Kazuhiro Fukumori

Temperature-responsive glass coverslips with an ultrathin poly(N-isopropylacrylamide) layer

A temperature-responsive cross-linked polymer gel was covalently grafted onto glass coverslips by electron beam irradiation. The grafted thickness and amount of polymer as well as the surface wettability increased with the initial monomer concentration. When the monomer concentration was 5 wt.%, the grafted polymer density was 0.84microgcm(-2), and cells adhered and spread on the surface at 37 degrees C, but detached at 20 degrees C. In contrast, when the monomer concentration was 35 wt.%, the polymer density was 1.28microgcm(-2), and the surfaces were cell repellent even at 37 degrees C. These results show a remarkable contrast to those obtained from temperature-responsive polymer-grafted tissue culture polystyrene dishes, since various types of cells showed temperature-dependent cell adhesion/detachment when the grafted density was around 2microgcm(-2) on these surfaces. We discuss the possible molecular mechanisms underlying this discrepancy.

Characterization of Ultra‐Thin Temperature‐Responsive Polymer Layer and Its Polymer Thickness Dependency on Cell Attachment/Detachment Properties

Ultra thin poly(N-isopropylacrylamide) (PIPAAm) modified glass coverslips (PIAPAm-CS) using electron beam irradiation exhibited a clear relationship between the polymer thickness and thermal cell adhesion/detachment behavior. The polymer thickness dependency and the characteristic of ultra thin PIPAAm layer, has been illustrated in terms of the molecular motion of the modified PIPAAm chains. PIPAAm-CSs surfaces with various area-polymer densities and thicknesses were characterized by AFM and protein adsorption assay. The newly obtained results gave a further insight into the illustration. Finally, the future application of intelligent surfaces was discussed for fabricating tissue and organ.

Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing

Cell-sheet-engineering based regenerative medicine is successfully applied to clinical studies, though cell sheets contain uniformly distributed cells. For the further application to complex tissues/organs, cell sheets with a multi-cellular pattern were highly demanded. Micro-contact printing is a quite useful technique for patterning proteins contained in extracellular matrix (ECM). Because ECM is a kind of cellular adherent molecules, ECM-patterned cell culture surface is capable of aligning cells on the pattern of ECM. However, a manual printing is difficult, because a stamp made from polydimethylsiloxane (PDMS) is easily deformed, and a printed pattern is also crushed. This study focused on the deformability of PDMS stamp and discussed an appropriate stamping force in micro-contact printing. Considering in availability in a medical or biological laboratory, a method for assessing the stamp deformability was developed by using stiffness measurement with a general microscope. An automated stamping system composed of a load cell and an automated actuator was prepared and allowed to improve the quality of stamped pattern by controlling an appropriate stamping force of 0.1 N. Using the system and the control of appropriate stamping force, the pattern of 8-mm-diameter 80-μm-stripe fibronectin was fabricated on the surface of temperature-responsive cell culture dish. After cell-seeding and cell culture, a co-culture system with the micro-pattern of both fibroblasts and endothelial cells was completed. Furthermore, by reducing temperature to 20 °C, the co-cultured cell sheet with the micro-pattern was successfully harvested. As a result, the method would not only provide a high-quality ECM pattern but also a breakthrough technique to fabricate multi-cellular-patterned cell sheets for the next generation of regenerative medicine and tissue engineering.

Modulation of cell adhesion and detachment on thermo-responsive polymeric surfaces through the observation of surface dynamics.

Various thermo-responsive polymeric surfaces were evaluated in terms of cell adhesion/detachment and surface analysis. Three kinds of thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) surfaces were prepared by an electron beam irradiation (PIPAAm-EB), a reversible addition fragmentation polymerization (PIPAAm-RAFT), and a redox polymerization (PIPAAm-Redox). Although cell adhesion and detachment on surfaces of PIPAAm-EB and PIPAAm-RAFT were able to be modulated by altering their surface characters with changing the amounts of polymers, the adhesion and detachment were hardly controlled on PIPAAm-Redox surfaces, even though the amounts of polymers on the surface were able to be modulated. Atomic force microscopy (AFM) probed the interactions between AFM tip and the polymeric surface for further investigating a different conformation of polymeric surface. The modification of AFM tip surface coated with octadecyltrichlorosilane was found to change the interaction between the thermo-responsive surface and the tip. Adhesion force analysis clearly showed changes in the hydrophilic/hydrophobic characters of three kinds of thermo-responsive surfaces immediately after a change in temperature. From the kinetics study of AFM, PIPAAm-EB and PIPAAm-RAFT surfaces became hydrophilic less than 30 min after temperature decrease, but PIPAAm-Redox surfaces required 120 min to become hydrophilic after temperature reduction. These results indicated that a faster conformational change triggered cell detachment and a slow conformation change hardly affected cell detachment. Therefore, polymeric conformation on solid substrate was an important factor for modulating cell adhesion and detachment.

