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S. Peng, L. Cong, Z. Zhang, D. Sun; Labeling Newborn Rabbit Retinal Pigment Epithelial Cells With Vital Dye CFDA-SE-- An in vitro Study. Invest. Ophthalmol. Vis. Sci. 2008;49(13):471.
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To establish a simple, reliable culture method for newborn rabbit retinal pigment epithelium cell (RPE) and test whether the vital dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) would be a suitable marker for transplanted RPE.
Newborn rabbit RPE was isolated with dispase and cultured on microporous filter-supports. RPE was incubated with varying concentrations of CFDA-SE for different time periods. The optimal labeling condition was defined based on the fluorescence intensity, the viability of labeled RPE and its plating efficiency. Flow cytometric analysis and fluorescence microscopy were used to monitor the fluorescence intensity with time. The characteristics of labeled and unlabeled cells were demonstrated by immunocytochemical methods using markers MNF116 and S-100, at 1 and 4 weeks. Mixed cultures of labeled rabbit RPE and human RPE were performed to demonstrate if there was transfer of dye among cells. Transepithelial resistance (TER) and ZO-1 expression were determined when the labeled and unlabeled RPE were co-cultured on laminin-coated transwell filter.
An incubation of 1 min at 37°C with a concentration of 20 µmol/L of CFDA-SE was optimal for RPE labeling. The intense of fluorescence of labeled RPE would decrease to 20% over 4 weeks in vitro. Immunostaining experiments showed no obvious difference between cells labeled for 1 week and those labeled for 4 weeks. There was no transfer of dye from labeled rabbit RPE to non-labeled human RPE in mixed culture process. The TER of co-culture monolayers had reached a maximum of 438 Ω X cm2 and robust expression of ZO-1 were observed in co-culture models.
Newborn rabbit RPECs can be obtained purely by the method of using dispase. The fluorescent dye CFDA-SE can be used as a new tracer of newborn rabbit RPE transplantation owing to its many advantages, such as convenience, safety and long-term labeling.
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