Acceleration of cell and cell sheet detachment from temperature-responsive culture dishes

(1)开发用于快速细胞释放的温度响应性培养表面：细胞从共价接枝有温度响应性聚合物的温度响应性培养表面脱离需要表面水合和细胞活性（例如接触力）两者。聚苯乙烯广泛用作细胞培养的基底。但它是疏水的，不能提供接枝聚合物水合所需的水。将温度响应性聚合物接枝到微孔膜上，通过微孔补充水分，加速细胞脱离。细胞片脱离时的加速作用比单细胞脱离时更明显。(2)细胞因子对细胞脱附的调控：我们的前期工作揭示了细胞利用细胞骨架和ATP产生的收缩力，最近发现了几种调控细胞骨架重组的分子和途径。这些因素，包括ATP dbcAMP，生长因子，和钙离子载体进行了检查细胞和细胞片脱离加速。然而，没有得到产生显著加速度的适当条件。(3)通过使用支撑膜缩短细胞网过程：为了防止细胞片收缩或折叠，使用柔性支撑膜。优化了操作条件，使分离过程减少到无载体膜时的十分之一。

(1) Development of temperature-responsive culture surfaces for rapid cell release : Cell detachment from temperature-responsive culture surfaces, which is covalently grafted with temperature-responsive polymer, requires both of surface hydreation and cellular activity such as conractile forces. Polystyrene is widely used for cell culture basal substrate. But it is hydrophobic and can not provide water needed for hydration of grafted polymer. By grafting temperature-responsive polymer onto microporous membranes, water was suppled though the pores and cell detachment acceleration was achieved. The acceleration was more significant in cell sheet detachment than in single cell detachment.(2) Regulaton of cell detachment with cytokines : From our previous works, it has been revealed that cells utilize thier contractile forces exerted by cytoskeleton and ATP.Recently, several molecules and pathways which regulate cytoskeletal reorgnization have been identified. These factors including ATP dbcAMP, growth factors, and calcium ionophore were examined for cell and cell sheet detachment acceleration. However the apropriate condition for the significant acceleration was not obtained.(3) Shortening of detachmenet process by utilizing supporter membranes : In order to prevent cell sheet shrinkage or folding, flexible supporter membranes are used. The procedures were optimized and the process was reduced to one tenth of that without supporter membranes.