Tissue reconstruction in 3D-spheroids from rodent retina in a motion-free, bioreactor-based microstructure.

While conventional rotation culture-based retinal spheroids are most useful to study basic processes of retinogenesis and tissue regeneration, they are less appropriate for an easy and inexpensive mass production of histotypic 3-dimensional tissue spheroids, which will be of utmost importance for future bioengineering, e.g. for replacement of animal experimentation. Here we compared conventionally reaggregated spheroids derived from dissociated retinal cells from neonatal gerbils (Meriones unguiculatus) with spheroids cultured on a novel microscaffold cell chip (called cf-chip) in a motion-free bioreactor. Reaggregation and developmental processes leading to tissue formation, e.g. proliferation, apoptosis and differentiation were observed during the first 10 days in vitro (div). Remarkably, in each cf-chip micro-chamber, only one spheroid developed. In both culture systems, sphere sizes and proliferation rates were almost identical. However, apoptosis was only comparably high up to 5 div, but then became negligible in the cf-chip, while it up-rose again in the conventional culture. In both systems, immunohistochemical characterisation revealed the presence of Müller glia cells, of ganglion, amacrine, bipolar and horizontal cells at a highly comparable arrangement. In both systems, photoreceptors were detected only in spheroids from P3 retinae. Benefits of the chip-based 3D cell culture were a reliable sphere production at enhanced viability, the feasibility of single sphere observation during cultivation time, a high reproducibility and easy control of culture conditions. Further development of this approach should allow high-throughput systems not only for retinal but also other types of histotypic spheroids, to become suitable for environmental monitoring and biomedical diagnostics.

Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium.

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.