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-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.

RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner

“…We have a well-characterized system of human multipotential retinal progenitor cell lines in which retinal cell differentiation can be monitored in response to neurotrophins [10,11,23,[42][43][44] or 3-D culture models of growth [10]. Our results show that in monolayer culture, photoreceptor differentiation occurs in response to bFGF and TGFα in a density-dependent manner confirming the studies of the default model of cone photoreceptor differentiation established by Belecky-Adams et al [22].…”

“…Antibodies AaNat, rhodopsin 1D4, Nr 2 e 3 , and GNB3 were procured from Chemicon (Temecula, CA, USA) and used at a dilution of 1:200 for immunohistochemistry and at 1:1,000 for western blot analysis. Actin antibody from Sigma-Aldrich Corp., St. Louis, MO, USA was used at a 1:500 dilution [10,11,42].…”

“…Other suggestions are transplantation of partially differentiated progenitors to transplantation of 3-D constructs of retina generated from progenitors in a 3-D bioreactor culture [7,[10][11][12]. Others are proposing transplantation of RPE induced from pluripotent stem cells, which could differentiate into photoreceptors in vivo [13].…”

“…In the present study, we have explored the role of RPE-secreted factors in monolayer cultures (conditioned media on a nontransformed human retinal cell line) [43,44] characterized in monolayer culture [23,38] and 3-D culture [10,11,42].…”

Gene Therapy Progress and Prospects: In tissue engineering

“…81 Effects of a simulated microgravity environment on TE constructs of various cell types shows the importance of micromechanical stimuli on cell differentiation and gene expression. [82][83][84][85][86][87] Likewise, application of mechanical stimuli during growth has a large effect on the structure and mechanical properties of the resultant constructs. 88 Until highly controlled tests are conducted that encompass all of the above factors, it will be impossible to deduce which material or combination of materials is ideal for use in TE scaffolds for a given clinical objective.…”