This research work received financial supportfrom the European Union (transMed; H2020-MSCA-765441), the German research council (DFG; PA1751/8-1, 10-1) and the China scholarship council.

Animal protocols compliant with &#167;4 of the German law of animal protection were reviewed and approved by the T&#252;bingen University committee on animal protection (Einrichtung f&#252;r Tierschutz, Tier&#228;rztlichen Dienst und Labortierkunde; Registration No. AK02/19M). In this study, retinas were obtained from wild-type (WT) and&#160;rd1&#160;mice, the latter being a well characterized model for hereditary retinal degeneration15. Mice were housed under standard white cyclic lighting, had free access to food and water, and were used irrespective of gender.
1. Checklist
<ol>
	<li>To ensure sterile conditions and avoid contaminations, clean, and disinfect the laminar air flow hood with 70% ethanol. Be sure to let the ethanol evaporate completely, to prevent intoxication of the retinal cultures.</li>
	<li>Autoclave tools (e.g., scissors, forceps, and ophthalmic microscope scraping spoon) before use.</li>
	<li>Prepare the following media in advance under a laminar-flow hood, under sterile conditions: Basal R16 medium (BM) (can be stored at 4 &#176;C for 4 weeks), BM with 20% fetal calf serum (FCS) (same day use), BM with 0.12% proteinase K (44 mAnson U/mg) solution (same day use) and complete R16 medium with supplements (CM) as described by Romijn16&#160;(can be stored at 4 &#176;C for 3 weeks) (see Tables S1, S2, and S3).</li>
	<li>Preheat the proteinase K solution at 37 &#176;C to activate it and use it in step 2.5.</li>
</ol>
2. Preparation
<ol>
	<li>Sacrifice rd1/WT animal at post-natal day (P) 5 by decapitation. For animals older than P11, use CO2 and/or cervical dislocation, as per the local animal protection regulations.</li>
	<li>Depending on the age of the animal, prior to enucleation, if needed, open the eye lids using forceps and very carefully separate the eye lids, without touching or scratching the eye below.</li>
	<li>Rapidly enucleate the eyes under a stereoscope using curved forceps.</li>
	<li>Incubate the eyes in BM for 5 min at room temperature (RT).</li>
	<li>Incubate the eyes in preheated BM, with 0.12% proteinase K at 37 &#176;C for 15 min.</li>
	<li>Perform the following steps inside a laminar air flow hood to ensure sterile conditions. To inactivate proteinase K, transfer eyes to BM containing 20% FCS and incubate for 5 min at RT.</li>
	<li>Dissect the eyes under a stereoscope, aseptically, in a Petri dish containing fresh BM at RT. Initiate the dissection as soon as possible after the enucleation. The longer this time is, the harder it is to dissect the retina, the eyes becoming very soft.
	<ol>
		<li>With forceps, hold the eye from the optic nerve. Using fine scissors, make a small incision in the cornea creating 2 edges from where the cornea, the choroid and the sclera can be gently peeled using 2 pairs of fine forceps (Figure 1 steps A&#8211;C). Alternatively, use a narrow-gauge cannula to make a first incision into the cornea and then insert one of the scissor blades into the opening.</li>
		<li>Grasp the lens with fine forceps. Place a second pair of forceps perpendicularly to the first ones so that the first forceps are between the 2 shanks of the second one. Pull to extract the lens from the eye cup. If the vitreous and the ciliary body are still attached to the retina, remove them carefully (Figure 1 step D). 
		NOTE: Steps 2.7.1 and 2.7.2 need practice and ensure caution to not damage the retina.</li>
		<li>Cut the retina perpendicular to its edges in four points, creating a four-leaf clover shape (Figure 1 steps E&#8211;F).</li>
		<li>Using a pipette with broadly cut base of a 1 mL tip, hold the retina in a hanging drop of medium and transfer it to a culture dish filter insert placed in a 6-well culture plate. The RPE layer should face the membrane (Figure 1 step G).</li>
		<li>Using a pipette, carefully remove the excess medium from the insert.</li>
		<li>From the sides of the well, add 1 mL of CM per well and incubate in a sterile incubator at 37 &#176;C with 5% CO2. Do not submerge the retina in the medium as this will reduce oxygenation and cause tissue degeneration. The explant should remain at the interface between liquid and air, covered only by a thin film of liquid created by the surface tension of water.</li>
	</ol>
	</li>
	<li>Leave the retinal explant undisturbed for the first 48 h to facilitate recovery after the explantation procedure.</li>
