Application of ultra thin hydrophilic polymer layer to acceleration of cell sheet recovery from temperature-responsive culture dishes Yoshikatsu Akiyama, Masayuki Yamato, Akihiko Kikuchi and Teruo Okano Institute of Advanced Biomedical and Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku Tokyo 162-8666 Japan Abstract: Slow recovery of cell sheet from T 20t poly(N-isopropylacrylamide) (PIPAAm) grafted TCPS Water molecules surfaces is not so practical in clinical field. To solve this problem regarding the slow recovery, based on the hypothesis 22_ that hydrophilic basal surfaces promote hydration of TCPS Lowring PIPAAm (hydrated) PIPAAm chains with ultra thin thickness, we successfully PIPAAm (dehydrated) temper ture prepared PIPAAm grafted culture dishes exhibiting rapid 200 cell and cells sheet recovery. Sequential grafting appropriate amount of PAAm and PIPAAm a (PIPAAm-PAAm-TCPS) attained more rapid cell and cell sheet recovery in comparison with PIPAAm grafted onto hydrophobic TCPS. Characterization of TCPS Loweing TCPS PIPAAm-PAAm-TCPSs surfaces revealed that appropriate temperature amount of the grafted PIPAAm and PAAm is necessary to PydrophAiPAAm aaer attain such rapid recovery of cell sheet, suggesting that this Fig. 1. Illustration of spontaneous cell sheet recovery from (upper) difference results from different properties of the basal PIPAAm grafted TCPS surfaces and (bottom) PIPAAm grafted onto surfaces and different mobility of the grafted polymer hydrophilic surfaces (PAAm-TCPS). chains between hydrophobic TCPS and hydrophilic PAAm. In addtion t als suppot thehypotesis.to accelerate the hydration of the grafted PIPAAm, in In addition it also support the hypothesis. previous reports, we attained rapid recovery of the cultured 1. INTRODUCTION cells and cell sheet, incorporating hydrophilic moiety such Previously, we reported that, to prepare as PEG and 2-caroboxylisopropylacrylamide into the grafted poly(N-isopropylacrylamide) PIPAAm grafted TCPS PIPAAm network.2' The incorporated hydrophilic (PIPAAm-TCPS) showing cell attachment and detachment molecules should form water releasing channels. properties in response to temperature, grafting 20 nm In the present study, based on these results, we thickness of PIPAAm layer is a key factor.' The degree of hypothesized that PIPAAm chains grafted onto not the chain mobility at the outermost surfaces is dependent on hydrophobic but hydrophilic polymer layer as basal degree of mobility of the grafted polymers chains in the substrate are more mobile and possibly hold more amount of vicinity of the interfaces of TCPS. At the interfaces of the water molecules than those onto hydrophobic TCPS, and basal TCPS surfaces, the grafted PIPAAm chains are consequently, the basal hydrophilic polymer layer can strongly restricted and dehydrated by immobile and accelerate hydration of the grafted PIPAAm chains (Fig. 1 hydrophobic TCPS surfaces. Thus, such immobile and bottom)). Owing to the facilitated hydration of the grafted dehydrated polymer chains progressively promote restriction PIPAAm chains, cultured cells are more rapidly detached and dehydration of the grafted PIPAAm chains at the from the surface of PIPAAm grafted onto the hydrophilic outermost surfaces, and provided the outermost surfaces polymer. In order to verify this hypothesis, we prepared with cell adhesive properties at 37°C, when the grafted layer PAAm-TCPS as a model hydrophilic basal surface, using is extremely ultra thin (ca 20 nm). In contrast, for thicker electron beam irradiation. As a second step, PIPAAm was layer, the grafted PIPAAm chains at the outermost surfaces grafted onto the hydrophilic PAAm-TCPSs are not subjected to the progressive immobility and (PIPAAm-PAAm-TCPS). These surfaces were dehydration intensively. Consequently, resulting characterized by XPS, contact angle measurement and outermost surfaces are cellrepellentsurfaces. FT-IR / ATR. We also further investigated cell Cells are detached from PIPAAm-TCPS with 2Onm attachment, detachment properties and cells sheet recovery thickens of the grafted PIPAAm by lowering temperature. in response to temperature, using PIPAAm-PAAm-TCPSs In this case, spontaneous cell sheet detachment from and, finally, we discussed the phenomenon in terms of PIPAAm is relatively a slow process, occurring gradually molecular motion of the grafted polymers chains and role of from the sheet periphery toward the interior (Fig. 1 (upper)). the grafted PAAm for the grafted PIPAAm chains. Thus, significant incubation time at reduced temperature is required to lift up an intact cell sheet completely. In order 2. EXPERIMENTAL 978-1l-4244-]l 858-9/07/$25.00 ©2007 IEEE -173 -