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Novel thermoresponsive polymer brush surfaces for harvesting cell sheet were fabricated by the surface-initiated RAFT polymerization of N-isopropylacrylamide (IPAAm) on azoinitiator-immobilized glass substrates in the presence of dithiobenzoate compound as a chain transfer agent (CTA). The chain length of the grafted PIPAAm on the surface was controlled by changing CTA concentration. Additionally, PIPAAm graft density on the surface was successfully regulated by grafting from azoinitiator-immobilized surfaces with various densities. By adjusting both the chain length and the density of grafted PIPAAm, a series of thermoresponsive polymer brush surfaces were prepared to regulate cell adhesion/detachment behavior by solely temperature change across the PIPAAm's lower critical solution temperature of 32 degrees C. PIPAAm brush surfaces were successfully optimized to recover the cell sheets of bovine carotid artery endothelial cells. Additionally, the immunostaining study revealed that the cell sheets can be recovered with their intact extracellular matrix (ECM) from PIPAAm surfaces, indicating that the cell sheets can be effectively transplanted to damaged tissues and organs.

Well-defined amphiphilic diblock copolymers comprising thermoresponsive polymer segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PID) and hydrophobic polymer segments, poly(benzyl methacrylate) (PBzMA), were synthesized by controlled living radical polymerization. Terminal derivatization of PID segments to either hydroxyl or phenyl groups was achieved through reactions of coupling agents with thiol groups exposed by cleavage of terminal dithiobenzoate groups. Diblock copolymers formed core-shell type polymeric micelles with thermoresponsive outer shells. Hydrodynamic micellar diameters ranged from 12 to 31 nm, controlled by varying PID chain lengths. Differences in PID terminal groups did not affect the critical micelle concentration or micellar diameters. However, these groups demonstrated a significant influence on the micellar thermoresponses. Hydroxylated PID/PBzMA micelles exhibited a phase transition of approximately 40 degrees C, independent of PID molecular weights. Even though molecular weights and compositions of PID chains were equivalent except for terminal groups, micelles having the outermost surface phenyl groups exhibited drastically lower phase transition temperature shifts, especially for micelles with low molecular weight PID chains.

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Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripe-like micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called "cell sheet" only by reducing temperature below PIPAAm's lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.

Well-defined diblock copolymers comprising thermoresponsive segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(IPAAm-co-DMAAm)) and hydrophobic segments of poly(d,l-lactide) were synthesized by combination of RAFT and ring-opening polymerization methods. Terminal conversion of thermoresponsive segments was achieved through reactions of maleimide or its Oregon Green 488 (OG) derivative with thiol groups exposed by cleavage of polymer terminal dithiobenzoate groups. Thermoresponsive micelles obtained from these polymers were approximately 25 nm when below the lower critical solution temperature (LCST) of 40 degrees C, and their sizes increased to an average of approximately 600 nm above the LCST due to aggregation of the micelles. Interestingly, the OG-labeled thermoresponsive micelles showed thermally regulated internalization to cultured endothelial cells, unlike linear thermoresponsive P(IPAAm-co-DMAAm) chains. While intracellular uptake of P(IPAAm-co-DMAAm) was extremely low at temperatures both below and above the micellar LCST, the thermoresponsive micelles showed time-dependent intracellular uptake above the LCST without exhibiting cytotoxicity. These results indicate that the new thermoresponsive micelle system may be a greatly promising intracellular drug delivery tool.

In some parts of native tissues, the orientation of cells and/or extracellular matrixes is well organized. We know that because anisotropy produces important tissue functions, an appropriate anisotropy needs to be designed to biomimetically construct complex tissue. Here, we show the unique features of anisotropic myoblast sheets for organizing the three-dimensional (3D) orientation of myoblasts and myotubes. Utilizing a micropatterned thermoresponsive surface, human skeletal muscle myoblasts were aligned on the surface, and manipulated as a single anisotropic cell sheet by reducing the culture temperature. Consequently, layering of anisotropic myoblast sheets using gelatin gel allowed 3D myotube constructs to be built up with the desired anisotropy. We also discovered a surprising myoblast behavior. An anisotropic cell sheet placed on top of other cell sheets in fabricating thick tissue was able to change the cell orientation in several layered cell sheets underneath. This self-organization is believed to provide the uniqueness required in designing 3D cell orientation architecture for reconstructed muscle tissue.

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