A hybrid combination of in vitro cultured buccal mucosal cells using two different methodologies, complementing each other in successfully repairing a stricture-inflicted human male urethral epithelium.

BACKGROUND: Autologous buccal mucosa cell transplantation has emerged as a promising treatment strategy for urethral stricture disease. However, ambiguity has persisted regarding the optimal cell type and culture conditions that aid successful urethral repair. Clinical study of our previously reported cell-based endoscopic approach, the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS), demonstrated durable epithelial regeneration and long-term urethral patency. The present work provides mechanistic insights supporting the BEES-HAUS approach of combining two-dimensional (2D) monolayer-cultured fibroblast-like cells and three-dimensional (3D) thermo-responsive gelation polymer (Festigel)-cultured cells. METHODS: Human buccal tissues (n=22) were cultured in two methods; one portion using the monolayer method (2D), and the other in 3D using Festigel. Flow cytometry for phenotype markers and ELISA for IGF-1 were carried out. RESULTS: 3D Festigel-cultured cells acquired an epithelial phenotype, with AE1/AE3 expression up to day 21, while 2D cultures yielded fibroblast-like CD140b-positive/AE1-AE3-negative cells. IGF-1 secretion was significantly higher in 2D cultures than 3D Festigel (p < 0.05), indicating a supportive paracrine role. These findings explain the complementary contribution of epithelial integration and IGF-1-mediated support observed as successful clinical outcome of the BEES-HAUS procedure. CONCLUSION: This study, a first of its kind, clarifies the rationale and advantages of combining 3D Festigel-expanded epithelial cells with the paracrine effect of IGF-1-secreting 2D fibroblast-like cells in a single transplantation strategy, thereby explaining the successful clinical outcomes reported in BEES-HAUS. Further research on this hybrid cell combination is recommended to expand this approach for regenerating and repairing other tissues and organs.