2021 Fiscal Year Research-status Report
A novel design methodology for the construction of functionalized human cell monolayer on an egg
| Project/Area Number |
21K12667
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| Research Institution | Kyushu University |
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Principal Investigator |
黄 文敬 九州大学, 工学研究院, 特任助教 (00633413)
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| Co-Investigator(Kenkyū-buntansha) |
張 宏 東海大学, 工学部, 特定研究員 (80774257)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | Bubble injector / Epithelial cell removal / CAM |
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| Outline of Annual Research Achievements |
The application of porous membrane for the partisioning of cellular microenvioronment is crucial for a microphysiological system. Membrane permeability is important. However, the permeability of commercially available plastic membranes is low. Therefore, this research aims to locally remove the monolayer of epithelial cells on chick chorioallantoic membrane (CAM) and apply the CAM as a porous membrane for human cell culture, which connects to chick embryo beating heart.
In FY2021, I mainly focused the method for the removal of chick epithelial cells on CAM using bubble injector, a tool used for the generation of high-speed microbubbles, which was developed by Prof. Yamanishi, Kyushu University. The results were presented at Conference on Frontiers in Bioengineering of the Japan Society of Mechanical Engineers (JSME). The following experiments have been conducted. First, establishment of research envioronment for this project including equipments for chick embryo incubation and platforms for the fabrication of the artificial eggshell. Second, application of bubble injector for local cell removel on the CAM. Third, confirmation of the methods for the analysis of CAM structures (evaluation of cell removal) including immunohistochemical staining method.
Further, I also confirmed the fabrication protocals of nanosheet in Department of Mechanical Engineering, Kyushu University, which is necessary for the construction of cellular microenvioronment on CAM in FY2022.
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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
According to the application form of the project, the followings are what I should do in FY2021. First, removing cells within a 6-mm area on CAM. Second, evaluation of cell removal. Third, a try of human cell culture.
I have clarified the power conditions of electrically-induced microbubbles for the removal of chick epithelial cells on CAM. After exposure to the microbubbles, the CAM was extracted and Hematoxylin-eosin (HE) staining was conducted to confirm the removal of chick epithelial cells on the top of the CAM. Further, I also cultured human umbilical vein endothelial cells and confirmed the availability of anti-CD31/PECAM-1 (endothelial cell marker). The culture conditions including temperature and CO2 suitable for both human endothelial cells and chick embryos are under investigation.
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| Strategy for Future Research Activity |
According to the application form of the project, the followings are what I should do in FY2022. I will culture human endothelial cells on the CAM with local removal of chick epithelial cells. Then I will apply the shear flow induced by the bubble injector to functionalize the human endothelial cells. Nanosheets will be used for the wrapping of the cellular microenvioronment after the loading of human cells on CAM.
Two factors will be investigated in detailed in FY2022. The first is the rejection reaction (if any) resulted from the co-culture of human umbilical vein endothelial cells and the chick epithelial cells. The interaction between the two cell types will be investigated. The effects of the interaction on the formation of a monolayer of human endothelial cells on the CAM will also be elucidated. The second is the relationship among the strength of fluid flow, the deformation of CAM and the detachment of loaded human endothelial cells.
Indeed, removal of epithelial cells on a CAM makes the CAM a wound model. The maximum area with epithelial cell removel leading to the death of chick embryo is still unknown. A large area with chick cell removal will benefit the culture of human cells on the CAM. Therefore, I will also investigate the upper limit of the area for the optimization of the model in FY2022.
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