title: Towards a defined, serum- and feeder-free culture of stratified human oral mucosal epithelium for ocular surface reconstruction.
abstract: OBJECTIVE
  Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium.
  
  
  METHODS
  In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration.
  
  
  RESULTS
  In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values.
  
  
  CONCLUSIONS
  In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.
authors.affiliation: Institute of Biomedical Technology, University of Tampere, Tampere, FinlandBioMediTech, Tampere, FinlandDepartment of Eye, Ear, and Oral Diseases, Tampere University Hospital, Tampere, FinlandSILK, Department of Ophthalmology, University of Tampere, Tampere, FinlandEye Center, Tampere University Hospital, Tampere, Finland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Ilmarinen
authors.forename: Juhana
authors.initials: J
authors.lastname: Laine
authors.forename: Kati
authors.initials: K
authors.lastname: Juuti-Uusitalo
authors.forename: Jura
authors.initials: J
authors.lastname: Numminen
authors.forename: Riitta
authors.initials: R
authors.lastname: Seppänen-Suuronen
authors.forename: Hannu
authors.initials: H
authors.lastname: Uusitalo
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
mesh_terms: D000328:Adult; D015415:Biomarkers; D018929:Cell Culture Techniques; D003316:Corneal Diseases / Q000601:surgery*; D016895:Culture Media, Serum-Free; D017097:Electric Impedance; D004847:Epithelial Cells / Q000637:transplantation*; D004848:Epithelium; D061252:Feeder Cells; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology* / Q000378:metabolism; D062793:Occludin / Q000378:metabolism; D019651:Plastic Surgery Procedures; D023822:Tissue Engineering; D055815:Young Adult; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; C568985:OCLN protein, human; D062793:Occludin; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein