culturing is another bioprinting technique that holds great promise for liver tissue research. One such method involves the formation of hepatocyte spheroids, aggregates with a diameter of 100–200 μm from individual hepatocytes ^[32]. These spheroids have been found to maintain their liver-specific functions and improve the hepatocyte survival time compared to traditional 2D cultures, making them a valuable in vitro model for liver metabolism and cytotoxicity research. The most commonly used method for creating these spheroid aggregates is the suspension drop technique ^[33], which involves dropping hydrogels containing functioning cells into a culture dish and allowing them to aggregate by gravity. This process creates spheroids of a predetermined size that can be maintained in culture for extended periods. Studies have shown that these spheroid aggregates resemble in vivo liver tissue at the proteome level and exhibit significant glycogen storage and albumin production, as well as robust expression of necessary metabolic and excretory enzymes, for up to two weeks ^[34]. Furthermore, researchers such as Elise et al. ^[35] have used droplets of hydrogel to construct primary human liver microtissues that can be cultivated for over nine days, and their vitality can be measured by relative adenosine triphosphate (ATP) concentration. Liver microspheres created through scaffold-free 3D culturing techniques have proven to be effective for hepatotoxicity research. Despite being in culture for up to nine days, these microspheres have shown good cell viability, with high levels of proteomics and metabolomics. One other method of generating these spheroids is using cell rejection plates containing magnetic nanoparticles on the surface, which impede cell attachment and use magnetic force to form the spheroids quickly. This technique is suitable for hepatotoxicity research ^[36]^[37]. Another approach, as demonstrated by Tostoes et al. ^[38], involves using an automated perfusion bioreactor to cultivate human hepatocyte spheroids that can be maintained for over four weeks, while retaining high levels of liver-specific markers, such as albumin, and CYP enzyme expression, as well as cell viability. This makes them suitable for repeated-dose drug toxicity testing over long periods.

The construction and design of spheroids using either the hanging drop or cell exclusion plate method are closely tied to the desired functionality and purpose of cell cultures in vitro. Of particular note is the superior capability of scaffold-free hydrogel spheroids produced through 3D bioprinting technology when compared to traditional 2D cultures. These spheroids can regulate the spatial distribution of critical hepatocytes and decellularized extracellular matrix factors, replicate in vivo physiochemical microenvironments, facilitate the long-term culture of hepatocytes with physiologically relevant molecular phenotypes, and sustain liver-like functions similar to those found in vivo, all of which make them ideal for disease research and drug testing applications.

4.2. Disease Models

Modeling disease liver tissue is crucial in comprehending the underlying pathological mechanisms of liver diseases and evaluating the potential of drugs. The existence of hepatocellular carcinoma and liver fibrosis can gravely jeopardize human health; therefore, the establishment of a robust in vitro model is imperative for conducting in-depth research on the pathophysiology of liver diseases. These models can be used to assess the toxicity of different drug doses, simulate the disease state of the human liver in a laboratory setting for comprehensive drug screenings, improve the efficiency of pre-drug testing, and accurately predict clinical outcomes. This research examines the current models for liver cancer, liver fibrosis, and toxicity testing.

4.2.1. Liver Cancer Models

Liver cancer is a primary global health concern that poses a significant threat to individuals. As per the 2021 statistics from the World Health Organization (WHO), it is the third most common cause of cancer-related deaths globally. Every year, approximately one million people are diagnosed with liver cancer worldwide ^[39]. Hepatocellular carcinoma is usually associated with liver cirrhosis, resulting from prolonged liver damage that may cause significant impairment of liver function ^[1]. Several factors, including alcohol-related hepatitis, viral hepatitis, and other liver disorders, can increase the likelihood of developing liver cancer ^[40]; hence, developing a reliable in vitro liver model is crucial for conducting extensive research on carcinogenesis and providing personalized treatment options. Mao et al. ^[41] used a composite hydrogel system made of gelatin-sodium alginate-matrix gel to print patient-derived primary tumor cells in a three-dimensional format. After a few days of culture, tumor markers, stem cell markers, fibrosis indices, and liver-specific protein expressions were measured. The tumor-related bioactivities and pharmacodynamic parameters were found to be more similar to those observed in patients compared to the results obtained from two-dimensional control cultures. This 3D-printed model confirmed the primary tumor cells’ robust proliferation properties and invasive metastatic capacity and accurately replicated the original tumor’s critical biological and genetic features. Therefore, the in vitro liver cancer model provides a valuable tool for studying surrogate carcinogenesis and evaluating anti-cancer treatment resistance.

