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

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(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.