A 0.5-T pure-in-plane-field magnetizing holder for in-situ Lorentz microscopy.

A side-entry specimen holder capable of applying a 0.5-tesla in-plane magnetic-induction field for in-situ transmission electron microscopy was developed. Three miniaturized electromagnets with 300 × 300-µm pole area and 180-µm pole gap are stacked along the electron-beam path in the holder. The middle magnet is used for magnetizing the specimen, which is inserted into the pole gap by using a 40-µm-width cantilever for atomic-force microscopy. The upper and lower magnets are used to keep the electron beam parallel to the optical axis. Magnetic-field magnitude was determined on the basis of experimentally measured electron-deflection angles and induction-field profiles along the electron-beam path calculated by finite element electromagnetic simulation. Magnetization reversal in 300-nm-thick Nd-Fe-B magnets from the saturated state was in-situ observed by using the holder and a 1-MeV cold-field-emission transmission electron microscope. The observation revealed that domain-wall pinning occurred in different manners at the c-plane and non-c-plane grain boundaries. The holder was thereby shown to be useful for analysis of magnetization-reversal behaviors of hard magnetic materials.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

Development of a cell sheet transportation technique for regenerative medicine.

PURPOSE: A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. MATERIAL AND METHODS: We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. RESULTS: During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. CONCLUSION: The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies.