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대사공학과 생물직교화학을 이용한 세포의 표지화 및 생체 내 추적 기술

Title
대사공학과 생물직교화학을 이용한 세포의 표지화 및 생체 내 추적 기술
Other Titles
Cell Labeling and Tracking using Metabolic Glycoengineering and Bioorthogonal Chemistry
Author
윤화인
Alternative Author(s)
Yoon, Hwa In
Advisor(s)
조용우
Issue Date
2016-08
Publisher
한양대학교
Degree
Doctor
Abstract
Establishment of appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining etabolic glycoengineering and bioorthogonal chemistry. First, cells were treated with unnatural sugars as Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz to generate unnatural azide groups (-N3) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with cyclooctyne modified dye or nanoparticles through bioorthogonal copper-free click chemistry. Importantly, bioorthogonal labeling method presented strong signal intensity with relatively low cytotoxicity and the amounts of azide groups and cyclooctyne modified agent could be easily controlled by feeding different amounts of Ac4ManNAz and cyclooctyne modified agent to the cell culture system. In chapter 2, DBCO-Cy5-labeled chondrocytes seeded on 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-Cy5-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. This cell labeling and tracking technology had minimal effect on cartilage formation in vivo. In chapter 3, endothelial progenitor cells were isolated from differentiated human embryonic stem cell to introduce unnatural azide groups on the cells by metabolic glycoengineering. High sensitivity of Ac4ManNAz and DBCO-Cy5 allowed the longterm cell tracking, and distinct signals from the labeled cells lead to a clear image for cell tracking compared DiD groups in the hindlimb ischemia model. Transplanted hESC-ECs could act as an alternative source of cells for therapeutic angiogenesis to treat peripheral ischemia. Optimized labeling method showed low cytotoxicity, had minimal effect on therapeutic angiogenesis, and yielded high contrast in vitro and in vivo images for efficient cell tracking. In chapter 4, as nanoparticle-based stem cell labeling and tracking for various imaging applications, glycol chitosan nanoparticles (CNPs) was used as a template nanoparticle for different imaging agents for diverse molecular imaging of stem cells, fluorescent dye, Cy5.5 (optical imaging), iron oxide particles (MR imaging) and gold particles (CT imaging) were chemically labeled on the CNP surface or physically loaded into CNPs. Bioorthogonal chemistry can highly increase the signals of NPs labeled cells for greater in vivo imaging sensitivity, long-term stability without cytotoxicity, reduced proliferation, and differentiation abilities.
URI
http://dcollection.hanyang.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000094046https://repository.hanyang.ac.kr/handle/20.500.11754/125451
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > FUSION CHEMICAL ENGINEERING(융합화학공학과) > Theses (Ph.D.)
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