Esophageal regenerative therapy using cell sheet technology

Abstract

We have been conducting research on esophageal regenerative therapy using cell sheet technology. In particular, in the endoscopic field, we have pushed forward clinical research on endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to esophageal ulcer after endoscopic submucosal dissection (ESD). We started research in this direction using animal models in 2004 and performed clinical research in 2012 in collaboration with Nagasaki University and Karolinska Institute. Although in full-circumferential cases it was difficult to prevent esophageal stricture after ESD, there were no complications and stricture could be suppressed. The cell sheet technology is still in its infancy. However, we are convinced that it has a high potential for application in various areas of gastrointestinal science. In this review, we focus on the pre-clinical and clinical trial results obtained and on the theoretical aspects of (1) stricture prevention, (2) esophageal tissue engineering research, and (3) endoscopic transplantation, and review the esophageal regenerative therapy by cell sheet technology.

Keywords: CMC, carboxymethyl cellulose; CPC, cell-processing center; Cell sheet technology; EBD, endoscopic balloon dilation; ECM, extracellular matrix; EMR, endoscopic mucosal dissection; ESD, endoscopic submucosal dissection; Endoscopic submucosal dissection (ESD); Endoscopic transplantation; Esophageal stricture; GMP, good manufacturing practice; OMECS, oral mucosal epithelial cell sheet; PGA, polyglycolic acid; PIPAAm, poly(N-isopropylacrylamide); PVDF, polyvinylidene difluoride; Regenerative medicine; SEMS, self-expandable metallic stent; TAC, triamcinolone; Tissue-engineered oral mucosal.

Fig. 1

Technology of temperature-responsive cell culture dishes. The surface of temperature-responsive dishes is hydrophobic at 37 °C, which allows cell adhesion, spreading, and proliferation. The same surface becomes hydrophilic once the temperature is reduced to <32 °C, and all the cells cultured on it are detached without the need for proteolytic enzymes such as trypsin. Cells cultured until confluence are harvested as a single contiguous cell sheet. (a) Culture condition on temperature-responsive dishes at 37 °C. (b) Harvest of the cell sheet with attached protein (after decreasing the temperature). Adapted with permission from Elsevier [7].

Fig. 2

Treatment of the artificial ulceration after esophageal ESD by transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture inserts. (a) Biopsies were taken from the patient's own oral buccal mucosal tissue. Oral epithelial cells were isolated from the tissue by proteolytic enzyme treatment. (b) 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. (c) Oral mucosal epithelial cell sheets (23 mm in diameter) were harvested by reducing the culture temperature to 20 °C. Scale bar = 50 micron. (d) 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. Adapted with permission from Elsevier [7].

Fig. 3

Endoscopic transplantation of cell sheets. (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 center of the esophagus 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 endoscope 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 by 180° using a pair of endoscopic forceps so that the side of the membrane containing the cell sheet faced the ulceration site. (e) The cell sheet was placed directly onto the esophageal ulceration site. (f) The cell sheet was held in place for approximately 10 min. Adapted with permission from Elsevier [7].

Fig. 4

Clinical research workflow of cell sheets delivered by air. Oral mucosal tissues were removed from patients at Nagasaki University and transported to Tokyo Women's Medical University by air. These tissues were cultured in our laboratory and then transported back to Nagasaki University Hospital for cell sheet transplantation.