Preparation of cell culture dishes immobilized nanobrush for regenerative medicine and preservation and culture of stem cells

由聚氧聚乙烯（PEO，亲水段）-聚氧聚丙烯（PPO，疏水段）-聚氧聚丙烯（PEO，亲水段）三嵌段共聚物Pluronic组成的三嵌段共聚物具有两亲性，是一种具有低临界溶解温度（LCST）的温度响应聚合物。已知具有亲水和亲水结构域的表面比仅具有亲水结构域的表面表现出生物相容性。然而，具有垂直于表面的两亲结构域的生物相容性材料尚未得到研究。本课题设计了具有多个片段长度（分子量）和两亲性含量的两亲三嵌段共聚物（pluronic），并通过表面反应将其接枝到细胞培养皿上，从而研制出由两亲三嵌段共聚物制备的细胞培养皿（纳米片段细胞培养皿）。本课题的目的之一是研究由两亲性三嵌段共聚物制备的纳米级细胞培养皿的最佳分子设计。本课题的第二个目标是在不使用胰蛋白酶的情况下，研究纳米刷（由两亲性三嵌段共聚物组成，具有LCST）接枝的细胞培养皿上培养的细胞脱离情况，并研究纳米刷细胞培养皿的优化设计。具有70%亲水性结构域和10000道尔顿分子量的三嵌段共聚物在培养皿上可以有效地保存造血干细胞。纳米刷密度高的培养皿不适合培养和保存造血干细胞。几种细胞外基质（ecm、明胶、胶原、层粘连蛋白、玻璃体连接蛋白、纤维连接蛋白和基质）通过共价键和包被法固定在聚苯乙烯培养皿上。不同培养皿培养的间充质干细胞（MSCs）表面标记物的表达具有可比性。明胶培养皿和玻璃体连接素培养皿培养的间充质干细胞在分化培养基中倾向于向成骨细胞分化。然而，在ecm移植培养皿上培养的MSC并没有促进成骨细胞的分化。在ecm移植培养皿上培养的间充质干细胞促进向神经细胞的分化。特别是基质和层粘连蛋白培养皿培养的间充质干细胞向神经细胞的分化程度较高。在ECM和pluronic接枝培养皿上培养的MSCs可以通过降低培养皿温度（≤4℃）从培养皿中分离

The triblock copolymer Pluronic, composed of polyethylene oxide(PEO, hydrophilic segment)-polypropylene oxide(PPO, hydrophobic segment)-PEO(hydrophilic segment) triblocks, exhibits amphiphilic properties, and is temperature responsive polymer having low critical solution temperature(LCST). The surface having hydrophilic and hydrophilic domains is known to show biocompatibility than the surface having only hydrohilic domain. However, the biocompatible materials having amphiphilic domains in perpendicular to the surface have not yet investigated. In this project, amphiphilic triblock copolymer(pluronic) having several segment length(molecular weight) and amphiphilic content was designed and grafted on cell culture dishes by surface reaction, and thus, the cell culture dishes prepared from amphiphilic triblock copolymer was developed(cell culture dishes having nanosegments). One of the goal of this project is to investigate the optimal molecular design of cell culture dishes having nanose … More gments, which was prepared from amphiphilic triblock copolymers.The second goal of this project is that detachment of the cells cultured on cell culture dishes grafted nanobrush, which is composed of amphiphilic triblock copolymer having LCST, was investigated without using trypsin in cell detachment and that the optimal design of cell culture dishes having nanobrush was investigated. Preservation of hematopoietic stem cells were efficiently possible on the dishes immobilized triblock copolymer having 70% hydrophilic domain and 10, 000 dalton of molecular weight. The dishes having high density of nanobrush were not suitable to culture and preserve the hematopoietic stem cells.Several extracellular matrices(ECMs, gelatin, collagen, laminin, vitronectin, fibronectin, and matrigel) were immobilized on polystyrene dishes by covalent bonding and coating method. The surface marker of mesenchymal stem cells(MSCs) cultured on different dishes immobilized ECMs show comparable expression. MSCs cultured on gelatin and vitronectin-coating dishes tended to differentiate into osteoblasts in differentiation medium. However, MSC cultured on ECM-grafted dishes did not promote to differentiate into osteoblasts. MSCs cultured on ECM-grafted dishes were found to promote differentiation into neural cells. Especially, MSCs cultured on matrigel-and laminin-grafted dishes showed higher differentiation into neural cells. MSCs cultured on ECM and pluronic grafted dishes can be detached from the dishes by decreasing temperature of dishes at 4℃ Less