Human Embryonic Stem Cell Culture Using Oligopeptides

• Main Authors: Yi-Tung Lu, 陸依彤
• Other Authors: Akon Higuchi
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/jqphu4

碩士 === 國立中央大學 === 化學工程與材料工程學系 === 104 === This Master thesis, ”Human Embryonic Stem Cell Culture Using Oligopeptides” contains two studies; Part 1, Human Embryonic Stem Cells Culture in Medium Containing Specific Oligopeptides for Replacement of TGF-1 and FGF-2 under Xeno-free Culture Conditions and Part 2, Culture and Detachment of Human Embryonic Stem Cells on Biomaterials Immobilized with Thermoresponsive Nanobrush. In Part 1, most of current hPSC medium contains animal-derived proteins including growth factors. The medium containing animal-derived components generates barriers for the clinical usage of hPSCs cultured. Here, xeno-free and feeder-free culture medium was developed, which avoids to use the growth factors of TGF-β1 or FGF-2 for the culture of hPSCs. Because growth factors are extremely expensive and should be stored at -20 degree due to low stability of protein at room temperature, specific oligopeptides were selected for TGF-β1 and FGF-2 replacement on hPSC culture, which can be stored at room temperature. If the culture medium can be stored in room temperature or 4 degree for long time, it is convenient to store and use the culture medium of hPSCs and the price of the culture medium is expected to become inexpensive. In this study, WA09 (H9) human embryonic stem cells (ESCs) were cultured on Matrigels in the culture medium developed in this study and investigated cell attachment ratio, expansion rate and differentiation ratio to evaluate what conditions are the best for hPSCs culture using the culture medium containing the oligopeptide for TGF-β1 or FGF-2 replacement. It is concluded that the oligopeptides for replacement of TGF-β1 can be used for hPSC culture in the chemical-defined and xeno-free conditions. In Part 2, the surface prepared using thermoresponsive polymers with low critical solution temperature (LCST) is an attractive candidate for stem cell culture because stem cells can be detached from the thermoresponsive surface without applying an enzymatic digestion method and, instead, by decreasing the temperature of culture medium, which enables cell aggregates or cell sheets to be obtained in culture medium. In this study, the thermoresponsive nano-brush surfaces were designed for the culture of human pluripotent stem cells (hPSCs). Using RAFT polymerization, three coating copolymers having polystyrene were prepared, which were polystyrene copolymers with (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm, (b) biocompatible polyethylene glycol methacrylate (PEGMA), and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin) could be conjugated via carboxylic acid of PAA. The optimal surface composition for human embryonic stem cells (hESCs, WA09) detached by decreasing temperature of the culture medium was investigated. hESCs were successfully cultured on the thermoresponsive surface, the most rapid detachment conditions of hESCs was to keep dishes cool using the copper plate below the culture dishes in the refrigerator (7-8 degree) where higher detachment ratio (50%) of hESCs was achieved within 30 minutes compared to other conditions. Moreover, hESCs could be detached nearly 100% combined with roller and 1-2 times pipetting. Currently, hESCs are culturing continuously on the thermoresponsive surface by the partial detachment process where hESCs can maintain their pluripotency on the thermoresponsive surface coated with these copolymers and can easily detach from the thermoresponsive surface by decreasing the temperature. In future, we are designing to shift to a novel 3D culture system to scale up for clinical application.