Vision Research 46 (2006) 4464–4471
0042-6989/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
Photoreceptor vitality in organotypic cultures of mature vertebrate
retinas validated by light-dependent molecular movements?
Boris Reidela, Wilda Orismeb, Tobias Goldmanna, W. Clay Smithb, Uwe Wolfruma,¤
a Institute of Zoology, Department of Cell and Matrix Biology, University of Mainz, Germany
b Department of Ophthalmology, University of Florida, Gainesville, FL 32610-0284, USA
Received 14 June 2006; received in revised form 24 July 2006
Vertebrate photoreceptor cells are polarized neurons highly specialized for light absorption and visual signal transduction. Photore-
ceptor cells consist of the light sensitive outer segment and the biosynthetic active inner segment linked by a slender connecting cilium.
The function of mature photoreceptor cells is strictly dependent on this compartmentalization which is maintained in the specialized reti-
nal environment. To keep this fragile morphologic and functional composition for further cell biological studies and treatments we estab-
lished organotypic retina cultures of mature mice and Xenopus laevis. The organotypic retina cultures of both model organisms are
created as co-cultures of the retina and the pigment epithelium, still attached to outer segments of the photoreceptor cells. To demonstrate
the suitability of the culture system for physiological analyses we performed apoptotic cell death analyses and veriWed photoreceptor via-
bility. Furthermore, light-dependent bidirectional movements of arrestin and transducin in photoreceptors in vivo and in the retinal cul-
tures were indistinguishable indicating normal photoreceptor cell-biologic function in organotypic cultures. Our established culture
systems allow the analysis of mature photoreceptor cells and their accessibility to treatments, characteristic for common cell culture.
Furthermore, this culturing technique also provides an appropriate system for gene delivery to retinal cells and will serve to simulate gene
therapeutic approaches prior to diYcult and time-consuming in vivo experiments.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Signal transduction; Photoreceptors; Light-dependent movements; Organotypic retina culture; Mouse; Xenopus
In the vertebrate eye, the retina is responsible for light
perception and also for the Wrst processing steps in vision.
The retina consists of diVerent neuronal cell types which are
organized in diVerent layers (Fig. 1G and H). The photore-
ceptor cells are localized within the innermost layer of neu-
ronal retina. Rod and cone photoreceptor cells are highly
polarised sensory neurons which consist of morphologi-
cally and functionally specialized compartments. The light-
sensitive photoreceptor outer segment is linked with the
inner segment and the cell body by a slender non-motile cil-
ium, the so-called connecting cilium (Fig. 1I). The outer
segment is arranged as hundreds of stacked membrane
disks and contains all the components of the visual trans-
duction cascade. The inner segment compartment mainly
houses all organelles for biosynthesis of proteins and
energy production, necessary for maintenance of the cell
function (Fig. 1I).
Photoexcitation of the visual pigment rhodopsin activates
the visual heterotrimeric G-protein transducin, leading to
cGMP hydrolysis in the cytoplasm and closing of cGMP-
gated channels in the plasma membrane of the outer segment
(Molday & Kaupp, 2000). Arrestin binding to activated
phosphorylated rhodopsin (R¤P) terminates the visual trans-
duction by preventing further binding of transducin to R¤P.
A fast light adaptation of photoreceptor cells relies on
?This work was supported by Deutsche Forschungsgemeinschaft
(DFG) (UW); Pro Retina Deutschland e.V. (BR, UW) and the FAUN
Stiftung, Nürnberg, Germany (UW), National Eye Institute (WCS), and
Karl Kirchgessner Foundation (WCS).
*Corresponding author. Fax: +49 6131 39 23815.
E-mail address: email@example.com (U. Wolfrum).
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
Fig. 1. Dissection and preparation of organotypic retina culture. (A) Eyeball with optic nerve prepared from a sacriWced mouse. (B) Cutting oV the optic
nerve under slight tension with forceps. (C) Incision of sclera by gently inserting scissor between the retinal pigmented epithelium and the sclera. (D) Com-
plete incision of sclera around eyeball to each side reaching the cornea. (E) Incisions in the retinal cup, previously removed from sclera, cornea, lens, vitre-
ous, iris, and hyaloid vessel. (F) Flattening of spread retina on culture membrane attached to nylon spacer. (G) DiVerential interference contrast picture of
a retinal cryosection indicating the orientation of retinal explant with pigmented epithelium attached to the culture membrane. (H) Schematic representa-
tion of retina and cell organization. (I) Schematic representation of vertebrate photoreceptor cell. OS, outer segment; IS, inner segment; ONL, outer
nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 13.2?m.
