RESUMO
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.
Assuntos
Técnicas de Cultura de Células/instrumentação , Células Epiteliais/citologia , Engenharia Tecidual/métodos , Células 3T3 , Animais , Automação , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Claudina-1/metabolismo , Desenho de Equipamento , Células Alimentadoras , Gases , Imuno-Histoquímica , Queratina-3/metabolismo , Camundongos , Microscopia de Contraste de Fase , Oxigênio/química , Permeabilidade , Fosfoproteínas/metabolismo , Porosidade , CoelhosRESUMO
We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.
Assuntos
Automação , Técnicas de Cultura de Células/métodos , Córnea/fisiologia , Células Epiteliais/citologia , Células Epiteliais/transplante , Epitélio Corneano/citologia , Regeneração , Animais , Humanos , Modelos Animais , Coelhos , Procedimentos de Cirurgia Plástica , Temperatura , Meios de TransporteAssuntos
Eletrocoagulação/instrumentação , Hemostasia Cirúrgica/instrumentação , Microcirurgia/instrumentação , Neuroendoscópios , Procedimentos Neurocirúrgicos/instrumentação , Instrumentos Cirúrgicos , Procedimentos Cirúrgicos Vasculares/instrumentação , Animais , Eletrocoagulação/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Hemostasia Cirúrgica/métodos , Humanos , Micromanipulação/instrumentação , Micromanipulação/métodos , Microcirurgia/métodos , Miniaturização , Procedimentos Cirúrgicos Minimamente Invasivos/instrumentação , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Procedimentos Neurocirúrgicos/métodos , Ratos , Estresse Mecânico , Procedimentos Cirúrgicos Vasculares/métodosRESUMO
OBJECTIVE: Microneurosurgery can be performed less invasively with the recent advances in neuronavigation and neuroendoscopy. For even less invasive microneurosurgery, we have developed a telecontrolled micromanipulator system. METHODS: The NeuRobot telecontrolled micromanipulator system was developed. With the use of this system, surgical simulations were performed with a human cadaveric head. RESULTS: The system consists of four main parts, i.e., a micromanipulator (slave manipulator), a manipulator-supporting device, an operation-input device (master manipulator), and a three-dimensional display monitor. Three 1-mm forceps and a three-dimensional endoscope, which could be remotely controlled with three degrees of freedom (rotation, neck swinging, and forward/backward motion), were installed in the slave manipulator. All surgical procedures were accurately performed with this system. CONCLUSION: The use of telecontrolled manipulator systems in neurosurgery is very promising, and we are convinced that this system will facilitate more accurate, less invasive microneurosurgery. The details of the NeuRobot system and preliminary results are presented.