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.

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

[Development of liquid phase immunoassay system using magnetic nanoparticles].

Biological immunoassay is a major detection system of biological materials from patient samples. Our group has been developing a highly sensitive immunoassay system using magnetic nanoparticles made from Fe3O4. Since unbound magnetic markers randomly move in solvent due to Brownian motion, there is no magnetic signal from unbound magnetic markers; therefore, the separation of bound from unbound markers (B/F separation) is not required. This advantage means that the detection time is greatly decreased in comparison with a normal method using fluorescent/enzyme reagent. In this paper, we describe the configuration of the developed system and demonstrate the performance of the detection of magnetic nanoparticles.

A young woman with clear cell adenocarcinoma of the uterine cervix.

In May 1999, an 18-year-old woman visited a physician because of vaginal bleeding and the excretion of large clots from the vagina. A vaginal tumor was discovered and the patient was referred to our outpatient department. Vaginal examination showed a bleeding, tumor, approximately 6 cm in size, protruding from the cervical os and filling the vagina. The cytological finding of the uterine cervix was class V, and the histological diagnosis by punch biopsy was clear cell adenocarcinoma (CCAC) of the uterine cervix. The patient initially received neoadjuvant chemotherapy (NAC) with intraarterial injections of 8 mg/m(2) of mitomycin, 270 mg/m(2) of etoposide, and 380 mg/m(2) of carboplatin. Although the NAC reduced the size of the tumor, it failed to produce favorable pathological changes and was therefore deemed ineffective. A radical abdominal hysterectomy and pelvic lymphadenectomy were performed on October 12. Macroscopic findings showed a tumor, 6 cm in diameter, growing from the right side of the uterine cervix, with a fragile, necrotic surface. Pathological diagnosis was CCAC of the cervix (pT2a, N0, M0). The patient was discharged from our hospital without any postoperative chemotherapy or radiation therapy. No signs of recurrence have been detected since. We reviewed the literature on CCAC patients in Japan up to the present and compared the data with the data reported in a review of CCAC in the Netherlands. While there were similarities between the patients in the two countries in the patients' pattern of growth and the poor prognosis of the tumors, there was a significant difference between the countries in the patients' history of diethylstilbestrol (DES) exposure. These results suggest that menarche and menopause may play roles in promoting carcinogenesis, or alternatively, that a subpopulation of women are subject to genetic or exogenous risk factors other than DES.