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
feedback mechanisms based on changes in the intracellular
free Ca2+-concentration which aVect several steps in the
visual transduction cascade (Palczewski, Polans, Baehr, &
Ames, 2000). However, massive bidirectional translocation of
transduction proteins, in particularly of arrestin and of trans-
ducin, between the functional compartments of photorecep-
tor cells contribute to a much slower adaptation of rod
photoreceptor cells (Sokolov et al., 2002).
Light-induced exchanges of signal cascade components
were Wrst noted about two decades ago (Broekhuyse, Tol-
huizen, Janssen, & Winkens, 1985; Brann & Cohen, 1987;
Philp, Chang, & Long, 1987; Whelan & Mc Ginnis, 1988)
and are currently of prominent interest in the Weld (e.g.
(Hardie, 2002; Gießl, Trojan, Pulvermüller, & Wolfrum,
2004; Burns & Arshavsky, 2005)): upon illumination, 80%
of transducin moves in minutes from the outer segment to
the inner segment and the cell body of rod photoreceptor
cells. The G-protein subunits return to the outer segments
in the dark in a longer time course of hours. In contrast,
arrestin translocates under these light conditions in a recip-
rocal direction. The intersegmental translocations of arres-
tin and transducin are thought to contribute to long term
adaptation of rod photoreceptor cells (Sokolov et al., 2002;
Strissel, Sokolov, Trieu, & Arshavsky, 2006). Nevertheless,
the molecular and cellular mechanisms underlying these
adaptive movements of arrestin and transducin still remain
elusive (Chen, 2005; Strissel et al., 2006).
In cell biology, molecular intracellular movements and
their association with the cytoskeleton are studied mainly
in well-deWned and accessible cultured systems. The accessi-
bility of cultured cells allows the application of chemical
reagents and well-deWned external stimulation. However, so
far no applicable method exists to cultivate diVerentiated
photoreceptor cells without loosing their compartmentali-
zation. After dissociation from their retinal environment,
photoreceptor cells lose their functional integrity in pri-
mary cell culture (Fintz et al., 2003; Leveillard et al., 2004).
Cultivation of photoreceptor cells in their native environ-
ment the retina may solve this problem. However, in most
attempts, organotypic cultures of mature vertebrate retinas
which contain diVerentiated compartmentalized photore-
ceptor cells survived only for very short times, e.g. several
hours (Ogilvie, 2001). Here, we show that a co-culture of
the neuronal retina and retinal pigment epithelium not only
survives for several days, but also keeps the physiological
conditions for molecular movements between the outer and
inner segment of the fragile photoreceptor cells. After intro-
ducing this culturing system in rodents we also adapted the
organotypic culture to the retina of Xenopus laevis, a well-
accepted model organism in retinal cell biology.
C57BL/6J mice were maintained on a cycle of 12h of light (200 lux)
and 12h of darkness, with food and water ad libitum. In light adaptation
experiments mice were illuminated (200 lux), or kept in the dark before
dissection of eyes and Wxed with 4% formaldehyde in soerensen’s phos-
Wild-type Xenopus frogs were obtained from Xenopus Express (Plant
City, FL). Frogs were either light-adapted for 60 min (800 lux), or kept in
the dark and subsequently Wxed with 3.7% formaldehyde in 73% methanol.
All animals used in these experiments were cared for and handled
according to the Association for Research in Vision and Ophthalmology
(ARVO) statement for the Use of Animals in Vision and Ophthalmic
Research and according to institutional animal care and use guidelines.
