Off-the-Shelf Cell Sheets as a Pleural Substitute for Closing Visceral Pleural Injuries

During pulmonary resections, removal of visceral pleura is frequently required, resulting in lung air leakage (LAL) and bleeding. Especially persistent LAL after pulmonary surgery has negative consequences. Current surgical procedures are ineffective in closing these visceral pleural injuries. Previously, the authors' laboratory has developed a novel and effective LAL sealant using tissue-engineered cell sheets harvested from temperature-responsive culture dishes. The clinical application of fresh fibroblast sheets (FSs) is limited by several problems related to the cell culture period, mass production, preservation, and transportation. Therefore, cryopreservation of FSs and feasibility of off-the-shelf FSs for repairing visceral pleural defects were investigated. Over 3 to 6 months, harvested skin-derived FSs in Dulbecco's modified Eagle's medium supplemented with 10% dimethyl sulfoxide were stored in an atmosphere of liquid nitrogen. The amounts of cytokines (basic fibroblast growth factor [bFGF] and vascular endothelial growth factor) released from frozen-thawed FSs were determined. bFGF levels were significantly elevated in frozen-thawed FSs compared with fresh FSs. After a visceral pleural injury model was created, a frozen-thawed skin-derived FS was transplanted directly to the defect. One month after transplantation, the frozen-thawed FS remained on the pleural surface, resulting in permanent closure, suggesting that cells in the off-the-shelf FS had the ability to proliferate and release various cytokines. Frozen-thawed FSs were useful for closing LALs during pulmonary surgery as an off-the-shelf technique and would be used as a pleural substitute.