Exhibition at the MPS World Summit 2024

Abstract details: Chronic liver diseases present a global health challenge with limited treatments due to unclear mechanisms. Current liver disease research relies on animal models, but species differences limit their significance. Here, we have developed an "open liver organoid" model incorporating chimeric PXB hepatocytes, human induced pluripotent stem cells (hiPSC)-derived hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs), which are the three key types of liver cells involved in liver disease progression [1, 2]. 

To simulate the human liver structure, these cells were cultured in hierarchical layers using an oxygen-permeable-bottom plate for efficient oxygen transfer. hiPSC-derived HSCs were embedded into a thin collagen gel to preserve their quiescence [3]. Compared to prevalent 2D liver models, our "open liver organoid" better captures liver tissue heterogeneity by incorporating three distinct cell types. In relative to spheroids, the “open organoid” structure of our model ensures uniform substance diffusion and facilitates streamlined analysis without the need for structural breakdown. 

Our results demonstrated the superior effectiveness of our oxygen-permeable-bottom plate in culturing three types of liver cells in thick layers while preserving their functions. In contrast, conventional tissue culture plates exhibited difficulty in maintaining cell functions under similar conditions. Additionally, albumin secretion in our model was found to be higher and more stable compared to hepatocyte sandwich culture over 14 days. Finally, steatosis was successfully reproduced in the model with excessive glucose. Our model exhibited significantly greater lipid accumulation compared to hepatocyte sandwich culture, emphasizing the key role of cell-cell interactions in studying liver diseases. In summary, our model shows promise for studying chronic liver diseases and developing targeted therapeutic interventions. The "open liver organoid" structure, with a more uniform transfer of nutrients, also holds potential for future integration with perfusion systems.

Abstract details: To perform perfusion culture on Microphysiological Systems, it is usually necessary to allow cells to settle from the time after cell seeding. However, human hepatocytes are known to have a high oxygen demand. Therefore, in the microscopic space on the chip, the oxygen and nutrients in the culture medium may be depleted before the cell attachment. PXB-Shizuku medium[1] can maintain the three-dimensional cell morphology characteristic of hepatocytes even at low cell densities, thus enabling the maintenance of the activity of drug-metabolizing enzymes. We considered that the use of PXB-Shizuku medium could maintain hepatic function even when the number of cells in the chamber was reduced to the point where oxygen supply was sufficient. Therefore, we examined whether the morphology and function of cells could be maintained by seeding hepatocytes in Chip-Shop reaction chambers at low density and culturing them in PXB-Shizuku medium. Hepatocytes were planted in chips at a cell density of 30% of the optimal cell density. After four hours of incubation in a humidified atmosphere of 5% CO2 and 95% air at 37℃, connect pumps to the chambers and PXB-Shizuku was delivered at 0.5 µL/min for 72 hours. The results showed that despite the lower-than-normal seeding density, the cells were not elongated and maintained the paving stone shape characteristic of hepatocytes. Overall, the results suggested that PXB-Shizuku medium can be used to culture hepatocytes with MPS devices. We are currently investigating drug metabolism activity and the ability to form capillary bile ducts and will report on these findings as well.

Abstract details: Background: In vitro-cultured human hepatocytes (HH) serves as a crucial study platform for enhancing our understanding of liver biology, pathophysiology, and drug metabolism and toxicity. However, the rapid quality deterioration, which is thought to be attributed to cellular stress associated with the procurement processes and the inadequacy of in vitro environment, impedes the versatility.  The objective of this study is to establish a novel culture approach to enhance the utility of the two-dimensional HH culture system.

Methods: HH, either cryopreserved primary HH (PHH) or HH isolated from humanized liver chimeric mice (HLCM-HH), were first evaluated the viability and the platability to determine an optimal cell seeding density. Then, HH plated on collagen coated plate at the optimal confluency were subjected to a screening of culture medium supplements necessary for the prevention of the quality deterioration, cell fate recovery, and functional maintenance.

Results: HLCM-HH demonstrates superior platability, defined as the percentage of viable cells adhering to the culture dish, compared to PHH. The optimal seeding confluency ranges from 90-130%, accounting for both platability and the average dimensions of HH. The screening identified insulin, dexamethasone, vitamin C, and DMSO as crucial, with DMSO being most critical. DMSO supplementation promotes the restoration of cellular polarity, which was lost during cell isolation, along with the expression of hepatocyte marker genes. The restoration process takes 5-7 days; thereafter HH exhibit morphology and marker gene expression comparable to those observed in human liver tissue. Nevertheless, DMSO strongly inhibits multiple enzymes indispensable for liver function, including those involved in xenobiotics and alcohol metabolism, while its withdrawal results in a rapid cell fate deterioration. Our screening identified DMSO2 as a substitute for DMSO, supporting fate maintenance while relieving HH from functional inhibitions. For instance, with long-term ethanol treatment, HH cultured with DMSO2-supplemented medium, but not DMSO, exhibit lipid accumulation and expression of CYP2E1, representing an in vitro mimicry of alcohol fatty liver.