2.2. Retina culture
The retina culture system was established according to the experimen-
tal procedures previously published (CaVe, Visser, Jansen, & Sanyal, 1989)
and altered as described in the following. In brief, intact eyes of postnatal
day 12–14 C57/BL6J mice were immediately removed from sacriWced ani-
mals and incubated with 1.2mg/ml Proteinase K (Sigma–Aldrich, Ger-
many) for 15min at 37°C. Proteinase K activity was stopped by
transferring eyes to culture medium containing 10% fetal calf serum
(10ml) for 5min. After rinsing the eyes three times in serum-free culture
medium (5ml), retinas were dissected in basal culture medium by remov-
ing sclera, ocular tissue, and the hyaloid vessel under preservation of the
pigmented epithelium (Fig. 1). Retinas were spread with the retinal pig-
mented epithelial cells facing down (Fig. 1F and G) on ME 25/31 culture
membranes (Schleicher and Schuell, Germany), and cultured in Dul-
becco’s ModiWed Eagle’s Medium with F12 supplement (DMEM-F12)
and 10% fetal calf serum, L-glutamine, penicillin and streptomycin
(Sigma–Aldrich, Germany) and maintained at 37 °C with 5% CO2. Speci-
mens of postnatal day (pn) 12 to 14 were cultured for at least 1 day (12h
light-emitting diodes (LED) 200 lux/12h dark) before start of light condi-
tioning. Tissues from litter-matched specimens of pn 14 were used as
In dark to light studies, cultured retinas and control mice were dark-
adapted for 4h and then exposed to 200 lux of light for 30min by light-
emitting diodes (LED). In light to dark studies, cultured retinas as control
mice were exposed to light of 400 lux for 60min before darkening. Since
the optic apparatus is removed, retina cultures were illuminated with less
light intensity than control animals.
2.3. Dissection and preparation of Xenopus tissue
Retinas from Xenopus eyes were dissected and prepared as described
for mouse eyes till the stage when lens is removed (Fig. 1E). Retinas of
Xenopus were cultured with the retina surrounding the lens. Eyes of con-
trol animals were Wxed with 3.7% formaldehyde in 73% methanol, were
rehydrated through a graded series of methanol in phosphate-buVered
saline, equilibrated overnight in 30% sucrose, and then embedded in OCT
medium (Sakura FineTek, Torrance, CA). Cryosections (12?m) were pro-
cessed for immunocytochemistry as previously described (Peterson et al.,
2.4. TUNEL staining
For TUNEL staining, the “In Situ Cell Death Detection Kit” (Boeh-
ringer Mannheim, Germany) was used per instructions. The incubation
with the “TUNEL-mixture” for 1h at 37°C in a humid chamber was ter-
minated by multiple washes in PBS. Dried sections were mounted in Mow-
iol (HOECHST 4.88, Hoechst, Germany).
2.5. Antibodies and Xuorescent dyes used on mouse tissue
AYnity-puriWed polyclonal rabbit antibodies against the ?-subunit of
the G protein were obtained from Biomol Research Laboratories, Inc.
(PA) and used on mouse retina slices. Mouse antibodies directed to arres-
tin (clone 3D1.2) were applied on mouse retina slices as previously charac-
terized (Nork, Mangini, & Millecchia, 1993). Xenopus retina slices were
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
immunocytochemical incubated using an anti-arrestin monoclonal anti-
body (1:50 xAr1-6). Labeling was detected with an anti-mouse-FITC con-
jugate (1:100). Slides were imaged with a Xuorescence microscope (Zeiss,
AxioVision Release 4.4).
2.6. Fluorescence microscopy
Cultured retinas, as eyes of control mice were cryoWxed in melting iso-
pentane, cryosectioned and treated as described (Reiners et al., 2003;
Wolfrum, 1991). Secondary antibodies conjugated to Alexa 488 or Alexa
568 (Molecular Probes), sections were mounted in Mowiol 4.88 (Far-
bwerke Hoechst, Frankfurt, Germany), containing 2% n-propyl-gallate.
There was no reaction observed in control sections. Mounted retinal sec-
tions were examined with a Leica DMRP microscope. Images were
obtained with a Hamamatsu ORCA ER charge-coupled device camera
(Hamamatsu, Germany) and processed with Adobe Photoshop (Adobe
3. Results and discussion
Previous studies indicated that photoreceptor cells lose
their compartmentalization and functional integrity in pri-
mary cell culture (Fintz et al., 2003; Leveillard et al., 2004).
In the present work, we cultivated photoreceptor cells in
their native environment in organotypic retina cultures.
