| 摘要: | 在這份論文「利用寡肽培養人類胚胎幹細胞」中分成兩個部分，Part 1為「人類胚胎幹細胞在無異種培養的條件下使用特定的寡肽取代原本在培養液中TGF-β1或FGF-2的蛋白質」，而Part 2為「人類胚胎幹細胞在熱敏感奈米片段材料上的培養與脫附效率」。在Part 1中，現在大部分的培養液裡的生長因子含有從動物獲得的蛋白質，而這些含有從動物中獲取的成分成為多功能幹細胞(hPSCs)培養在臨床應用上的限制。在這研究裡，於避免使用生長因子TGF-β1或FGF-2的情況下，發展無異種和無滋養層的培養液，因為生長因子在室溫下較不穩定必須貯存在-20度下，需花費較多在貯存方面，所以在這裡選用TGF-β1或FGF-2的寡肽取代物，可被貯存於室溫下。如果培養液可被長久貯存於室溫或是4度的環境下，用於培養hPSCs的培養液更為方便也更為節省。於研究中，使用此培養液培養人類胚胎幹細胞(hESCs)WA09在Matrigel上，且計算細胞貼附率，增值速度以及分化率找出使用此含有TGF-β1或FGF-2的寡肽取代物培養液的最佳條件。我們找到在只有化合物和無異種的條件下，培養液中含有TGF-β1的寡肽取代物較適合用於培養hPSCs。而在Part 2裡，利用低臨界溶解溫度(LCST)的熱敏感材料製備培養皿去培養幹細胞且降低培養液溫度使得細胞脫附，可維持細胞聚集和片狀，取代使用酵素讓細胞從培養皿上脫附。在這裡設計熱敏感材料去培養人類多功能幹細胞，利用RAFT高分子合成法，三種具有和polystyrene的共聚物為(a)熱敏感性的poly(N-isopropyl acrylamide), PNIPAAm (b) 生物相容性的polyethylene glycol methacrylate (PEGMA) 和(c)可藉由polyacrylic acid (PAA)的carboxylic acid共軛寡肽(oligo-vitronectin)，調配藉由降低培養液的溫度對培養hESCs最理想的表面成分。將培養皿置於銅板上且放進7-8度的冰箱中，30分鐘之內hESCs可達高脫附率50%相比於其他冷卻方式。更進一步的是，hESCs的脫附率可達到將近100%當使用roller和1至2次的pipetting。現在藉由降低溫度使部分細胞脫附，hESCs可被持續的培養在熱敏感材料上，且維持其多功能性。未來，我們正在設計從2D培養到3D培養在臨床上的應用。;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. |
