生体様の代謝と輸送を再現する肝組織マイクロデバイス

2012 Fiscal Year Annual Research Report

• Project/Area Number: 13F03713
• Research Institution: The University of Tokyo
• Principal Investigator: 酒井 康行 東京大学, 生産技術研究所, 教授
• Co-Investigator(Kenkyū-buntansha): PERRY Guillaume 東京大学, 大学院工学系研究科, 外国人特別研究員
• Keywords: CRCAN-ON-CHID / LIVER

Research Abstract

The liver is one of the most important organ for drug metabolism and elimination through biliary excretion. For drug screening applications, it is really important to keep a microenvironnment as close as possible to in-vivo, with high hepatic cells (hepatocytes) metabolism capabilities and self-oragnization to form bile canaliculi. Reach this microenvironment mainly depends on hepatocytes oxygenation, nutrient supply so we can also play with the microdevice design to control these parameters and hepatocytes organization. Up to date, there is only one method in vitro to evaluate bile excretion, which is realized by patterning μwelis on a collagen gel and overlay with Matrigel. However this culture method needs an external system to recover bile. In our study, we would like to achieve an in-vitro model close to in-vivo using two ways. In the first one is adapted from the classical approach already microwell in well plate. We will focus on improving assay device which give up to now the best results. We want to control the oxygen diffusion with a μwell made in PDMS and the cell organization using Matrigel or a liver endothelial cell line (TMNK1). The second way is to used a microfluidic device which could be closer to in vivo conditions but will have to overcome challenges in term of long-term cell viability. Based on the previous works realized in Sakai lab, the purpose of the proposed research is to develop a microsystem preserving the high metabolic capability of hepatocytes and enabling continuous bile recovery using the classical μwell or a microfluidic device. Two approaches will be investigated in parallel. A key transversal research point to these two topics will be the development of successful co-culture protocol to enhance control and reproducibility.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

With the first approach using PDMS microwells, we designed a v-shape μwell in order to harvest all inocolated. Then, we enhanced cell's adhesion inside the μwells and looked for the good culture conditions using Extracellular Matrix or co-culture with liver endothelial cells to get the good morphology (semi-spheroid) for bile recovery. In parallel, the microfluidic device was designed in order to promote cells assembly as liver cord and to recover bile. Altought, some cells were assembled as wanted, it appear that some cells are also damaged by the shear stress induced by the low flow rate.

Strategy for Future Research Activity

In μwell the last experiments are very promising. We will continue the experiments using Extracellular Matrix and the coculture with endothelial cells to get bile canaliculi formation, good hepatic fonctions and a long viability. We want to control the location of the bile caniculi to be able to recover bile throught a micromanipulator or a microdevice. In the case of the microfluidic device, new designs will be realized in order to achieve, with reproducibility, the cell assembly as a liver cord. Then, we will have to optimize the perfusion parameters to obtain a high viability for this microfluidic culture system and hepatocytes with high metabolism capabilities.