4.2.2. Fatty Liver Model

The incidence of non-alcoholic fatty liver disease (NAFLD), the primary cause of metabolic liver disease leading to liver failure, has been on the rise in recent decades. To better understand the pathophysiology of NAFLD and its progression, it is crucial to develop reliable in vitro models for drug discovery and liver pathophysiology research ^[42]. To that end, a few microfluidic models have been created to study NAFLD, such as the one by Du et al. ^[43], who co-cultured HepG2 and HUVECS cells in gelatin spheres and transferred them to microfluidic chips. This model was then tested by exposing the cells to FFA-containing palmitic and oleic acids to stimulate the development of NAFLD and by measuring key metabolic parameters, such as albumin and urea secretion, CYP enzyme activity, and lipid droplet growth. Despite the promising results from these studies, there is still room for design and modeling improvement, given the small size of the liver chip ^[44] and the complex fabrication procedures required for each microstructure. Soon, it is expected that advances in 3D printing technology will lead to the creation of a 3D-printed fatty liver model.

4.2.3. Liver Fibrosis Model

Various factors, such as viral hepatitis, fatty liver, and excessive alcohol consumption, can cause liver fibrosis. Despite its widespread prevalence and debilitating effects, there are no effective treatments for fibrosis, and current treatments mainly focus on managing symptoms ^[45]. Bioprinted liver fibrosis models have the potential to provide a platform for studying the disease progression and tissue restoration. Sofia et al. ^[46] co-cultured HepRG and HSC in 3D spheres in 96-well plates for 21 days. After 14 days of exposure to the pro-fibrotic chemicals propanolol and methotrexate, the liver models displayed fibrotic characteristics, including HSC activation, collagen secretion, and collagen deposition, demonstrating that the response to liver fibrosis involves multiple cells within the organ, not just single cell cultures. In their study, Marie et al. ^[47] utilized a 3D-bioprinting-assisted photocuring method to embed HepaRG, LX-2, and HUVECs in GelMA and co-cultured them. They observed that liver cells in the co-culture system secreted TGFβ-1, which triggered the expression of ACTA2 and COL1A1 in LX-2 cells and subsequently led to the accumulation of fibrotic collagen. Furthermore, LX-2 cells exhibited a dormant state initially in the 3D culture model, and only after being treated with TGFβ-1 did they become activated. Until now, although researchers have managed to create the cell line successfully, the maximum survival duration of the current models is limited to just 14 days, which is inadequate for investigating the chronic toxicity of drugs. However, Marie et al. ^[47] have developed a model that can be maintained for up to four weeks, making it an excellent tool for drug testing and genetic toxicity studies.

4.2.4. Model of Drug-Induced Liver Damage

Before a new medication can be released to the market, it must undergo trial phases to assess its potential toxicity and predict the human response accurately. However, the existing model systems for toxicity prediction can be unreliable due to significant species differences, particularly in the specific expression of liver-related enzyme genes ^[48]. In contrast, bioprinted liver organoid models have demonstrated stable differentiation and proliferation for up to two weeks, with high cell viability and liver marker expression comparable to human liver cells ^[49]. When exposed to an acetaminophen environment, the bioprinted liver-like organ model showed a decreased cell viability of 21–51% (p < 0.05) and elevated levels of the damage marker miR-122 in the culture medium, suggesting its potential as a substitute for human organs in toxicity testing ^[50]. Hence, the bioprinted liver-like organ model’s ability to predict toxicity and its standardization in creation makes it a promising candidate for toxicity testing.

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