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M. Rieke, E. Gottwald, P. G. Layer; Retinal Neurospheres, Produced on a Cellchip Device for Bioreactor Applications. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5775.
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The CellChip©® is designed as a platform for standardized production of artificial tissues, generation and differentiation of three-dimensional tissue constructs becomes possible in the controlled environment of a bioreactor. Here we compare the results of CellChip©® based tissue reconstruction, to our standard rotation culture system to produce mammalian retinal reaggregates
Dissociated retinal cells from gerbils at postnatal day 2-3 (P2-P3) were seeded on a CellChip©® (generous gift of E. Gottwald, FZ Karlsruhe) in an Petri dish. The Petri dish was free of cells or covered with a monolayer culture of gerbil RPE cells, without direct contact to the cells in the CellChip©®. The cells are cultured in a Medium composed of DMEM plus 10% fetal calf serum, 1% L-glutamine, 0,1% penicillin/streptomycin and 0,01% gentamycin, or in same medium conditioned by gerbil RPE cells from P3. Reaggregates were collected after 10-12 days in culture, before the spheres were fixed (PFA 4%) and cryosectioned. Reorganisation and differentiation were documented immunohistochemically with antibodies against specific cell-markers. Proliferation was investigated by BrdU supplementation and celldeath by TUNEL-assay.
Mammalian retinal precursor cells are able to reaggregate and form 3 dimensional neurospheres under stationary culture conditions. Sphere size is obviously regulated and limited. During cultivation time of 10-12 days the cells start to differentiate and form histotypic tissues of different organisational levels, depending on the culture conditions. Cocultivation with separated RPE cells leads to a laminar structure with clear IPL formation in the center of the spheres and an increase in proliferation.
We have established a stationary culture method to form histotypic organised mammalian retinal spheres from dissociated retinal precursor cells. The spheres grow on a CellChip©®, that allows the cultivation in a bioreactor for optimized culture conditions. Cocultivation with RPE cells leads to a enhanced nearly laminated structure, comparable to the in vivo situation. This cultivation system goes forward to become a interesting tool combined with life cell imaging and high troughput applications, to get deeper insight into tissue reconstruction or for future applications in biomedical testing.
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