Review
Recent Progress in Hepatocyte Culture Models and Their Application to the Assessment of Drug Metabolism, Transport, and Toxicity in Drug Discovery: The Value of Tissue Engineering for the Successful Development of a Microphysiological System

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Abstract

Tissue engineering technology has provided many useful culture models. This article reviews the merits of this technology in a hepatocyte culture system and describes the applications of the sandwich-cultured hepatocyte model in drug discovery. In addition, we also review recent investigations of the utility of the 3-dimensional bioprinted human liver tissue model and spheroid model. Finally, we present the future direction and developmental challenges of a hepatocyte culture model for the successful establishment of a microphysiological system, represented as an organ-on-a-chip and even as a human-on-a-chip. A merit of advanced culture models is their potential use for detecting hepatotoxicity through repeated exposure to chemicals as they allow long-term culture while maintaining hepatocyte functionality. As a future direction, such advanced hepatocyte culture systems can be connected to other tissue models for evaluating tissue-to-tissue interaction beyond cell-to-cell interaction. This combination of culture models could represent parts of the human body in a microphysiological system.

Introduction

The ultimate goal of preclinical research in drug discovery is to gain better predictive insights into drug metabolism and pharmacokinetics (DMPK), safety, and efficacy of new chemical and biological entities to enable successful clinical trials and delivery to patients. The use of human biological materials such as microsomes and hepatocytes, already described in the 1990s,1 has a long history in predicting drug disposition in humans. This methodology is based on a fundamental understanding of species differences in the DMPK profile of molecules.2 These efforts are believed to have contributed to a reduction of the attrition rate of phase-I studies in the period from 1991 to 2000 caused by issues in pharmacokinetics or bioavailability.3 The next obstacle to overcome in clinical trials is improving the attrition rates of phase-II studies caused by safety or lack of efficacy.4 Experience in the DMPK research field suggests that proactive use of human materials is a suitable option for improving clinical predictability from the viewpoint of toxicology and pharmacology.

The liver is the central organ for drug metabolism and plays a key role in the outcome of clinical pharmacokinetics, such as through the first-pass effect, hepatic clearance, and drug-drug interaction. A number of studies have used primary hepatocytes to predict these phenomena, preferably in suspension. For example, successful prediction of hepatic clearance of medicines that underwent metabolism has been reported.5, 6, 7 In addition, hepatocyte suspensions enabled the prediction of hepatic clearance in which the rate-limiting process was sinusoidal uptake.8, 9 However, there are some drawbacks of this experimental system. For example, they are known to rapidly lose their functionality,10, 11 so experiments that examine long-term exposure to chemicals cannot be performed. Disruption of cell polarity and tight junctions in the process of preparation of hepatocyte suspensions limits the evaluation of biliary excretion of chemicals. Taken together, development of hepatocyte culture systems that can maintain their physiological conditions for an extended period is the next expectation.

Tissue engineering is a growing research field and has provided useful culture systems. Sandwich-cultured hepatocyte (SCH) is a commonly used cell-based system developed in the 1990s.12, 13 Spheroid culture is a unique system that allows cells to be organized in a 3-dimensional (3D) manner.14, 15 An understanding of the current applications of cultured hepatocytes in drug discovery, in addition to future expectations of more advanced engineered culture models, is important to accelerating both the invention of new culture models and their rapid implementation in the pharmaceutical industry. Indeed, many pharmaceutical companies have been exploring these new technologies for implementation in drug discovery.16

In the present review, we summarize tissue engineering technologies used to improve liver functionality in culture. Next, we describe applications of the SCH system to drug discovery and then emphasize the importance of the interplay of metabolism and vectorial transport of medicines from the industrial viewpoint for elucidating their disposition in the body. In addition, we highlight the 3D-bioprinted liver tissue model and cocultured spheroid model as new culture models that permit long-term culture. Finally, we summarize future perspectives for the successful development of a microphysiological system represented as an organ-on-a-chip and human (or body)-on-a-chip. This review will cover the current challenges of these inventions as well as our expectations for their use in drug discovery to improve clinical predictability of DMPK profiles, safety, and efficacy of new drug candidates.

