98th Annual Meeting DOG 2000

K 28

Immunocytochemical analysis of protein expression in primary cultured adult human retinal pigment epithelial cells during subculture

M. Valtink, S. Ventura, A. Jalali Souson-Abadi, K. Engelmann

Background: Cultured retinal pigment epithelial (RPE) cells show a typical morphology, e.g. pigmentation and epithelial phenotype, only during early passages in vitro. During subculture, loss of these characteristics becomes evident. Transdifferentiation towards the expression of neuronal or mesenchymal characteristics have been discussed as reason for morphological alterations.

Methods: 22 primary cultures of human adult RPE cells were cultured under optimized conditions as previously described, and subcultured until the cells became senescent (after approx. 13 passages). In a first set of experiments, cells of passages P1-P3 were seeded at varying density and the expression of neuron-specific enolase (NSE), vimentin and fibronectin was examined in dependence on cell density. In a second set, cells were cultured until senescence and confluent cultures of each passage were examined for the expression of neuron-specific enolase (NSE), vimentin and fibronectin as well. Semiquantitative analysis was performed using a cytofluorometer.

Results: In the first experiment expression of fibronectin and vimentin increased until and then remained constant, while the expression of NSE still increased. In the second experiment no change in protein expression of fibronectin, vimentin or NSE depending on culture age could be observed. The results did not correlate with morphological alterations of the cells.

Conclusion: This study revealed no alteration in expression of enzymes (NSE), structural (vimentin) or matrix (fibronectin) proteins during subculture of RPE cells under the employed optimized conditions. It is therefore considerable that long-term cultured RPE cells can retain typical functions after transplantation despite changes in morphological appearance.

Universit├Ąts-Augenklinik, Martinistr. 52, D-20246 Hamburg, Germany