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Abstract
This study was undertaken to determine if retinal pigment epithelial (RPE) cells encased
in permselective hollow fibers survive in a tissue culture environment and secrete
a diffusible trophic factor(s) that may affect retinal cell survival in vitro. In
this study, RPE cells were isolated from 6- to 8-day-old Long-Evans rats, then loaded
into hollow fibers. The RPE-cell fibers were then cultured for at least one week in
serum-containing medium. These RPE-cell fibers were subsequently co-cultured with
cells isolated from retinas of day 2 Long-Evans rats in a defined medium. For at least
6 days in culture, opsin-positive cells were observed on the surface of larger flat
cells. Over 80% of the small, round cells immunostained for opsin. However, opsin-immunostained
cells were seldom seen in cultures with control fibers, that lacked RPE cells. In
addition, conditioned medium collected from either the RPE-cell fibers or cultured
RPE cells affected survival of opsin-positive retinal cells in culture in a manner
similar to that of the RPE-cell fibers. Furthermore, selected growth factors such
as epidermal, nerve and fibroblast growth factors, were unable to sustain retinal
cell survival and affect morphological development as seen in RPE-CM supplemented
cultures. In vivo companion developmental studies demonstrated that few opsin-positive
cell bodies were observed in retinas of day 2 Long-Evans rats, the age corresponding
to the stage of retinal cell isolation. In retinas of day 5 Long-Evans rats, the age
corresponding to the end point of the in vitro assay, a dramatic increase in the number
of opsin-immunostained cell bodies was noted, which corresponds to the developmental
sequence also seen in culture. Light and electron microscopic examination revealed
that the RPE cells cultured in the hollow tubes maintained an RPE-like structure for
several months, in that these cells contained melanosomes and extended microvilli
from their apical border and formed junctional complexes with adjacent cells. Results
of this study confirm our earlier findings that RPE cells secrete an apparent novel
factor(s) that affects retinal cell survival in vitro and, most significantly, the
described encapsulation/secretion mechanism may provide a convenient method to deliver
such factors for further in vivo testing of this phenomenon.