Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells.
Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells.
Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate.
Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.
Department of Ophthalmology (E.T., D.B., L.T.), Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Biophysics and Cell Biology (E.T., D.B., B.Z., G.V.), Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Operative Techniques and Surgical Research (E.T.), Institute of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Human Genetics, Faculty of Medicine (D.B.), University of Debrecen, Debrecen, Hungary; and MTA-DE Cell Biology and Signaling Research Group (B.Z., G.V.), Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Address correspondence to Lili Takács, M.D., Ph.D., Department of Ophthalmology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Nagyerdei krt. 98, Hungary; e-mail: email@example.com
The authors have no funding or conflicts of interest to disclose.
Supported by the TÁMOP 4.2.1./B-09/1/KONV-2010-0007 project.
Accepted January 26, 2016