S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Mechanical Aerospace Engineering (기계항공공학부) Theses (Ph.D. / Sc.D._기계항공공학부)
Cell Behavior Modulation using Biophysical Interactions
물리적 자극을 이용한 세포 거동 조절 및 활성 연구
- 공과대학 기계항공공학부
- Issue Date
- 서울대학교 대학원
- PDMS pillar array; Bacterial magnetic nanoparticle; Single-walled carbon nanotube; Cell migration; Mitochondria targeting; Self-degradation
- 학위논문 (박사)-- 서울대학교 대학원 : 기계항공공학부, 2015. 8. 이정훈.
- In this thesis, we report the effect of nano- micro- scale materials having various material property affecting cellular responses. For this purpose, micro-scale pillar array which has stepped structure with varying bottom diameter were fabricated by double step photolithography process. Single walled carbon nanotube and magnetic nanoparticle were also engineered to stimulate cellular behaviour. These materials were successfully embedded into cellular system to regulate its migration behaviour, morphology and viability. Our study could give a pioneer understanding regarding cellular response affected by nano- micro- scale systems.
First, we designed a new variation of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the focal adhesion area of a cell. This goal is achieved via a stepped micro pillar array device (SMPAD) in which the contact area with a cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was manufactured to be identical, whereas that of the bottom was changeable, to achieve the desired substrate rigidity. Fibronectin is immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behaviour are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness.
Next, we demonstrate that the nano-scale magnetic modulation of mitochondrial VDAC2, which is the only mammalian-specific isoform among VDAC isoforms, can contribute to protect the neurodegenerative disease attenuating the changes in the intracellular calcium levels that were induced by beta-amyloid. In this study, BMPs originated from Magnetospirillum sp. AMB-1 directly conjugated with VDAC2 antibody using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linker which is used to couple carboxyl groups to primary amines. BMPs-VDAC2 antibody complexes (BMPs-Ab) introduced into SH-SY5Y cells, human derived neuroblasts which are often used as in vitro models of neuronal function and differentiation. We investigated that the effect of magnetically modulated VDAC2 on the change of intracellular Ca2+ levels induced by Aβ. SH-SY5Y cells were loaded with 5 μM Fluo-3 AM for 30 min, and then the changes in the level of Ca2+ before and after treatment with Aβ were measured by 488-nm laser source to excite Fluo-3. BMPs-VDAC2 antibody complexes (BMPs-Ab) introduced into SH-SY5Y cells were successfully internalized into SH-SY5Y cells. We found that the capture of VDAC2 with BMPs-Ab was significantly decreasing the expressed intracellular calcium levels induced by Aβ. This magnetic modulation of VDAC2 considerably increases the proliferation and reduced Aβ-induced toxicity in SH-SY5Y. These results suggest that magnetic modulation of VDAC-2 is able to protect the neurodegenerative disease attenuating the changes in the intracellular calcium levels that were induced by Aβ.
Finally, we investigated a self-degradation route for single-wall carbon nanotubes (SWNTs) mediated by built-in peroxidase-like activity of bacterial magnetic nanoparticles (BMPs). Biocompatible BMPs originated from Magnetospirillum sp. AMB-1 were directly conjugated through covalent bonding to yield functionalized SWNTs (f-SWNTs) without any additional functionalized processes. Employing transmission electron microscopy (TEM) and Raman spectroscopy, we found that BMPs can act as effective built-in intrinsic peroxidase compare to other enzymatic methods for the degradation of SWNTs. For the possible application in neurobiology, f-SWNT-BMP hybrids were shown as an inhibitor to reduce formation of amyloid-beta (Aβ) fibrils which is considered as the key element behind Alzheimers disease. To conform this, we showed that neurotixicity of Aβ peptide affecting SH-SY5Y cell death is reduced in the presence of these hybrids. Our findings could offer a new approach of mitigating the toxic impact and neurobiological application of CNTs.