Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Morphology and Adhesion Complex of Cultured Epithelium, on Amniotic Membrane in Vitro and in Vivo

PURPOSE: The morphologic characteristics and adhesion complex formation of cultured limbal epithelium of rabbit on amniotic membrane, in vitro and in vivo. METHODS: Rabbit limbal explants were cultured in vitro on amniotic membrane for 4 weeks. In the in vivo culture, the rabbit corneal epithelium was removed. Next, a tunnel was created at the limbus and, the edge of amniotic membrane was secured in the tunnel and cultured for 4 weeks. The proliferation of epithelium on the amniotic membrane was observed for 4 weeks at 1 week intervals. RESULTS: AE-5 immunohistochemical staining was positive and PAS staining was negative for cultured rabbit limbal epithelium, in vitro and in vivo. Hematoxylin and Eosin staining showed the morphologic characteristics of normal rabbit corneal epithelium in both groups. Transmission electron microscopy performed at an interval of 1 week showed adhesion complex by 3 weeks of in vitro culture, and no significant change was seen until week 4. The formation of the adhesion complex was shown starting at week 1 of in vivo culture and increased until week 4. CONCLUSIONS: The morphology of corneal limbal epithelium of rabbits cultured on amniotic membrane in vitro and in vivo, did not differ significantly compared with normal rabbit epithelium. In vivo culture resulted in more a normal-looking adhesion complex compared with the in vitro culture.

The Characteristics of Limbal Epithelial Cells Auto-Cultivated In Vivo on Amniotic Membrane in Rabbits

PURPOSE: To investigate the characteristics of the limbal epithelial cells auto-cultivated in vivo on amniotic membrane (LIVAMs) designed for the treatment of limbal stem cell deficiency. METHODS: We removed the epithelium of AM with a No.15 blade after it was blotted with 20% ethanol and made a 360 degrees stromal flap along the epithelial defect. We then mounted over-sized AM (1 mm larger in diameter than the defect) over the defect with the border of AM inserted under the flap, and performed interrupted suture with 10-0 nylon. A therapeutic contact lens was fitted over the AM and a temporary tarsorrhaphy was performed. To examine whether the limbal epithelial cells grew well onto AM, we observed the cornea after fluorescein dye staining using a slit lamp. To explore the characteristics of LIVAMs, we performed hematoxylin-eosin (H&E) staining, immunochemical staining with AK-2, AE-5, AM-3 monoclonal antibodies, and transmission electron microscopy. RESULTS: Three of four rabbits had successful epithelial growth on the amniotic membrane. The epithelial growth on the amniotic membrane was stained using immunohistochemical staining (AK-2, AE-5). Electron microscopy showed a structure similar to that of a normal corneal epithelium. CONCLUSIONS: The technique of auto-cultivation of limbal epithelial cells in vivo on amniotic membrane can be an efficient and convenient method and preserves the characteristics of limbal epithelial cells for the treatment of limbal stem cell deficiency.

The Study for Limbal Epithelial Cell Movement according to the Position of Limbal Transplant in Rabbits

PURPOSE: This study aimed to evaluate the limbal epithelial cell movement according to the position of limbal transplant in rabbits. METHODS: We performed autologous conjunctival limbal transplantation in rabbits, in situ (directed corneal end toward cornea and conjunctival end toward conjunctiva) or in versus (directed corneal end toward conjunctiva and conjunctival end toward cornea) manner. We performed 360o whole autologous conjunctival limbal transplantation in quadrant fashion. Twelve months later, we observed the gross cornea under a slit lamp, and then observed corneal tissue with PAS staining using light microscopy and immunohistochemical and immunofluorescence staining using monoclonal antibody. As mentioned above, the conjunctival limbal tissue had been transplanted in situ and in versus manner for confocal microscopy. However, before transplantation, we performed organ culture of the limbal epithelial cells with CellTracker(TM) Green CMFDA dye (Molecular Probes, USA). Two weeks later, we observed the corneal and limbal epithelial cells using the confocal microscope. RESULTS: The slit lamp examinations of the cornea showed no new vessels and clear in both specimens. There were no goblet cells and the corneal epithelial arrangement was normal under light microscopy. There were positive immunohistochemical stains for cornea-specific AK2 and AE5, and negative immunofluorescence stains for goblet cell-specific AM3 patterns in both in situ and in versus specimens. CellTracker(TM) Green CMFDA dye up-taken epithelial cells were not presented in the conjunctival epithelial side, but were in the corneal epithelial side in both specimens. CONCLUSIONS: We confirmed that the epithelial cells with characteristics of corneal epithelium moved toward the corneal center at any positions of limbal transplants in rabbits.

Effect of cellular activation of ultra low temperature preserved limbal epithelial cells of rabbit cultured in vitro / 眼科

0.05).The morphological characteristics were similar and positive on HE staining and AE5 immunohistochemical staining.PAS staining were negative in two groups.Conclusion The cellu- lar biological activations of limbal epithelial cells are decreased in the ultra low temperature preservation condition,but the affect are limit and can't change the property of corneal limbal epithelial cells and application for ocular surface reconstruction.(Ophthalmol CHN,2008,17:108-112)