The liver, the largest organ in the human body, is a multi-functional organ with diverse metabolic activities that plays a critical role in maintaining the body and sustaining life. Although the liver has excellent regenerative and recuperative properties, damages caused by chronic liver diseases or viral infection may lead to permanent loss of liver functions. Studies of liver disease mechanism have focused on drug screening and liver tissue engineering techniques, including strategies based on in vitro models. However, conventional liver models are plagued by a number of limitations, which have motivated the development of 'liver-on-a-chip' and microplatform-based bioreactors that can provide well-defined microenvironments. Microtechnology is a promising tool for liver tissue engineering and liver system development, as it can mimic the complex in vivo microenvironment and microlevel ultrastructure, by using a small number of human cells under two-dimensional (2D) and three-dimensional (3D) culture conditions. These systems provided by microtechnology allow improved liver-specific functions and can be expanded to encompass diverse 3D culture methods, which are critical for the maintenance of liver functions and recapitulation of the features of the native liver. In this review, we provide an overview of microtechnologies that have been used for liver studies, describe biomimetic technologies for constructing microscale 2D and 3D liver models as well as liver-on-a-chip systems and microscale bioreactors, and introduce applications of liver microtechnology and future trends in the field.

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Alcohol is one of the main causes of liver diseases, and the development of alcoholic liver disease (ALD) treatment methods has been one of the hottest issues. For this purpose, development of in vitro models mimicking the in vivo physiology is one of the critical requirements, and they help to determine the disease mechanisms and to discover the treatment method. Herein, a three-dimensional (3D) ALD model was developed and its superior features in mimicking the in vivo condition were demonstrated. A spheroid-based microfluidic chip was employed for the development of the 3D in vitro model of ALD progression. We co-cultured rat primary hepatocytes and hepatic stellate cells (HSCs) in a fluidic chip to investigate the role of HSCs in the recovery of liver with ALD. An interstitial level of flow derived by an osmotic pump was applied to the chip to provide in vivo mimicking of fluid activity. Using this in vitro tool, we were able to observe structural changes and decreased hepatic functions with the increase in ethanol concentration. The recovery process of liver injured by alcohol was observed by providing fresh culture medium to the damaged 3D liver tissue for few days. A reversibly- and irreversibly-injured ALD model was established. The proposed model can not only be used for the research of alcoholic disease mechanism, but also has the potential for use in studies of hepatotoxicity and drug screening applications.

In situ embedding of sensitive materials (e.g., cells and proteins) in silk fibers without damage presents a significant challenge due to the lack of mild and efficient methods. Here, we report the development of a microfluidic chip-based method for preparation of meter-long silk fibroin (SF) hydrogel fibers by mimicking the silkworm-spinning process. For the spinning of SF fibers, alginate was used as a sericin-like material to induce SF phase separation and entrap liquid SFs, making it possible to shape the outline of SF-based fibers under mild physicochemical conditions. L929 fibroblasts were encapsulated in the fibric hydrogel and displayed excellent viability. Cell-laden SF fibric hydrogels prepared using our method offer a new type of SF-based biomedical device with potential utility in biomedicine.

High-aspect ratio micro- and nano-structures have been used for the production of a variety of applications. In this paper, we describe a simple and cost-effective approach to fabricate an arrayed microarchitecture with an ultra-high aspect ratio using soft materials. The shapes and sizes of the honeycomb structure can be easily modulated by changing the dimensions and position of the base mould pattern and the pressure. The honeycomb structure is used to prepare a drug delivery patch and a microwell array to form cell spheroids without cell loss. The honeycomb structures prepared using natural ECM (collagen–Matrigel) materials are successfully fabricated. The hepatocytes and endothelial cells are seeded and co-cultured in the ECM-based micro-honeycomb to prepare a 3D liver model successfully mimicking an ultrastructure of liver and providing enhanced liver function.

The early and accurate prediction of the hepatotoxicity of new drug targets during nonclinical drug development is important to avoid postmarketing drug withdrawals and late-stage failures. We previously established long-term expandable and functional human-induced pluripotent stem cell (iPSC)-derived liver organoids as an alternative source for primary human hepatocytes. However, PSC-derived organoids are known to present immature fetal characteristics. Here, we treated these liver organoids with microbial short-chain fatty acids (SCFAs) to improve metabolic maturation based on microenvironmental changes in the liver during postnatal development. The effects of the three main SCFA components (acetate, propionate, and butyrate) and their mixture on liver organoids were determined. Propionate (1 µM) significantly promoted the CYP3A4/CYP3A7 expression ratio, and acetate (1 µM), propionate (1 µM), and butyrate (1 µM) combination treatment, compared to no treatment (control), substantially increased CYP3A4 activity and albumin secretion, as well as gene expression. More importantly, mixed SCFA treatment accurately revealed troglitazone-induced hepatotoxicity, which was redeemed on a potent CYP3A4 inhibitor ketoconazole treatment. Overall, we determined, for the first time, that SCFA mixture treatment might contribute to the accurate evaluation of the CYP3A4-dependent drug toxicity by improving metabolic activation, including CYP3A4 expression, of liver organoids.

There is an increasing interest in organ-level 3D tissue constructs, owing to their mirroring of in vivo-like features. This has resulted in a wide range of preclinical applications to obtain cell-or tissue-specific responses. Additionally, the development and improvement of sophisticated technologies, such as organoid generation, microfluidics, hydrogel engineering, and 3D printing, have enhanced 3D tissue constructs to become more elaborate. In particular, recent studies have focused on including complex compartments, i.e., vascular and innervation structured 3D tissue constructs, which mimic the nature of the human body in that all tissues/organs are interconnected and physiological phenomena are mediated through vascular and neural systems. Here, the strategies are categorized according to the number of dimensions (0D, 1D, 2D, and 3D) of the starting materials for scaling up, and novel approaches to introduce increased complexity in 3D tissue constructs are highlighted. Recent advances in preclinical applications are also investigated to gain insight into the future direction of 3D tissue construct research. Overcoming the challenges in improving organ-level functional 3D tissue constructs both in vitro and in vivo will ultimately become a life-saving tool in the biomedical field.

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