使用微載體培養貼附性細胞已被證明是能實現擴增幹細胞及哺乳動物細胞重要方法之一，微載體脫附細胞時需考慮胰蛋白酶孵育時間，時間過長會造成細胞傷害及黏附力過強之細胞不易脫落等缺點。為了改善細胞於微載體之脫附率，本實驗開發 (1) 可溶解還原型微球、(2) 可溶解感溫還原型微球。已成功使用明膠通過油包水乳化法以雙硫鍵作為交聯劑造球，調整不同雙硫鍵濃度成功將明膠網絡穩定形成可溶解還原型明膠微球，並將NIPAM與烯丙基胺(Allylamine)以可逆加成-斷裂鏈轉移（RAFT）反應聚合溫敏性嵌段共聚物，結構中嵌入帶正電且親水的Allylamine。水接觸角顯示略微親水，利於細胞貼附增殖並提升最低臨界溶液溫度(LCST)為33.4 °C，趨近人體溫度37°C，適合細胞培養環境中應用。藉由化學法(設計及合成雙硒鍵表面交聯劑)及物理法塗層溫敏性共聚物修飾微球表面，形成可溶解型微球具有還原及溫度雙重敏感特性，感溫高分子改質於微球表面後，協助微球在37oC之穩定性，另外表面感溫共聚物改質後貼附率提升11%、脫附比例上升45.8%，細胞脫附時加入還原劑切斷雙硫鍵於30分鐘內成功瓦解，收回的細胞經檢測均為活細胞，初步證實可溶解還原型微球、可溶解感溫還原型微球具有助於細胞貼附擴增、傳遞活細胞並釋放之潛力。

The use of microcarriers to culture adherent cells has been proven to be one of the important ways to amplify stem cells and mammalian cells number. When separating cells with microcarriers, it is necessary to consider the trypsin incubation time, which will cause cell damage and the cells with strong adhesion are not easy to fall off. In order to improve the detachment rate of cells on microcarriers, we developed (1) soluble microspheres and (2) soluble thermosensitive microspheres. In this study, gelatin had successfully formed microspheres through water-in-oil (w/o) emulsion method with disulfide bond as crosslinking agent. Adjusting the concentration of disulfide bonds stabilized the gelatin network to form soluble reduceable gelatin microspheres and polymerized with NIPAM and allylamine through reversible addition fragmentation chain transfer (RAFT) reaction to form thermosensitive block copolymers embedded with positively charged and hydrophilic allylamine. Water contact angle showed slightly hydrophilic, making this microsphere suitable for cell adhesion and proliferation. The lower critical solution temperature (LCST) was raised to 33.4℃ close to the human body temperature 37℃, which is suitable for cell culture applications. By using chemical method (design and synthesis of diselenide bond surface crosslinker) and physical method to coat thermosensitive copolymer onto the surface of the microspheres, the soluble microspheres exhibited dual sensitivity to reducing agent and temperature. The thermosensitive polymers on the surface of the microspheres improved the stability of the microspheres at 37℃. In addition, the attachment rate increased by 11% and the detachment rate increased by 45.8% after the surface modification. Furthermore, the disulfide bond was cleaved by reducing agent when cell separation within 30 min and the recovered cells were all alive. The potential of soluble, thermosensitive and reduceable microspheres for cell attachment, proliferation, delivery and release of living cells was preliminarily confirmed.

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