A Kinetic Pump Integrated Microfluidic Plate (KIM-Plate) with High Usability for Cell Culture-Based Multiorgan Microphysiological Systems.

Microphysiological systems (MPSs), including organ-on-a-chip (OoC), have attracted attention as a novel method for estimating the effects and side effects of drugs in drug discovery. To reproduce the dynamic in vivo environment, previous MPSs were connected to pump systems to perfuse culture medium. Therefore, most MPSs are not user-friendly and have poor throughput. We aimed to develop a kinetic pump integrated microfluidic plate (KIM-Plate) by applying the stirrer-based micropump to an open access culture plate to improve the usability of MPSs. The KIM-Plate integrates six multiorgan MPS (MO-MPS) units and meets the ANSI/SBS microplate standards. We evaluated the perfusion function of the kinetic pump and found that the KIM-Plate had sufficient agitation effect. Coculture experiments with PXB cells and hiPS intestinal cells showed that the TEER of hiPS intestinal cells and gene expression levels related to the metabolism of PXB cells were increased. Hence, the KIM-Plate is an innovative tool for the easy coculture of highly conditioned cells that is expected to facilitate cell-based assays in the fields of drug discovery and biology because of its usability and high throughput nature.

Selective elimination of tumorigenic hepatic stem cells using hybrid liposomes.

To avoid the risk of tumorigenesis after cell transplantation, tumorigenic stem cells should be selectively eliminated from induced pluripotent cells, embryonic stem cells, and somatic stem cells. We previously reported the presence of tumorigenic stem cells in human fetal hepatocyte-induced hepatoblasts after sodium butyrate (SB) treatment. In this study, we aimed to investigate the selective elimination of tumorigenic stem cells in human hepatoblasts using hybrid liposomes (HLs) prepared by sonicating a mixture of 90 mol% l-α-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene (n) dodecyl ether (C12 (EO)n, n = 23) in a buffer solution. Flow cytometric analysis revealed that the number of hepatoblasts increased by around 12-18 times in SB-treated cells compared to non-treated cells. In the colony formation assay, colonies of tumorigenic stem cells were observed in a soft agar plate after SB treatment. HL treatment for 48 h resulted in a remarkable decrease in the number of colonies. HLs also induced apoptosis of tumorigenic stem cells by activating caspase-3. Flow cytometry showed a significant accumulation of HLs, including fluorescent lipids, in tumorigenic hepatic stem cells. The reappearance of tumorigenic stem cells was suppressed even in subsequent subcultures of HL-treated cells. High CYP3A4 activity was observed in a three-dimensional in vitro assay. These results suggest that HL treatment could specifically eliminate tumorigenic hepatic stem cells. Incubation with HLs can be an effective culture method to maintain the quality of stem cells and reduce the risk of tumorigenesis after cell transplantation.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

High density culture of pancreatic islet-like 3D tissue organized in oxygen-permeable porous scaffolds with external oxygen supply.

Transplantation of macroencapsulated pancreatic islets within semipermeable membranes is a promising approach for the treatment of type 1 diabetes. Encapsulation beneficially isolates the implants from the host immune system. Deleteriously however, it also limits oxygen supply to the cells. This creates challenges in loading islets at the amount and density required to meet the practical demands of clinical usage. To overcome this challenge, we investigated the feasibility of using macroporous scaffolds made of an oxygen-permeable polymer, poly(dimethylsiloxane) (PDMS) by culturing pancreatic islet-like three-dimensional tissue made of a rat pancreatic beta cell line on the scaffolds. With external oxygenation, the density and function of cells on the PDMS scaffold were more than three times and almost two times higher than those without oxygenation, respectively. This suggests that the oxygenation afforded by the PDMS scaffolds allows for high-density loading of islet tissue into the devices.

Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells.

PURPOSE: Cancer stem cells (CSCs), also known as tumor-initiating cells, are involved in tumor progression, metastasis, and drug resistance. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the inhibitory effects of HL on the growth of CSC subpopulations in liver cancer cells (HepG2) in vitro. METHODS: HLs composed of 90 mol% L-α-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23) dodecyl ether were prepared by sonication. Cell viability was determined by the trypan blue exclusion assay. In liver cancer cells, CSCs were identified by the presence of the cell surface marker proteins CD133 and EpCAM by flow cytometry. A soft agar colony formation assay was performed using HepG2 cells pretreated with HLs. RESULTS: HLs selectively inhibited liver cancer cell growth without affecting normal hepatocytes. Additionally, HLs induced apoptosis of HepG2 cells by a"ctivating caspase-3. Notably, the CD133(+)/EpCAM(+) CSC sub-population of liver cancer cells treated with HLs was reduced. Furthermore, HLs markedly decreased the number of colony-forming cells. Finally, we confirmed the fusion and accumulation of HLs into the cell membranes of CSCs using a fluorescently labeled lipid (NBDPC). Significant accumulation of HL/NBDPC into the CSCs (particularly EpCAM(+) cells) occurred in a dose-dependent manner. CONCLUSION: These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy.