Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage ma...Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.展开更多
For regenerative medicine, clarification of in vivo migration of transplanted cells is an important task to secure the safety of transplanted tissue. We had prepared tissue-engineered cartilage consisting of cultured ...For regenerative medicine, clarification of in vivo migration of transplanted cells is an important task to secure the safety of transplanted tissue. We had prepared tissue-engineered cartilage consisting of cultured chondrocytes with collagen hydrogel and a biodegradable porous polymer, and we clinically applied it for treatment of craniofacial anomaly. To verify the safety of this tissue-engineered cartilage, we had syngenically transplanted the tissue-engineered cartilage using chondrocytes harvested from EGFP-transgenic mice into subcutaneous pocket of wild type mice, and investigated localizations of transplanted chondrocytes in various organs including cerebrum, lung, liver, spleen, kidney, auricle, gastrocnemius, and femur. After 8 to 24 weeks of the transplantation, accumulation of cartilaginous matrices was observed in tissue-engineered cartilage, while EGFP-positive transplanted chondrocytes were localized in this area. Otherwise, no EGFP was immunohistochemically detected in each organ, suggesting that subcutaneously-transplanted chondrocytes do not migrate to other organs through the circulation. In cartilage tissue engineering using cultured chondrocytes, risk for migration and circulation of transplanted cells seemed negligible, and that ectopic growth of the cells was unlikely to occur, showing that this is safe technique with regard to the in vivo migration of transplanted cells.展开更多
文摘Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.
文摘For regenerative medicine, clarification of in vivo migration of transplanted cells is an important task to secure the safety of transplanted tissue. We had prepared tissue-engineered cartilage consisting of cultured chondrocytes with collagen hydrogel and a biodegradable porous polymer, and we clinically applied it for treatment of craniofacial anomaly. To verify the safety of this tissue-engineered cartilage, we had syngenically transplanted the tissue-engineered cartilage using chondrocytes harvested from EGFP-transgenic mice into subcutaneous pocket of wild type mice, and investigated localizations of transplanted chondrocytes in various organs including cerebrum, lung, liver, spleen, kidney, auricle, gastrocnemius, and femur. After 8 to 24 weeks of the transplantation, accumulation of cartilaginous matrices was observed in tissue-engineered cartilage, while EGFP-positive transplanted chondrocytes were localized in this area. Otherwise, no EGFP was immunohistochemically detected in each organ, suggesting that subcutaneously-transplanted chondrocytes do not migrate to other organs through the circulation. In cartilage tissue engineering using cultured chondrocytes, risk for migration and circulation of transplanted cells seemed negligible, and that ectopic growth of the cells was unlikely to occur, showing that this is safe technique with regard to the in vivo migration of transplanted cells.
