Abstract
BackgroundAutologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization.MethodsRat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs).ResultsCOMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs.ConclusionsLNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.
Highlights
Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency
Cultivated oral mucosal epithelial cells (COMECs) are obtained by co-culturing with limbal niche cells (LNCs) or 3T3 cells oral mucosal epithelial cells (OMECs) were expanded using the culture model described above (Fig. 1a)
LNCs support the growth of OMECs To further investigate the characteristics of OMECs, we examined the expression of several cell markers by immunocytochemistry
Summary
Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. Limbal stem cells (LSCs) play a vital role in maintaining a healthy ocular surface [1]. Significant damage to LSCs can lead to limbal stem cell deficiency (LSCD) characterized by pain, loss of vision, corneal neovascularization and conjunctivalization, chronic corneal inflammation, and subsequent scarring [2]. With improved stem cell technology, a variety of cell-based therapies have been suggested for LSCD to reconstruct the ocular surface [3]. Autologous cultivated limbal epithelial transplantation (CLET) has been reported as an effective and safe treatment [4, 5]. Autologous cultivated oral mucosal epithelial transplantation (COMET) was proposed to treat bilateral LSCD and achieved satisfactory results [7,8,9,10]
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