2016 Fiscal Year Annual Research Report
Development of a microfluidic biochip for hepatic induced pluripotent stem cell differentiation
| Project/Area Number |
16F16715
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| Research Institution | The University of Tokyo |
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Principal Investigator |
酒井 康行 東京大学, 大学院工学系研究科(工学部), 教授 (00235128)
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| Co-Investigator(Kenkyū-buntansha) |
LEREAU-BERNIER MYRIAM 東京大学, 大学院工学系研究科(工学部), 外国人特別研究員
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| Project Period (FY) |
2016-07-27 – 2018-03-31
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| Keywords | ヒトiPS細胞 / hepatic differentiation / iver-on-chip / drug screening |
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| Outline of Annual Research Achievements |
Advanced progresses in stem cell differentiation are challenging issues. Stabilization and homogeneous mature hepatocyte patterns with induced pluripotent stem (iPS) cells are urgently required to move from lab to industrial and clinical applications. The present project aims to develop, from hepatocytes derived from human iPS (hiPS), a new culture model to achieve liver maturation patterns. This bioreactor is divided into two modules: the dialysis compartment for nutrient mass transfer and growth factor concentrations and the microfluidic compartment to cultivate, differentiate and mature iPS cells. In order to optimize the hepatic differentiation of hiPS cells, two different protocols have been tested: both are derived from a previously published differentiation protocol in Petri dishes, with four steps leading from hiPS to hepatocytes. In Petri, both protocols lead to hepatocytes after 20 days. We are also investigating the possible effect of hiPS density at the time of initiation of hepatic differentiation on the more or less matureness of hepatocytes at the end of the protocol. In bioreactor, we are still facing problem of adhesion of cells at the hepatoblast step of the differentiation protocol. As inoculation density may be the key point, experiments are ongoing to produce a large number of hepatoblasts in order to be able to inoculate bioreactors with a large number of cells. In parallel, we are trying to inoculate hiPS cells directly in the bioreactor and perform the differentiation protocol inside the device in order to avoid the critical detachment step.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
One first objective of this project was to optimize the hepatic differentiation of hiPS cells in a bioreactor which has been developed in the laboratory. This bioreactor is divided into two modules: the dialysis compartment for nutrient mass transfer and growth factor concentrations and the microfluidic compartment to cultivate, differentiate and mature iPS cells.
Two different differentiation protocols have been tested: both are derived from a previously published differentiation protocol in Petri dishes, with four steps leading from hiPS to hepatocytes. In Petri format, both protocols are leading to hepatocytes after 20 days. However, it seems that one is more efficient in producing mature hepatocytes than the other. Moreover, in order to optimize the differentiation protocol, we are also investigating the possible effect of hiPS density at the time of initiation of hepatic differentiation on the more or less matureness of hepatocytes at the end of the protocol.
In the bioreactor, cells have so far been inoculated at the end of step 3. However, at this point, we are still facing problem of adhesion of cells. As several detachment protocol have been tried with the same negative results, it seems that the inoculation density is a crucial factor. Experiments are ongoing to produce a large number of hepatoblasts in order to be able to inoculate bioreactors with a large number of cells. In parallel, we are trying to inoculate hiPS cells directly in the bioreactor and perform the differentiation protocol inside the device in order to avoid the critical detachment step.
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| Strategy for Future Research Activity |
For next year, the first objective is to achieve differentiation inside the bioreactor, either by detaching the hepatoblast-like cells from Petri dishes and inoculating them at a high density, or by performing the entire differentiation protocol inside the device. Once done, biological assays to validate the hepatic differentiation will include investigations of mRNA levels analyzed by RTqPCR , hepatic biomarkers and metabolism’s performance (using drug cocktail assay with CYP450 substrates).
The second objective will be to further optimize hepatic maturation in the bioreactor using two techniques. Firstly, matrigel will be loaded in the cell culture compartment after oncostatin M differentiation. Secondly, taking into account the importance of the microenvironment created by the liver non-parenchymal cells in the hepatic endoderm formation, we believe co-cultures in biochip will help to mimic the environment of liver and promote hepatocyte maturation. We propose to investigate the effect of flow rate stimulation in microfluidic cultures during hepatic differentiation via additional assays to evaluate the proportion of differentiated cells in hepatocytes and in non-parenchymal cells by specific hepatocyte and endothelial staining (albumin for hepatocytes, van Willbrand for endothelial cells) and by FACS analysis of the cell population, and test the co-cultures in the microfluidic biochip by adding iPS non-parenchymal cells after oncostatin M differentiation step. Finally, iPS biochips will be compared with (i) Petri iPS and (ii) Petri human primary hepatocytes cultures.
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