Cultivation of neonatal retinas is a standard method for the
analysis of retina diVerentiation and for pharmacologic and
electrophysiologic studies on the developing neuronal ret-
ina network (Jablonski, 2003; CaVe, Soderpalm, Holmqvist,
& van Veen, 2001; Perez-Leon et al., 2003). However, in
neonatal retinas, the photoreceptor diVerentiation is still in
progress and the compartmentalization of photoreceptor
cells is not complete. So far, most attempts to culture
mature vertebrate retinas have failed and the diVerentiated
photoreceptor cells survived only for very short times, e.g.
several hours (Ogilvie, 2001). To extend the life span of the
retinal cells in the organotypic culture of maturate retinas,
we customized culturing conditions. Our investigations
revealed that cyclic light during culturing contributes to
visible maintenance of the retina morphology. The stability
of the fragile photoreceptor outer segments was enhanced
by co-culturing with attached pigment epithelium cells
immobilized on the nitrocellulose cultivation membrane
(Fig. 1G and H). Mouse ages of post natal days (PN) 12–14
have been estimated to be a good compromise between
photoreceptor integrity and outer segment length. This
improvement in culturing of maturate retinas results in
enhanced viability and retain physiological activity ex vivo.
Viability of the organotypic retina culture was assessed
by TUNEL staining after diVerent culture time periods
(Fig. 2). Fig. 2A shows a (positive) control slice treated
with DNase, where all nuclei of retinal cells are stained by
the TUNEL method. In retinas cultured for 1–3 days
there are only a few TUNEL positive nuclei stained in the
outer nuclear layer (Fig. 2B–D). After 7 days of culturing,
numerous nuclei in almost every layer of cultured retinas
are stained indicating a high amount of apoptotic cells
(Fig. 2E). This evaluation of the viability state of the cul-
tured retinal explants from mice by TUNEL staining,
conWrmed a low rate of apoptotic retinal cells during the
Wrst three days of culturing. Present results indicate that
at least for the Wrst three days ex vivo, the organoptypic
retina culture is a viable system suitable for physiological
3.1. Light-dependent translocation of arrestin and transducin
also occurs in organotypic cultures of the mouse retina
Light-driven translocation of arrestin and transducin
between the inner and outer segment compartment had
Fig. 2. Viability assessment of retinas cultured for diVerent time periods. (A) Tunel staining of nuclei in a longitudinal cryosection of DNase-treated mouse
retina. (B) Tunel staining of nuclei in a longitudinal cryosection of cultured retina after one day ex vivo. (C) Tunel staining of nuclei in a longitudinal cryo-
section of cultured retina after two days ex vivo. (D) Tunel staining of nuclei in a longitudinal cryosection of cultured retina after 3 days ex vivo. (E) Tunel
staining of nuclei in a longitudinal cryosection of cultured retina after seven days ex vivo. OS, outer segment; IS, inner segment; ONL, outer nuclear layer;
OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 13.2?m.
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
been described two decades ago (Philp et al., 1987; Whelan
& Mc Ginnis, 1988). Although the phenomenon was inten-
sively investigated during the last years (Pulvermüller et al.,
2002; Sokolov et al., 2002; Wolfrum, Giessl, & Pulvermul-
ler, 2002; Peterson et al., 2003; Mendez, Lem, Simon, &
Chen, 2003; Nair et al., 2004), the molecular processes
Fig. 3. Light-dependent translocation of transducin and arrestin in mammalian rod photoreceptor cells. (A) Immunocytochemical localisation of trans-
ducin in a longitudinal cryosection through a light-adapted mouse retina. (B) Immunocytochemical localization of transducin in a longitudinal cryosec-
tion through a cultured light-adapted mouse retina. Transducin can be localized in inner segments of photoreceptor cells in retinas from light-adapted
animals and in inner segments of light-adapted cultured retinas. (C) Schematic rod photoreceptor cells (PRC) with visualized transducin localisations in
red. OS, outer segment; CC, connecting cilium; IS, inner segment; N, the nucleus; S, synapse. (D) Immunocytochemical localisation of transducin in a lon-
gitudinal cryosections through a dark-adapted mouse retina. (E) Immunocytochemical localisation of transducin in a longitudinal cryosection through a
cultured dark-adapted mouse retina. Transducin can be localized in outer segments of photoreceptor cells in retinas from dark-adapted animals as in
outer segments of dark-adapted cultured retinas. (F) Immunocytochemical localization of arrestin in a longitudinal cryosection through a light-adapted
mouse retina. (G) Immunocytochemical localisation of arrestin in a longitudinal cryosection through a cultured light-adapted mouse retina. Arrestin can
be localized in outer segments of photoreceptor cells in retinas from light-adapted animals as in outer segments of light-adapted cultured retinas. (H)
Schematic rod photoreceptor cells (PRC) with visualized arrestin localisations in green. (I) Immunocytochemical localization of arrestin in a longitudinal
cryosection through a dark-adapted mouse retina. (J) Immunocytochemical localisation of arrestin in a longitudinal cryosection through a cultured dark-
adapted mouse retina. Arrestin can be localized in inner segments of photoreceptor cells in retinas from dark-adapted animals as in inner segments of
dark-adapted cultured retinas. Scale bars, 7.5?m. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web ver-
sion of this paper.)
