Takeshi Ohki

Fabrication of human oral mucosal epithelial cell sheets for treatment of esophageal ulceration by endoscopic submucosal dissection

BACKGROUND Esophageal stenosis is one of the major complications of aggressive endoscopic resection. Tissue-engineered epithelial cell grafts have demonstrated effectiveness in promoting re-epithelialization and suppressing inflammation causing esophageal scarring and stenosis after endoscopic submucosal dissection (ESD) in an animal model. OBJECTIVE To confirm the reproducibility and efficacy of a human oral mucosal epithelial cell (hOMEC) sheet cultured on temperature-responsive surface in conformity with Good Manufacturing Practice guidelines. DESIGN A preclinical study. SETTING Good Manufacturing Practice grade cell-processing center, animal laboratory. SUBJECTS Canine esophageal ulcer models, which were made by ESD. INTERVENTIONS Oral mucosal specimens were obtained from 7 healthy volunteers. MAIN OUTCOME MEASUREMENT Fabricated and transplanted hOMEC sheets were subjected to histological analysis. RESULTS The reproducibility of the fabrication of hOMEC sheets was confirmed. In this method, animal-derived materials such as 3T3 feeder layer and fetal bovine serum were successfully excluded from the culture condition. Furthermore, the environment of the culture room and safety cabinet in the cell-processing center was maintained for obtaining sterility assurances during the fabrication. Transplanted hOMEC sheets after ESD were observed to graft onto canine esophageal ulcer surfaces. LIMITATIONS Small number of subjects, animal model. CONCLUSIONS Cultured hOMEC sheets were fabricated without animal-derived materials and demonstrated efficacy as a medical device that promotes re-epithelialization of an esophageal ulcer after ESD.

Fabricated autologous epidermal cell sheets for the prevention of esophageal stricture after circumferential ESD in a porcine model.

BACKGROUND Endoscopic submucosal dissection (ESD) is an accepted treatment for early esophageal carcinoma. However, resection of a large mucosal area, as with circumferential ESD, induces severe stricture formation. OBJECTIVE To evaluate the efficacy of cultured autologous epidermal cell sheets to prevent severe esophageal constriction after circumferential ESD. DESIGN Animal study. SETTING University institute. INTERVENTION Eight pigs underwent circumferential esophageal ESD while under general anesthesia. In 4 pigs, fabricated autologous epidermal cell sheets were endoscopically transplanted to the central ESD sites immediately after the ESD. The other 4 pigs underwent circumferential ESD only. Necropsy and histological assessment were performed at 1 and 2 weeks post-ESD. MAIN OUTCOME MEASUREMENTS Weight gain, degree of mucosal constriction, and histological assessments. RESULTS All pigs in the control group showed severe esophageal constriction after 2 weeks. The control and transplanted groups had weight gains of -10.3% and 0.3% (P = .03), respectively, and the mean degrees of constriction were 88% and 56% (P < .01), respectively. Early re-epithelialization and mild fibrosis in the muscularis were observed in the transplanted group. LIMITATIONS Animal study, small sample size. CONCLUSIONS Fabricated autologous skin epidermal cell sheets would be useful in preventing severe esophageal constriction after circumferential ESD.

Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine.

Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L‐isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory‐grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature‐responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets. Copyright

Fabrication and validation of autologous human oral mucosal epithelial cell sheets to prevent stenosis after esophageal endoscopic submucosal dissection.

Objectives: Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. Methods: The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. Results: Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. Conclusions: The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.

Collision avoidance method for mobile robot considering motion and personal spaces of evacuees

In the case of disasters such as earthquakes or Nuclear/Biological/Chemical(NBC) terrorist attacks, mobile robots, called “rescue robots,” that can work in dangerous environments instead of rescue crews in rescue missions, can be of great help. However, realizing such robot systems requires many types of technologies. In particular, path planning is an important technology that provides a mobile robot with autonomous navigation to a target destination with collision avoidance. To avoid evacuees, the robot should consider the motion of people in the near future. In this research, we propose a collision avoidance method that estimates the motions and personal spaces of the evacuees. The method consists of three steps: “estimation,” “conversion,” and “planning.” In the estimation step, the future positions of evacuees are estimated by considering their planned motions and personal spaces. Then, in the conversion step, a time axis is added to construct a 3D time-space coordinate system. Finally, in the planning step, a distance-time transform is applied to plan a safe 3D path from the robots current position to the desired goal. The proposed method has been implemented on our rescue robot simulator, and some simulation experiments were conducted to verify its usefulness.

Usefulness of clip traction in the early phase of esophageal endoscopic submucosal dissection

Background:  The usefulness of clip traction in endoscopic submucosal dissection (ESD) for early esophageal carcinoma was investigated.

Application of regenerative medical technology using tissue-engineered cell sheets for endoscopic submucosal dissection of esophageal neoplasms.

We have developed a technique for endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to an esophageal ulcer following endoscopic submucosal dissection (ESD). The epithelial cell sheets successfully prevented esophageal stricture after ESD. Key technology is that epithelial cell sheets cultured from oral mucosal tissue and attached proteins can be harvested using cell sheet technology and can be transplanted to a wound site without the use of adhesive material. This regenerative procedure can promote the epithelialization of ulceration safely and effectively. In the near future, the development of advanced endoscopic treatment of regenerative medicine shows promise.

Prevention of esophageal strictures after endoscopic submucosal dissection

Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have recently been accepted as less invasive methods for treating patients with early esophageal cancers such as squamous cell carcinoma and dysplasia of Barretts esophagus. However, the large defects in the esophageal mucosa often cause severe esophageal strictures, which dramatically reduce the patients quality of life. Although preventive endoscopic balloon dilatation can reduce dysphagia and the frequency of dilatation, other approaches are necessary to prevent esophageal strictures after ESD. This review describes several strategies for preventing esophageal strictures after ESD, with a particular focus on anti-inflammatory and tissue engineering approaches. The local injection of triamcinolone acetonide and other systemic steroid therapies are frequently used to prevent esophageal strictures after ESD. Tissue engineering approaches for preventing esophageal strictures have recently been applied in basic research studies. Scaffolds with temporary stents have been applied in five cases, and this technique has been shown to be safe and is anticipated to prevent esophageal strictures. Fabricated autologous oral mucosal epithelial cell sheets to cover the defective mucosa similarly to how commercially available skin products fabricated from epidermal cells are used for skin defects or in cases of intractable ulcers. Fabricated autologous oral-mucosal-epithelial cell sheets have already been shown to be safe.

Local Path Planner for Mobile Robot in Dynamic Environment based on Distance Time Transform Method

Abstract For unknown field explorations in disaster areas, mobile robots that can replace human workers in dangerous environments can greatly improve disaster response efforts by reducing additional risk to human life. However, realizing such robot systems requires various technologies. In particular, path planning is quite important because mobile robots in the real world are surrounded by dynamic obstacles such as people, which may hinder a robot’s activities. In this research, we propose a collision avoidance method for a mobile robot in dynamic environments, considering the near-term motion and “personal space” of dynamic obstacles. Our method consists of the following three steps: “estimation,” “conversion,” and “planning.” In the estimation step, dynamic and static obstacles are recognized and their future positions are estimated from their previous motions. Next, in the conversion step, a time axis is added to construct a 3-D time–space coordinate system. Finally, in the planning step, a distance–time transform is applied to plan a safe 3-D path from the robots current position to the desired goal. The proposed method has been implemented on our mobile robot and mobile robot simulator and experiments were conducted to verify its usefulness.

Regenerative medicine: tissue-engineered cell sheet for the prevention of post-esophageal ESD stricture.

Induced pluripotent stem (iPS) cells have captured the worlds attention and directed an unprecedented focus on regenerative medicine. The potential of iPS cells to aid in the development of new treatments for various diseases is exciting, and researchers are only beginning to discover their potential benefits for humans. iPS cells are more effective if they are interconnected with tissues; however, new technologies are needed to create and transplant these tissues. This study introduces a new connection between endoscopy and regenerative medicine in gastroenterology through specifically addressing how cell sheet technology can be a viable method of tissue creation and transplantation.