RESUMO
Replacement of damaged cells is a promising approach for treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP); however, availability of donor tissue for transplantation remains a major obstacle. Key factors for successful engineering of a tissue include the identification of a neural cell line that is: homogeneous but can be expanded to give rise to multiple cells types; is nontumorigenic, yet capable of secreting neurotrophic factors; and is able to form three-dimensional (3D), differentiated structures. The goal of this study was to test the feasibility of tissue engineering from a multipotential human retinal cell line using a NASA-developed bioreactor. A multipotential human retinal precursor cell line was used to generate 3D structures. In addition, retinal pigment epithelium (RPE) cells were cocultured with neural cells to determine if 3D retinal structures could be generated in the bioreactor with cells grown on laminin-coated cytodex 3 beads. Cell growth, morphology, and differentiation were monitored by light and scanning electron microscopy, Western blot analysis, and analysis of glucose use and lactate production. The neuronal retinal precursor cell line cultured in a bioreactor gave rise to most retinal cell types seen in monolayer culture. They formed composite structures with cell-covered beads associated with one another in a tissue-like array. The beginning of layering and/or separation of cell types was observed. The neuronal cell types previously seen in monolayer cultures were also seen in the bioreactor. Some of the retinal cells differentiate into photoreceptors in the bioreactor with well-developed outer segment-like structures, a process that is critical for retinal function. Moreover, the neuronal cells that were generated resembled their in vivo phenotype more closely than those grown under other conditions. Outer segments were almost never seen in the monolayer cultures, even in the presence of photoreceptor-inducing growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor (TGF-alpha). Muller cells were occasionally seen when retinal, RPE cells were cocultured with retinal cells in the bioreactor. These have never been seen in this retinal cell line before. Cells grown in the bioreactor expressed several proteins specific for the retinal cell types: opsin, protein kinase C-alpha, dopamine receptor D4, tyrosine hydroxylase, and calbindin.