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
underlying these movements still remained elusive. In the
present study, we investigated the suitability of the organo-
typic retina culture for the analysis of physiological pro-
cesses ex vivo, in particular light-dependent protein
To validate whether retinas remain in the physiological
condition for the analysis of light-driven protein move-
ments during cultivation, in a Wrst set of experiments, we
compared the movement of transducin in retinas of mice
and explanted cultured mouse retinas (Fig. 3). After fully
light (>1/2h) and dark adaptation (>3h), the endpoints of
light-induced molecular translocations (Elias, Sezate, Cao,
& McGinnis, 2004), we found no diVerences between the
in vivo and the ex vivo condition. ImmunoXuoresce cyto-
chemistry revealed that during light adaptation, transducin
translocated to the inner segment of photoreceptors in the
cultured retina as in the control animal (Fig. 3A and B).
Likewise, after dark adaptation, transducin was found in
the outer segment compartment in both the cultured retina
and control animal (Fig. 3D and E).
The localization of visual arrestin has been described to
be reciprocal to that of transducin in light and dark adapta-
tion, respectively. Consequently, we also veriWed the move-
ment of arrestin in light-adapted and dark-adapted retina
cultures. Under light-adapted conditions, arrestin is local-
ized to the outer segment of photoreceptors in retinas of
intact animals as of cultured retinas (Fig. 3F–H). In con-
trast, arrestin localized to the inner segment during dark
adaptation for the retina from the control animal as well as
the compared explant (Fig. 3I and J). These normal move-
ments of both arrestin and transducin provide evidence for
the continuing physiological activity of the retina cultures.
3.2. Light-dependent translocation of arrestin in organotypic
cultures of the Xenopus retina
In a next set of experiments, we asked the question
whether the successful culturing technique developed for
mouse retinas is adaptable to other vertebrate species. To
expand the possibilities of analyses we transferred the cul-
turing technique to the amphibian X. laevis, a well-accepted
animal model in retinal cell biology. In contrast to the
mouse culture, we kept the lens during the dissection in the
retinal sphere to remain stability of the relatively thin Xeno-
Cultured mature retinas of X. laevis under dark or light
conditions as retinas of light treated mature frogs were ana-
lyzed for the localization of arrestin (Fig. 4). Arrestin local-
izations in fully light or dark-adapted photoreceptor cells
were indistinguishable between control maturate Xenopus
and previously studied Xenopus tadpoles (Peterson et al.,
2003; Peterson et al., 2005) and the ex vivo cultures. Photo-
receptor outer segments of light-adapted cultured retinas
and of control animals show a prominent localization of
arrestin in outer segments of photoreceptors (Fig. 4A and
B). In contrast, arrestin is mostly localized to inner seg-
ments of photoreceptors after dark adaptation of frogs or
cultured retinas, respectively (Fig. 4D and E).
We were able to show translocation of visual arrestin
also in photoreceptor cells of cultured frog retinas as in
light-adapted intact frogs. The amphibian photoreceptor
cell has slightly diVerent morphology and dimensions com-
pared to the mammalian photoreceptor like bigger outer
segments, which makes it a suitable organism to visualize
protein distributions in outer segments for example.
Fig. 4. Light-dependent translocation of arrestin in amphibian rod photoreceptor cells. (A) Immunocytochemical localisation of arrestin in a longitudinal
cryosection through a light-adapted Xenopus retina. (B) Immunocytochemical localisation of arrestin in a longitudinal cryosection through a cultured
light-adapted Xenopus retina. Arrestin can be localized in outer segments of photoreceptor cells in retinas from light-adapted animals as in outer segments
of light-adapted cultured retinas. (C) Schematic rod photoreceptor cells (PRC) with visualized arrestin localisations in green. OS, outer segment; CC, con-
necting cilium; IS, inner segment; N, nucleus; S, synapse. (D) Immunocytochemical localisation of arrestin in a longitudinal cryosection through a dark-
adapted Xenopus retina. (E) Immunocytochemical localization of arrestin in a longitudinal cryosection through a cultured dark-adapted Xenopus retina.
