<b>Establishment and characterization of multilayered fibroblast cell sheets from the canine oral mucosa, skin, and tail skin</b>

Background: Cell sheet therapy has been developed as an effective regenerative medicine to improve wound repair treatment and prevent postoperative complications. Fibroblasts are widely used to create cell sheet engineering because of their essential role in tissue regeneration and the acceleration of the wound healing process. Aim: The study aimed to establish the multilayered fibroblast sheets from canine fibroblast. Methods: The study used fibroblasts from three origin canine tissues, including oral mucosa, skin, and tail skin. A canine fibroblast sheet was produced using the large-numbers cell seeding method with Rho kinase inhibitor. The assessment included viability cells, histological evaluation of the sheet, and secretion of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) of the fibroblast sheet. Results: The three fibroblast groups successfully established a multilayered fibroblast sheet. Hematoxylin and eosin staining examination showed the thickest sheet in the oral mucosa fibroblast sheet. Immunohistochemical examination revealed that the multilayered cell sheet comprised fibroblasts expressed by vimentin. All multilayered fibroblast sheet groups secreted the VEGF and MCP-1. No differences were observed in the viability cells of each origin cell. Conclusion: The multilayered fibroblast sheets were successfully established from canine fibroblast. This finding provides the primary data to develop regenerative medicine in the veterinary field.