Naoko Senda

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. Copyright

Low cost, high resolution optical coherence tomography utilizing a narrowband laser diode

We developed a low cost, high resolution optical coherence tomography system utilizing a narrowband laser diode (LD), which is usually used in optical pickup for compact disc. To achieve high axial resolution even with the narrow bandwidth of the LD, we have constructed a free space interferometer including a phase-diversity detection system and a high numerical aperture (NA) objective. The axial and lateral resolution in the air was about 2.6 μm and 1 μm, respectively. The tomographic imaging of biological tissue was demonstrated, and the results showed that our OCT system enabled cellular-level imaging.

Spheroid imaging of phase-diversity homodyne OCT

Non-invasive 3D imaging technique is essential for regenerative tissues evaluation. Optical coherence tomography (OCT) is one of 3D imaging tools with no staining and is used extensively for fundus examination. We have developed Phase-Diversity Homodyne OCT which enables cell imaging because of high resolution, whereas conventional OCT was not used for cell imaging because of low resolution. We demonstrated non-invasive imaging inside living spheroids with Phase-Diversity Homodyne OCT. Spheroids are spheroidal cell aggregates and used as regenerative tissues. Cartilage cells were cultured in low-adhesion 96-well plates and spheroids were manufactured. Cell membrane and cytoplasm of spheroid were imaged with OCT.

Cell sheets image validation of phase-diversity homodyne OCT and effect of the light irradiation on cells

Optical coherence tomography (OCT) is one of powerful 3D tissue imaging tools with no fluorescence staining. We have reported that Phase-Diversity Homodyne OCT developed in Hitachi could be useful for non-invasive regeneration tissue evaluation test. The OCT enables cell imaging because of high resolution (axial resolution; ~2.6 μm, lateral resolution; ~1 μm, in the air), whereas conventional OCT was not used for cell imaging because of low resolution (10~20 μm). Furthermore, the OCT has advantage over other 3D imaging devices in cost because the light source and the objective were originally used as an optical pickup of compact disc. In this report, we aimed to assess effectiveness and safety of Phase-Diversity Homodyne OCT cell imaging. Effectiveness of OCT was evaluated by imaging a living cell sheet of human oral mucosal epithelial cells. OCT images were compared with reflection confocal microscopy (RCM) images, because confocal optical system is the highest resolution (<1 μm) 3D in vivo imaging technique. Similar nuclei images were confirmed with OCT and RCM, which suggested the OCT has enough resolution to image nuclei inside a cell sheet. Degree of differentiation could be estimated using OCT images, which becomes possible because the size of cells depends on distribution of differentiation. Effect of the OCT light irradiation on cells was studied using NIH/3T3 cells. Light irradiation, the exposure amount of which is equivalent to OCT, had no impact on cell shape, cell viability, and proliferation rate. It suggested that the light irradiation has no cell damage under the condition.

Human oral mucosal epithelial cell sheets imaging with high-resolution phase-diversity homodyne OCT

There is a need for development of non-invasive technique to evaluate regenerative tissues such as cell sheets for transplantation. We demonstrated non-invasive imaging inside living cell sheets of human oral mucosal epithelial cells by phase-diversity homodyne optical coherence tomography (OCT). The new method OCT developed in Hitachi enables cell imaging because of high resolution (axial resolution; ~2.6 μm, lateral resolution; ~1 μm, in the air). Nuclei inside cell sheets were imaged with sufficient spatial resolution to identify each cell. It suggested that the new method OCT could be useful for non-invasive cell sheet evaluation test.