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Impressum
Culture of primary adult human RPE and IPE on 3T3 feeder layers
Thumann G., Aisenbrey S., Kociok N., Kirchhof B.
Department of Ophthalmology, University of Cologne, Germany
Objective: To optimize culture conditions and stabilize phenotype,
primary human adult retinal and iris pigment epithelial cells from donor
eyes were co-cultured with post mitotic NIH 3T3 fibroblasts.
Method: To stop mitosis subconfluent NIH 3T3 fibroblasts were treated
with 10mg/ml mitomycin for 2 hours at 37°C and seeded into 4 well
culture plates and on chamber slides at a cell density of 2.5x104 cells
per dish or slide. Primary human IPE and RPE were plated over the 3T3
fibroblasts 24 hours to 4 days after plating the fibroblasts depending
on the availability of donor eyes. Cell adhesion and morphology were examined
by light microscopy.
Results: In the presence of post mitotic 3T3 fibroblasts, primary
human adult IPE and RPE cell adhere and spread on the culture dish within
12 hours after seeding even after prolonged post mortem times. Within
8-10 days after seeding the 3T3 fibroblasts died leaving pure cultures
of primary pigment epithelial cells.
Conclusion: Culture of human adult IPE and RPE cells in the presence
of non-dividing 3T3 fibroblasts results in a high plating efficiency even
in eyes of older donor and after prolonged postmortem times.