Section snippets

Categorization of Tissue Engineering Technologies for Hepatocyte Culture

Maintenance of the physiological conditions of cells and tissues in an in vitro environment is believed to be the critical factor for the development of a promising culture model. The use of extracellular matrix such as type-I collagen and extracts from Engelbreth-Holm-Swarm tumor in mice17 is a popular approach. A recent study reported that the stiffness of the culture surface also affects cellular functionality. You et al.18 reported that albumin secretion from primary rat hepatocytes is

Application of Sandwich-Cultured Hepatocyte Models; Biliary Excretion

The value of the SCH model is emphasized by bile canaliculi formation in the culture. The system appears to be widely recognized in the pharmaceutical industry as a number of applications for drug discovery have been reported (Table 2). This is presumably because the SCH system was considered to be a better tool for investigating the biliary excretion of compounds that are part of the drug disposition pathway. For example, Liu et al.12 reported the potential of the model in evaluating the

Application of Sandwich-Cultured Hepatocyte Models; Overall Hepatic Disposition

Hepatic disposition consists of sinusoidal uptake, metabolism, canalicular efflux, and sinusoidal efflux. Given that bile canaliculi formation has been shown in SCH, the system allows access to each component of hepatic disposition. Some research has focused on the interplay of these components, suggesting expansion of the applications of the SCH model. Lee at al.58 investigated the hepatic disposition of troglitazone using SCH and demonstrated biliary excretion of its sulfate and glucuronide.

Application of Sandwich-Cultured Hepatocyte Models; Hepatotoxicity

Further usage of the SCH model extends to the toxicology field, especially the evaluation of cholestasis, which is believed to be linked to impairment of bile acid flow. Based on preserved transporter function of bile acids such as sodium taurocholate cotransporting polypeptide and bile salt export pump in SCH, assays evaluating a series of flow of bile acids have been reported.97 Assays using SCH help clarify the effect of coincubated compounds on the sinusoidal uptake and biliary excretion of

Exploration of Better Applications of Advanced Culture Models

An appropriate assessment of hepatotoxicity that may be caused by new molecular entities is undoubtedly important as surveillance has highlighted hepatotoxicity as one of the major reasons for withdrawal of drugs from the market.103 Because this hepatotoxicity is not limited to cholestasis but includes a wide range of hepatic dysfunction, a number of approaches have been postulated to appropriately assess the hepatotoxic potential of compounds for reducing the risk during compound selection at

Benefits and Challenges of Advanced Hepatocyte Culture Models

The key aspects of advanced hepatocyte culture systems reviewed in this article, as well as the microphysiological system, are illustrated in Figure 3. The SCH system has a clear advantage in bile canaliculi formation, but drug-metabolizing enzyme activity may not be fully reflective of the in vivo situation,113 and the system is improved by combining with the other tissue engineering technologies listed in Table 1. The 3D-HLT model allows repeated exposure of chemicals, and this ability is

Further Investigation; Foundation for Successful Development of a Microphysiological System

Although the present review has focused on improved hepatocyte function in advanced culture systems, some culture systems have other purposes for investigating the function of nonparenchymal cells if such cells are incorporated into culture models. The 3D-HLT model is reported to respond to fibrotic agents such as methotrexate and thioacetamide, resulting in the progression of fibrosis, characterized by collagen deposition and elevation of ACTA2 and COL1A1 genes.109 Another case is the

Practical Considerations for the Use of Advanced Culture Models in Drug Discovery

Although expectations of the utility of advanced cell culture models in drug discovery are great, there are some key considerations to their adoption by the pharmaceutical industry. First, a basic characterization of new culture models is very important for their appropriate implementation in any situation in drug discovery. In the case of luminal breast epithelial cells, the inside-out cellular polarity is observed when they are in 3D culture in collagen gels. On the other hand, the cells

Conclusion

Tissue engineering technology provides many benefits to the traditional ways of cell culture and leads to advanced culture models of the liver rather than hepatocytes. The SCH system is a well-recognized model that benefits from extracellular matrices and enables the achievement of bile canaliculi formation in vitro. The application of SCH models is expanding from evaluation of the biliary excretion of chemicals to a more integrated readout, such as the interplay of metabolism and transport,

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