While tissue-engineering approaches of heart valves have made great strides towards creating functional tissues in vitro, the instruments used, named bioreactors, cannot efficiently integrate multiple stimuli to accurately emulate the physiological microenvironment. To address this, we conceptually designed and built a bioreactor system that applied a range of mechanical tension conditions, modulated matrix stiffness, and introduced biochemical signals in a combinatorial and high-throughput manner. Proof-of-concept experiments on PAVIC-seeded hydrogels were performed to assess the independent and combined effects of tensile strain, matrix stiffness and TGF-β1 on myofibroblast differentiation by measuring α-SMA expression, a marker that indicates a disease-associated phenotype. We found that matrix stiffness and TGF-β1 significantly increased α-SMA levels (p < 0.001), while the effect of mechanical strain was only significant on soft gels (~12 kPa) without TGF-β1. This study therefore demonstrated independent and integrated effects of multiple stimuli in regulating key cellular events in the aortic valve.

While tissue-engineering approaches of heart valves have made great strides towards creating functional tissues in vitro, the instruments used, named bioreactors, cannot efficiently integrate multiple stimuli to accurately emulate the physiological microenvironment. To address this, we conceptually designed and built a bioreactor system that applied a range of mechanical tension conditions, modulated matrix stiffness, and introduced biochemical signals in a combinatorial and high-throughput manner. Proof-of-concept experiments on PAVIC-seeded hydrogels were performed to assess the independent and combined effects of tensile strain, matrix stiffness and TGF-β1 on myofibroblast differentiation by measuring α-SMA expression, a marker that indicates a disease-associated phenotype. We found that matrix stiffness and TGF-β1 significantly increased α-SMA levels (p < 0.001), while the effect of mechanical strain was only significant on soft gels (~12 kPa) without TGF-β1. This study therefore demonstrated independent and integrated effects of multiple stimuli in regulating key cellular events in the aortic valve.

Yes / Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies. / Malaysia Ministry of Education (Fundamental Research Grant Scheme, FRGS Vot. 1482 and IGSP Vot. 679).

No / Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications. / (Science Fund Vot No.: 0201-01-13-SF0104 or S024) awarded by Malaysia Ministry of Science and Technology (MOSTI) and IGSP Grant Vot No. U679 awarded by Universiti Tun Hussein Onn Malaysia.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23752号 / 医博第4798号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM