Volume 143, Issue 3, Pages 582-588.e2 (September 2012) Prevention of Esophageal Stricture After Endoscopic Submucosal Dissection Using Tissue-Engineered Cell Sheets  Takeshi Ohki, Masayuki Yamato, Masaho Ota, Ryo Takagi, Daisuke Murakami, Makoto Kondo, Ryo Sasaki, Hideo Namiki, Teruo Okano, Masakazu Yamamoto  Gastroenterology  Volume 143, Issue 3, Pages 582-588.e2 (September 2012) DOI: 10.1053/j.gastro.2012.04.050 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Treatment of the artificial ulceration after esophageal ESD by transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture inserts. (1) Biopsy specimens were taken from the patient's own oral buccal mucosal tissue. Oral epithelial cells were isolated from the tissue by proteolytic enzyme treatment. (2) The epithelial cells were seeded onto temperature-responsive culture inserts without a 3T3 feeder layer and cultured with autologous serum for 16 days at 37°C. (3) Oral mucosal epithelial cell sheets (23 mm in diameter) were harvested by reducing the culture temperature to 20°C. Bar = 50 μm. (4) Autologous oral mucosal epithelial cell sheets on a support membrane were transplanted with endoscopic forceps onto the bed of the esophageal ulceration immediately after ESD. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Method of endoscopic cell sheet transplantation. (A) The cell sheet on a support membrane passed through the EMR tube. The membrane was passed down to the esophagus with the side attaching the cell sheet facing the inside and the side without the cell sheet facing the esophageal wall. (B) An entrance balloon was inflated to keep cavity dilated. (C) Operation of the scope continued as the entrance balloon maintained a wide working space by preventing air leakage. (D) When the cell sheet reached the ulceration site, the membrane was rotated 180 degrees with endoscopic forceps so that the side of the membrane containing the cell sheet now faced the ulceration site. (E) The cell sheet was placed directly onto the esophageal ulceration. (F) The cell sheet was held in place for about 10 minutes. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 A case with esophageal stricture prevented by the application of the transplantation of cell sheets (case 1). (A) A 70-year-old man underwent transplantation of 2 cell sheets. (B) One week after transplantation, epithelialization was observed at the center of the ulcerative site. (C) Three weeks after transplantation, epithelialization was endoscopically observed to be almost complete. (D) Four weeks after transplantation, complete epithelialization was observed without any evidence of stricture. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 A case with esophageal stricture prevented by the application of the transplantation of cell sheets (case 6). (A) A 55-year-old man underwent transplantation of 7 cell sheets in a spotted manner. (B) One week after transplantation, novel epithelia were regenerated from the transplanted sites. (C) Three weeks after transplantation, complete epithelialization was observed without any evidence of stricture. (D) Three weeks after transplantation, iodine staining also confirmed the complete epithelialization. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 The healing process of the ulcer after esophageal ESD without cell sheet transplantation and a small biopsy from the center of the ulceration. A and B show the wound healing process after ESD without cell sheet transplantation. (A) The markings of the esophageal tumor immediately before ESD. (B) Four weeks after esophageal ESD without cell sheet transplantation. The healed scar shrank and focused in one point in a radial fashion. Strong staining by iodine areas indicates glycogenic acanthosis. (C) Schematic drawing of wound healing without cell sheet transplantation. Without cell sheet transplantation, wound healing is mainly caused by the elongation of native esophageal epithelia around the wound and the contraction of the esophageal wall to reduce the ulcer size. (D) Immunohistochemistry of a small biopsy specimen from the center of the ulceration at 1 week with anti-pancytokeratin antibodies to detect transplanted epithelial cells in case 9. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 A case with esophageal stricture (case 4). (A) Very large esophageal ulceration (11 cm in diameter, involving almost a whole circumference, and expanding to the esophagogastric junction). (B) One week after transplantation of 7 cell sheets to the posterior wall. (C) The epithelial tissue of the posterior wall was thicker than that of the anterior wall. (D) The patient underwent endoscopic balloon dilation 21 times. Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 The surface of temperature-responsive dish is hydrophobic at 37°C which allows cell adhesion, spreading, and proliferation. However, the same surface becomes hydrophillic once the temperature is reduced to less than 32°C and all the cells cultured on it are detached without any need for proteolytic enzymes like trypsin. Confluently cultured cells are harvested as a single contiguous cell sheet. (1) Culture condition on temperature-responsive dishes at 37°C. (2) Harvest of cell sheet with attached protein (after decreasing of temperature). Gastroenterology 2012 143, 582-588.e2DOI: (10.1053/j.gastro.2012.04.050) Copyright © 2012 AGA Institute Terms and Conditions