Arrestin can be localized in inner segments of photoreceptor cells in retinas from dark-adapted animals as in inner segments of dark-adapted cultured ret-
inas. Scale bar, 22.5 ?m. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper.)
B. Reidel et al. / Vision Research 46 (2006) 4464–4471
To study the molecular mechanisms during adaptation in
consideration of diVerent molecular and morphological
features between the two photoreceptor types (mouse and
Xenopus), could give us new insights in photoreceptor func-
tion in general.
4. Conclusions and perspectives
Isolated photoreceptor cells in primary cultures lose
their compartmentalization (Fintz et al., 2003; Leveillard
et al., 2004). Also in cultured embryonic retinas photorecep-
tors do not develop complete compartmentalization, espe-
cially with regard to diVerentiate no distinct outer segments
(Stiemke & HollyWeld, 1994). To analyze protein localiza-
tion, like the light-dependent redistribution of arrestin and
transducin in photoreceptor cell compartments, we estab-
lished an organotypic retina culture of mature mice. In
these cultures it is possible to localize components in photo-
receptors in diVerentiated compartments of the inner and
Our present data show the retina culture system to be a
viable environment for photoreceptor cells and suitable for
the analysis of light-dependent protein movements between
photoreceptor cell compartments. This is principally sup-
ported by the ability of the cultured photoreceptors to still
react to diVerent light conditions by moving proteins of the
visual signal transduction in and out of cellular compart-
ments. As only fully adapted stages were analyzed, we can-
not exclude diVerences in kinetics or extents of protein
traYcking altered by the cultured conditions in comparison
to the in vivo-sate.
One of the major advantages in using organotypic retina
cultures is the accessibility of this ex vivo system. Our cul-
turing technique provides the opportunity to add and test
substances or apply gene transfer systems directly into the
culture medium. This includes for example pharmacologi-
cal treatments. In contrast to other culture systems, such as
eyecup cultures, treatments can directly reach the photore-
ceptors in the organotypic retina culture. In eyecup cul-
tures, added substances have to penetrate or pass many
layers of the retina from the vitreal side until they can reach
the photoreceptor cells. In our preparation, the retina is
exposed to added reagents on both the vitreal surface and
the RPE surface. The culturing of eyecups and retinas
could be compared with intraocular injections in either the
vitreous (to target ganglion cells) or the sub retinal space to
especially target photoreceptor and/or pigment epithelium
cells. Our organotypic retina culture gives us a model sys-
tem, which can be used to simulate sub retinal injections.
This kind of “in vivo-like” environment can also be utilized
for the simulation of gene therapeutic approaches. In pre-
liminary experiments, vector systems and their gene (repair)
constructs can be validated on the organotypic retina cul-
ture prior to injection into living animals. These proceed-
ings can reduce the amount of animal experiments to a
minimum and also decrease material consumption. For
example the extensive production of viruses for gene
delivery can be shortened, by expressing the gene constructs
with easier to handle transfection systems on retinal tissue
Until now, developed and diVerentiated photoreceptor
cells with distinct outer segments could only be studied
in vivo or ex vivo eyecup cultures, e.g. (Nair et al., 2005). In
contrast to eyecup cultures, our culturing system of mature
retinas provides more accessibility to photoreceptor cells.
Furthermore, the maintained attachment of the pigment
epithelium cells provided extended stability and vitality of
photoreceptor cells of mature retina culture. In the present
study, we introduced a technique of culturing mature reti-
nas and evaluated their physiological activity by visualizing
light-dependent protein translocations in fully compart-
mentalized photoreceptor cells. This organotypic culture of
retinas could provide a new powerful experimental tool to
answer extended cell biological questions which previously
could not be addressed experimentally.
The present work has been supported by ProRetina Ger-
many e.V. and the FAUN-Stiftung, Nürnberg, Germany.
The authors thank Donald R. Dugger for skilful technical
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