Generation of 3D Retina-Like Structures From a Human Retinal Cell Line in a NASA Bioreactor
Abstract: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-α). 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-α, dopamine receptor D4, tyrosine hydroxylase, and calbindin.
Document Type: Research Article
Affiliations: 1: *Department of Pathology, Morehouse School of Medicine, Atlanta, GA 30310-1495 2: ‡Department of Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310-1495 3: †Department of Medicine, Morehouse School of Medicine, Atlanta, GA 30310-1495 4: §Department of Microbiology, University of South Carolina, Medical School, Columbia, SC
Publication date: December 1, 2002
- Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.