Culture and Characterization of Oral Mucosal Epithelial Cells on a Fibrin Gel for Ocular Surface Reconstruction.
OBJECTIVE
To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD).


METHODS
Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin-eosin staining.


RESULTS
Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype.


CONCLUSIONS
Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.
a Cells for Sight, Transplantation and Research Programme, University College London Institute of Ophthalmology , London , UK .|b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.
Radhika
R
Sheth
a Cells for Sight, Transplantation and Research Programme, University College London Institute of Ophthalmology , London , UK .|b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.
Michael H
MH
Neale
a Cells for Sight, Transplantation and Research Programme, University College London Institute of Ophthalmology , London , UK .|b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.|c Moorfields Eye Hospital National Health Service Foundation Trust , London , UK.
Alex J
AJ
Shortt
b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.
Isobel
I
Massie
a Cells for Sight, Transplantation and Research Programme, University College London Institute of Ophthalmology , London , UK .|b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.
Amanda J
AJ
Vernon
a Cells for Sight, Transplantation and Research Programme, University College London Institute of Ophthalmology , London , UK .|b Ocular Biology and Therapeutics Division , University College London Institute of Ophthalmology , London , UK and.
Julie T
JT
Daniels
D000328:Adult; D000933:Antifibrinolytic Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D003316:Corneal Diseases / Q000601:surgery*; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D061252:Feeder Cells; D005337:Fibrin; D019084:Fluorescent Antibody Technique, Indirect; D005782:Gels; D006801:Humans; D053535:Keratin-13 / Q000378:metabolism; D053539:Keratin-19 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology*; D019651:Plastic Surgery Procedures; D054457:Tissue Scaffolds; D014148:Tranexamic Acid / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D000933:Antifibrinolytic Agents; D015415:Biomarkers; D005782:Gels; D053535:Keratin-13; D053539:Keratin-19; D053558:Keratin-3; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014148:Tranexamic Acid; D005337:Fibrin
Fibrin gels; limbal stem cell deficiency; ocular surface reconstruction; oral mucosal epithelial cells; tranexamic acid