Conclusion: The implementation of the stepwise culture configuration with DMSO and DMSO2 facilitates cell fate recovery and functional restoration, thereby significantly improving the versatility of HH for a broad spectrum of biomedical research activities.

Abstract details: An in vitro culture system consisting of hepatocytes and macrophages is a simple and important screening system for immune-mediated drug-induced liver injury (IDILI). However, current studies that utilize hepatic cell lines[1] and primary human hepatocytes[2] still need improvements in terms of in vivo relevant drug metabolism, donor-variability and rapid degradation during the culture. Therefore, optimization of the culture system, including meeting a high oxygen demand of hepatocytes[3] and contact characteristics of both cell types. In this study, high functional and reproducible human chimeric mouse hepatocytes (PXB-cells®) were cultured on oxygen-permeable polymethylpentene (PMP) plates in different culture formats, including a relay culture in which hepatocyte-conditioned medium with drugs was transferred to THP-1 macrophages and a coculture using a culture insert. 

 Compared to PXB cells cultured under normal culture conditions using a tissue culture polystyrene (TCPS) plate, PMP-cultured cells showed upregulation of CYP3A4 expression (4.2-folds). When hepatocyte-conditioned medium consisting of IDILI drugs; amiodarone (3 µM) and nevirapine (100, 200 µM) was transferred to THP-1 macrophages, only macrophages treated with conditioned medium from PMP-cultured hepatocytes showed highly increased IL-1b production, but not TNF-a production, suggesting that activation of inflammasome is highly related to inflammation. Also, there was no increase in inflammatory cytokines when the drugs were cultured with THP-1 macrophages alone, suggesting that drug metabolism by hepatocytes is a major driving force for inflammation. On the other hand, the coculture using a culture insert showed no increase in inflammatory cytokines when the same drugs were administered. 

 Further study will be conducted by evaluating damage-associated molecular patterns (DAMPs) produced by hepatocytes and their receptors on macrophages to compare the reproduction of the underlying mechanism of IDILI on both culture types, and an optimized culture format will be proposed based on the physiological reproducibility and usability of the culture system. 

Abstract details: Microphysiological systems (MPSs) including organ-on-a-chip (OoC) have attracted attention as a novel drug evaluation method in drug discovery. We focused on the operability of MPS and developed a kinetic-pump integrated microfluidic plate (KIM-Plate) as a platform device of MPS.

KIM-Plate was developed for commercialization in collaboration with Sumitomo Bakelite, a Japanese manufacturing company. The KIM-Plate has a simple structure consisting of open-type 24-well size cell culture chambers connected by microchannels (K. Shinha et al., Micromachines, 2021). The greatest advantage of the KIM-Plate was that users perform perfusion culture and coculture like conventional culture plates. In addition, differentiation and functional evaluation could be performed for each organ model by using commercially available culture inserts and cell desks. Therefore, the effects of coculture and perfusion could be evaluated in detail using highly conditioned organ models. The culture medium was perfused by rotating the stirrer bar with a magnetic stirrer motor of the kinetic-pump. Therefore, the KIM-Plate was an effortless operation for perfusion, as it only needed to be installed on a stirrer base with a stirrer motor. We found that the coculture of liver and intestine models with the KIM-Plate increased the metabolic function of the liver model. In addition, we confirmed that the drug effect on cancer cells was higher in perfusion culture using the KIM-Plate than in static culture using conventional well plates. Therefore, we focused on these effects and aimed to develop a hepatic MPS based on the KIM-Plate that can detect hepatotoxicity, a serious drug development problem, with high sensitivity in vitro.

We performed repeated dose tests of acetaminophen using chimeric mouse-derived human hepatocytes (PXB-cells) as a liver model. Hepatotoxicity induced by acetaminophen was detected at lower concentrations (more similar concentration to blood concentrations at drug administration) in perfusion culture using the KIM-Plate than in conventional well plate culture. This result suggested that the hepatic MPS could be a powerful evaluation tool for hepatotoxicity. In the future, we will evaluate the usefulness of the KIM-Plate as an evaluation tool for hepatotoxicity in vitro by studying the hepatotoxicity of various drugs.