Aim
To find out an animal-free, xeno-free culture method for human fetal retinal pigment epithelium (fRPE) cells aiming for cell-replacement therapy.
Conclusion
Our study indicates that culturing fRPE cells in 10% human AB serum-supplemented medium is more favorable compared with KSR, B27 and traditional FBS-supplemented mediums when fRPE cells are to be applied in cell-based therapy.
Methods
Human AB serum, knock-out serum replacement (KSR) and B27 were supplemented as a substitute of fetal bovine serum (FBS) in culture medium of human fRPE cells. Cell morphology was examined by light microscope and transmission electron microscope. Proliferation ability was detected by cell cycle analysis and examination of KI67 expression. Apoptosis was analyzed using FACS. The expression of RPE-specific markers was demonstrated by quantitative real-time polymerase chain reaction (qPCR), Western blot (WB) and immunocytochemistry. Paracrine function was determined using enzyme-linked immunosorbent assay method.
Results
Our results indicated that the optimum concentration of KSR was 15%, the optimum concentration of B27 was 2%, and the optimum concentration of human AB serum was 10%. fRPE cells cultured in 15% KSR and 2% B27 media showed repressed propagation and differentiation ability, and gradually lost epithelial morphology and RPE function. While fRPE cells cultured in 10% human AB serum media showed a typical cobblestone morphology with pigmentation, elevated proliferation ability, satisfying paracrine function and expressed RPE-specific markers.