Stamp-stiffness calibrated micro contact printing

Micro contact printing is a quite useful technique for patterning protein such as cellular adhesion molecular. Especially, the method is popular to fabricate substrates for cell patterning in tissue engineering. However, a manual printing is difficult, because the stamp made from polydimethylsiloxane (PDMS) is easily deformed, and a printed pattern is also crushed. This study focused on the stiffness of PDMS stamp and discussed a micro-contact-printing controlled stamping force for a higher quality of pattern than that of manual. Considering in availability in medical or biological laboratory, the measurement method of stamp stiffness was developed by using a general microscope. The proposed stamping method gave a high printing-quality with 2.5% error of stamping area. Stamping setup controlling stamping force was developed, and a protein was patterned on a cell-culture substrate by the setup. By using the protein-patterned substrate, two-cell-patterned co-culture was successfully performed.

Multiple micro-contact printing of extra cellular matrix with fine alignment

This study performed multiple micro-contact printing of extra-cellular matrix with a fine alignment system for the position and the posture of polydimethylsiloxane (PDMS) stamp. The position and the posture were adjusted with a 4-axis automated stage with a resolution of 1 μm or 0.0025 deg. The adjustment was achived by monitoring the surface shape of PDMS stamp measured by using a laser displacement sensor in the alignment system. After the adjustment, fluorescent-dye-conjugated fibronectin applied on the surface of PDMS stamp was printed onto a cell culture dish. As the result of multiple printing, two different patterns of fibronectin were located on the same dish within an error of 50 μm.

'Erratum to "Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing" [Biomaterials 35 (2014) 9802-10]'.

‘Erratum to “Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing” [Biomaterials 35 (2014) 9802e10]’ Nobuyuki Tanaka a, , Hiroki Ota , Kazuhiro Fukumori , Jun Miyake , Masayuki Yamato , Teruo Okano a, * a Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Womens Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan b Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan

Noncontact fine alignment for multiple microcontact printing

This study proposed a method for the fine alignment of polydimethylsiloxane (PDMS) stamp used in multiple microcontact printing. The procedure of alignment method was organized into three steps; (1) the fabrication of PDMS stamp with an alignment marker, (2) the position detection of PDMS stamp with a noncontact laser displacement sensor on an automated planar x-y axes stage, and (3) the position adjustment of PDMS stamp to target position with another automated planar x-y-θ axes stage. The procedure was able to perform without contact onto the surface of PDMS stamp just before the moment of contact printing. Therefore, the surface would be safe from molecular/bacterial contamination. This study implemented the procedure into a developed system for the alignment of PDMS stamp. After adjusting the position of PDMS stamps applied with fibronectin-binding-fluorescent dyes with three different colors, contact printing was performed. As a result, the error of position between two different patterns of fibronectin was within 33 μm. This method would be useful for generating a multi-type-cellular tissue in tissue engineering and regenerative medicine.

New facile method for preparing themperature-resopnsive cell culture surfaces using a thioxantone-based photoinitiator immobilized polystyrene surface

PIPAAm grafted polystyrene (PIPAAm-Pst) dishes were prepared using visible light irradiation and polystyrene dishes modified with thioxanethone-based photoinitiator. At optimized preparation conditions, PIPAAm-PSs fabricating cell sheets were successfully prepared by 5 mins visible light irradiation. After PIPAAm polymerization, micro-patterned temperature responsive polystyrene culture surface (MPTRCS) were generated by additional micro-patterned polymerization of PAAm. Endothelial cells were attached selectively PIPAAm region at 37°C. By lowing temperature, micro-pattered endothelial cells were detached from MPTRCS as continuously micro-pattered cell sheet.

Sociocytology Illuminated by Reconstructing Functional Tissue with Cell Sheet Based Technology

To create cell-dense functional tissues, the authors have developed a new class of tissue reconstructing technology, called “cell-sheet engineering”. In contrast to biodegradable scaffold-based three-dimensional (3D) tissue engineering, cell sheet-based bioassembler technology can reconstruct cell-dense tissues with extracellular matrix (ECM). For further mimicking living tissues and organs such as the liver and myotube, novel 3D bioassembler technologies are required for controlling the micro-scaled alignment of cells. This chapter summarizes the reconstruction of functional tissues with cell-sheet based technologies including (1) functional liver tissues using co-culture system of hepatocyte/nonparenchymal cells, (2) micro-patterned co-culture by microcontact printing systems with fine alignment, and (3) multi-layered oriented myotube tissues using anisotropic cell sheets and gelatin gel coated devices. Eventually, the fabrications of functional tissues and organs with bioassembler technology would lead to illuminating “sociocytology”.