Development of Frozen-thawed Allogenic Fibroblast Sheets for Esophageal Anastomotic Leakage

Abstract

Background & Aim

Gastrointestinal anastomotic leakage is a severe postoperative complication, and its effective preventive measures remain unestablished. We have developed allogeneic multilayered fibroblast sheets, which promote wound healing in various organs. When considering clinical applications, the cryopreserved cell sheets can be transplanted immediately after thawing. However, frozen-thawed allogenic fibroblast sheets have not been investigated for gastrointestinal anastomotic leakage. This study examined the prevention of anastomotic leakage by transplanting frozen-thawed allogeneic multilayered fibroblast sheets into an esophageal anastomosis model.

Methodology

Multilayered fibroblast sheets were prepared by culturing the oral mucosa of Sprague–Dawley rats for 9 days. The esophageal anastomosis model was established by transecting the esophagus of Wistar/ST rats and performing reanastomosis with three-point interrupted sutures. The multilayered fibroblast sheets were cryopreserved in an ultralow temperature freezer after freezing in a three-dimensional freezer. The rats were categorized into the control (suture only) and fibroblast sheet groups (in which two fibroblast sheets were transplanted) (Fig. 1). The incidence of anastomotic leakage and burst pressure was evaluated 3 and 5 days after transplantation. Furthermore, growth factors secreted by the fibroblast sheets and the collagen expression levels in the tissue were assessed.

Results

The incidence of anastomotic leakage was significantly lower in the fibroblast sheet group (allogeneic multilayered fibroblast sheet transplantation) than in the control group (suture only) (Table 1).Furthermore, the burst pressure was significantly higher in the fibroblast sheet group than in the control group (day 5: 78 ± 26 mmHg vs 124 ± 30 mmHg; p < 0.01).The mRNA expression level of type I collagen 5 days postoperatively was significantly higher in the fibroblast sheet group than in the control group (control group: 8.35 ± 3.77 vs cell sheet group: 19.58 ± 10.28; p = 0.05). Additionally, the quantitative analysis of type I collagen 5 days postoperatively revealed significantly higher levels in the fibroblast sheet group than in the control group (control group: 0.09 ± 0.04 μg vs fibroblast sheet group: 0.14 ± 0.03 μg; p = 0.04).

Conclusion

The transplantation of frozen-thawed allogeneic multilayered fibroblast sheets reduced the incidence of anastomotic leakage and burst pressure, indicating their potential to help prevent esophageal anastomotic leakage.