In this study, we evaluated the genomic stability of oral mucosal epithelial cells (OMECs) cultured in complex media (COM) and xenobiotic-free media (XF) to assess their potential clinical application for limbal stem cell deficiency (LSCD) treatments. OMECs serve as a promising autologous cell source for bilateral LSCD treatment, offering an alternative to limbal epithelial cells (LECs). However, genomic integrity is crucial to ensure the long-term success of transplanted cells. We performed micronucleus (MNi) tests and comet assays to compare DNA damage in OMECs cultured in both media types. The results indicated no significant differences in cell morphology, viability, or size between the two conditions. The MNi frequency was similar, with 5.67 and 6.17 MNi per 1,000 cells in COM and XF conditions, respectively. Comet assay results showed low levels of strand breaks (SBs) and oxidized DNA lesions in both media, with XF showing a slightly lower, albeit statistically insignificant, percentage of tail DNA for net Fpg-sensitive sites. Our findings suggest that OMECs can be effectively cultivated in either COM or XF media without inducing significant DNA damage, supporting the potential use of XF media in clinical settings to reduce contamination risks. This study underscores the importance of genomic stability in cultured cells for ocular surface transplantation, contributing valuable insights into optimizing culture conditions for safer and more effective clinical applications.

Biomedical Centre Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Department of Molecular Biology of Cancer Institute of Experimental Medicine of the Czech Academy of Sciences Prague Czech Republic

Department of Pathology 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Health Effects Laboratory Department of Environmental Chemistry and Health Effects NILU The Climate and Environmental Research Institute NILU 2007 Kjeller Norway

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University Prague Czech Republic

Laboratory of Biology and Pathology of the Eye Institute of Biology and Medical Genetics 1st Faculty of Medicine and General Teaching Hospital Charles University Czech Republic

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Thoft RA, Friend J.. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 1983;24:1442–3. PubMed

Genna VG, Maurizi E, Rama P, et al. Biology and medicine on ocular surface restoration: advancements and limits of limbal stem cell deficiency treatments. Ocul Surf 2025;35:57–67. PubMed

Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet (London, England) 1997;349:990–3. PubMed

Nakamura T, Inatomi T, Sotozono C, et al. Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease. Acta Ophthalmol Scand 2004;82:468–71. PubMed

Prabhasawat P, Ekpo P, Uiprasertkul M, et al. Long-term result of autologous cultivated oral mucosal epithelial transplantation for severe ocular surface disease. Cell Tissue Bank 2016;17:491–503. PubMed

Kengpunpanich S, Chirapapaisan C, Ngowyutagon P, et al. Comparative analysis of long-term results of three epithelial cell transplantation procedures for treating limbal stem cell deficiency. Ocul Surf 2024;32:71–80. PubMed

El-Hofi AH, Helaly HA.. Evaluation of limbal transplantation in eyes with bilateral severe ocular surface damage secondary to chemical injury. Clin Ophthalmol 2019;13:383–90. PubMed PMC

Cabral JV, Jackson CJ, Utheim TP, et al. Ex vivo cultivated oral mucosal epithelial cell transplantation for limbal stem cell deficiency: a review. Stem Cell Res Ther 2020;11:301. PubMed PMC

Barut Selver O, Yagci A, Egrilmez S, et al. Limbal stem cell deficiency and treatment with stem cell transplantation. Turk J Ophthalmol 2017;47:285–91. PubMed PMC

Schwab IR, Johnson NT, Harkin DG.. Inherent risks associated with manufacture of bioengineered ocular surface tissue. Arch Ophthalmol 2006;124:1734–40. PubMed

Galili U. Host synthesized carbohydrate antigens on viral glycoproteins as ‘achilles’ heel’ of viruses contributing to anti-viral immune protection. Int J Mol Sci 2020;21:6702. PubMed PMC

Sharma SM, Fuchsluger T, Ahmad S, et al. Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium. Stem Cell Rev Rep 2012;8:696–705. PubMed

Cabral JV, Voukali E, Smorodinova N, et al. Cultivation and characterization of oral mucosal epithelial cells on fibrin gel in a xenobiotic-free medium for the treatment of limbal stem cell deficiency. Exp Eye Res 2025;253:110300. PubMed

Attico E, Galaverni G, Torello A, et al. Comparison between cultivated oral mucosa and ocular surface epithelia for COMET patients follow-up. Int J Mol Sci 2023;24:11522. PubMed PMC

Brejchova K, Trosan P, Studeny P, et al. Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions. Exp Eye Res 2018;176:20–8. PubMed

Sharma N, Venugopal R, Mohanty S, et al. Simple limbal epithelial transplantation versus cultivated limbal epithelial transplantation in ocular burns. Ocul Surf 2024;34:504–9. PubMed

Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 2005;23:1357–66. PubMed

Lorenzo Y, Azqueta A, Luna L, et al. The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells. Carcinogenesis 2009;30:308–14. PubMed

Pathak M, Olstad OK, Drolsum L, et al. The effect of culture medium and carrier on explant culture of human limbal epithelium: a comparison of ultrastructure, keratin profile and gene expression. Exp Eye Res 2016;153:122–32. PubMed

Lorenzo Y, Haug Berg K, Ringvold A, et al. Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue. Acta Ophthalmologica 2018;96:834–40. PubMed PMC

Bonassi S, Znaor A, Ceppi M, et al. An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis 2007;28:625–31. PubMed

Fenech M, Holland N, Chang WP, et al. The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat Res 1999;428:271–83. PubMed

Collins A, Moller P, Gajski G, et al. Measuring DNA modifications with the comet assay: a compendium of protocols. Nat Protoc 2023;18:929–89. PubMed PMC

Malagutti-Ferreira MJ, Crispim BA, Barufatti A, et al. Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells. Braz J Med Biol Res 2023;56:e12713. PubMed PMC

Bruschweiler ED, Hopf NB, Wild P, et al. Workers exposed to wood dust have an increased micronucleus frequency in nasal and buccal cells: results from a pilot study. Mutagenesis 2014;29:201–7. PubMed

Jirsova K, Vesela V, Skalicka P, et al. The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells. Ocul Surf 2021;20:195–8. PubMed

Azqueta A, Runden-Pran E, Elje E, et al. The comet assay applied to cells of the eye. Mutagenesis 2018;33:21–4. PubMed

Jirsova K, Brejchova K, Krabcova I, et al. The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment. Curr Eye Res 2014;39:21–30. PubMed

Singam PK, Majumdar S, Uppala D, et al. Evaluation of genotoxicity by micronucleus assay in oral leukoplakia and oral squamous cell carcinoma with deleterious habits. J Oral Maxillofac Pathol 2019;23:300. PubMed PMC

Varga D, Johannes T, Jainta S, et al. An automated scoring procedure for the micronucleus test by image analysis. Mutagenesis 2004;19:391–7. PubMed

Titenko-Holland N, Jacob RA, Shang N, et al. Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate. Mutat Res 1998;417:101–14. PubMed

Bonassi S, Coskun E, Ceppi M, et al. The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutat Res 2011;728:88–97. PubMed

Moller P, Azqueta A, Boutet-Robinet E, et al. Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results. Nat Protoc 2020;15:3817–26. PubMed PMC

Vodenkova S, Azqueta A, Collins A, et al. An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity. Nat Protoc 2020;15:3844–78. PubMed

Daniels JT, Dart JK, Tuft SJ, et al. Corneal stem cells in review. Wound Repair Regen 2001;9:483–94. PubMed

Shibai-Ogata A, Kakinuma C, Hioki T, et al. Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging. Mutagenesis 2011;26:709–19. PubMed

Koehler C, Ginzkey C, Friehs G, et al. Ex vivo toxicity of nitrogen dioxide in human nasal epithelium at the WHO defined 1-h limit value. Toxicol Lett 2011;207:89–95. PubMed

Ventura C, Pinto F, Lourenco AF, et al. Assessing the genotoxicity of cellulose nanomaterials in a co-culture of human lung epithelial cells and monocyte-derived macrophages. Bioengineering (Basel) 2023;10:986. PubMed PMC

Møller P. Measurement of oxidatively damaged DNA in mammalian cells using the comet assay: Reflections on validity, reliability and variability. Mutat Res Genet Toxicol Environ Mutagen 2022;873:503423. PubMed

Baricevic M, Ratkaj I, Mladinic M, et al. In vivo assessment of DNA damage induced in oral mucosa cells by fixed and removable metal prosthodontic appliances. Clin Oral Investig 2012;16:325–31. PubMed

Dahl JA, Duggal S, Coulston N, et al. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. Int J Dev Biol 2008;52:1033–42. PubMed

Hirayama M, Satake Y, Higa K, et al. Transplantation of cultivated oral mucosal epithelium prepared in fibrin-coated culture dishes. Invest Ophthalmol Vis Sci 2012;53:1602–9. PubMed

Fenech M, Bonassi S.. The effect of age, gender, diet and lifestyle on DNA damage measured using micronucleus frequency in human peripheral blood lymphocytes. Mutagenesis 2011;26:43–9. PubMed

Moller P. Effect of age and sex on the level of DNA strand breaks and oxidatively damaged DNA in human blood cells. Mutat Res Genet Toxicol Environ Mutagen 2019;838:16–21. PubMed

Milic M, Ceppi M, Bruzzone M, et al. The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders. Mutat Res Rev Mutat Res 2021;787:108371. PubMed PMC