Takehiro Nishikawa

Honeycomb-patterned thin films of amphiphilic polymers as cell culture substrates

Abstract Recently, we have found honeycomb patterns with sub-micron line width that form during the non-equilibrium process of cast film formation. The honeycomb-patterned films were fabricated using four macromolecular compounds (amphiphilic copolymers containing lactose units or carboxyl groups as side-chains and polyion complexes composed of anionic polysaccharides). The specific binding of lactose by lectin confirmed that the lactose moieties contained in the honeycomb films work as biologically active ligands. Bovine serum albumin (BSA) labeled with fluorescein was covalently attached to the honeycomb films using water-soluble carbodiimide (WSC) as an activator. Using fluorescence imaging of the modified film, we could show that the proteins are immobilized on the honeycomb patterns. Adhesion of bovine aorta endothelial cells (ECs) to the honeycomb films indicates that the honeycomb structure works as an adhesive site for the cells.

Mesoscopic patterning of cell adhesive substrates as novel biofunctional interfaces

In this report, cell adhesion to honeycomb-patterned films is described with respect to the dimensions of the honeycomb structure. The honeycomb-patterned films can be fabricated by casting a dilute solution of amphiphilic polymers on solid substrates. The honeycomb structure is not homogeneous in all dimensions. Analysis of distribution of the honeycomb hole sizes demonstrates a gradual decrease in honeycomb hole diameter along the radius of the cast area. The largest holes were located near geometric center of the cast area. The diameter of the largest honeycomb holes in the cast area could be controlled by varying the cast volume of the polymer solution. Cell cultures on the honeycomb films demonstrated that cell adhesion could be inhibited at the outer region of the cast area. The extent of the inhibition of cell adhesion was influenced by the chemical properties of the polymers constituting the honeycomb films.

Nitric oxide release in human aortic endothelial cells mediated by delivery of amphiphilic polysiloxane nanoparticles to caveolae.

Microdomains such as lipid raft and caveolae are organized as functional compartments in plasma membrane of cells. In this study, we note the functional platform of caveolae with dual functions, internalization of external substances and cell signalings leading to nitric oxide release, and hypothesize that the switching of enzyme activity of endothelial nitric oxide synthase can be achieved by targeting caveolae with nanoparticles. We prepared polysiloxane nanoparticles and studied cellular uptake of the nanoparticles and its concomitant influence on the nitric oxide release in human aortic endothelial cells. We found that polysiloxane nanoparticles were endocytosed via caveolae in human aortic endothelial cells and that enhanced nitric oxide release was followed by the cellular uptake of the nanoparticles. Furthermore, we confirmed that endothelial nitric oxide synthase was activated during cellular uptake of the nanoparticles. These findings support our idea that delivery of the polymeric nanoparticles to endothelial cells can lead to the induction of nitric oxide release.

Stabilization of Micropatterned Polymer Films as Artificial Extracellular Matrices for Tissue Engineering

Abstract Honeycomb-patterned polymer films are formed upon self-organization of water microspheres stabilized by amphiphilic polymers. The honeycomb films can work as cell culture substrates. The films composed of heparin-cationic lipid complex, however, gradually lost the honeycomb structure when immersed in culture medium. Crosslinking of the films was carried to stabilize the honeycomb pattern. A bisazido derivative was used as a crosslinker connecting the constituent polymer of the honeycomb films. Morphological study of the immersed films revealed that the crosslinked films maintained the honeycomb structure in the phosphate buffer solution.

Honeycomb Films of Biodegradable Polymers for Tissue Engineering

Abstract : We report that microporous films (honeycomb films) can lead various types of cells to tissue formation. The honeycomb films were fabricated by applying a moist air to a spread polymer solution containing biodegradable polymers (poly(L-lactic acid) (PLLA) and poly((epsilon)-caprolactone) (PCL)) and an amphiphilic polymer. Hepatocytes were cultured on a self-supporting honeycomb film of PLLA. The hepatocytes formed a single layer of columnar shape cells with a thickness of 20 micrometer. The tissue formation of hepatocytes specifically occurred on the honeycomb film of PLLA not on a flat film of PLLA. Three dimensional tissue structures were formed when cells were cultured on both sides of the self supporting honeycomb film. Double layers of hepatocytes were obtained by the method. Striated tissues such as heart and blood vessel could be reconstructed by utilizing a stretched honeycomb film of PCL.