For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63+p40 positivity in cells ≤ 11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p <
0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.