Neural differentiation of human retinal pigment epithelial cells on alginate/gelatin substrate.

Abstract

Purpose: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells.

Methods: hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation.

Results: A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells.

Conclusions: These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.