	<li>Change the medium every second day (48 h). Discard 700 &#181;L of medium from each well and add 900 &#181;L of fresh CM to the well. In this way, the amount of medium lost by evaporation is recovered and the retinal explant keeps some of the neuroprotective factors produced in the previous 48 h.</li>
	<li>Incubate the removed medium in a separate and closed microcentrifuge tube along with the cultures to control and evaluate possible contamination (i.e., change in color of the medium). 
	NOTE: Retinal explants can be kept in culture for at least 2 weeks12.</li>
</ol>
3. After culturing
NOTE: Explants can be used for different experimental applications (western blot, histology, whole mounts, genetic analysis, electrophysiology). Depending on the application, organotypic retinal explants can be snap frozen, lysed, or prepared for cryosectioning. The steps below describe histological preparation.
<ol>
	<li>Perform a 45-min fixation with 4% paraformaldehyde (PFA), followed by gradual sucrose cryoprotection (10% sucrose for 10 min, 20% for 20 min and 30% for 2 h at room temperature (RT) or overnight (ON) at 4 &#176;C). Add these buffers directly in the well.</li>
	<li>Cut the membrane around the retinal explants.</li>
	<li>Embed both the membrane and retinal tissue in the medium for frozen tissue.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61868/61868fig01.jpg" /> 
Figure 1: Step-by-step procedure for the preparation of organotypic retinal explant cultures.&#160;(A) Mouse eyes are enucleated and transferred to a solution of proteinase K to allow separation of sclera and choroid from the retina and RPE. A small cut in the sclera/choroid layer is introduced. (B) Two forceps are used to peel the sclera/choroid layer. (C) The black choroid layer can be seen during the peeling. The underlining dark retinal pigment epithelium (RPE) remains attached to the eyeball. The sclera and choroid are removed along with the optic nerve. (D) The lens and vitreous are extracted with forceps.&#160; Remaining ciliary body is removed.&#160;(E) The retina retains a bowl-like shape. (F) To flatten the retina for culturing in a dish, 4 cuts in equal distance around the retina are made with a scissor, giving it a clover-like shape. The retina culture is transferred to a membrane culture insert in a 6-well plate with the use of a cut 1 mL pipette tip. The retina still retains some of the bowl-shape. However, upon removal of the excess liquid surrounding the retina, it will unfold to a planar structure. (G) In the culture membrane setup, the retina culture is resting on a porous polycarbonate (PC) membrane on top of a solution of complete R16 medium. To ensure viability, the culture must be kept in a humidified sterile incubator at 37 &#176;C with 5% CO2, and the medium should be replaced every 48 h.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig01large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure</a>

The authors have nothing to disclose.

The protocol presented describes organotypic explant cultures of mouse retina with intact RPE in defined R16 medium, free of serum and antibiotics. This protocol was originally developed starting in the late 1980s7,28&#160;and since then it has been continuously refined6,11,12. Notable applications include studies into the mechanisms of hereditary retinal degeneration and the identification of retinoprotective drugs23,29,30.
For a successful experiment, some important considerations need to be taken into account. Here are some important troubleshooting points to help enhance the quality of cultures. First, the retinal cultures may display excessive folding and/or rosette formation31. This can be caused by touching the retina with a forceps during the explantation procedure. Moreover, the ciliary body must be completely removed from the explant, as this can increase retinal folding during culture. Second, during the transfer of the retina to the well plate in a hanging drop, if the retina faces the membrane the wrong side down, keep it in the drop hanging from the pipette tip and very gently push the medium in and out of the tip (without detaching the hanging drop) to flip the retina around. Finally, if the RPE remains attached to the sclera and detaches from the retina, it is most likely caused by an insufficient predigestion of the sclera. This problem could be especially important when working with eyes from older animals or non-rodent species (e.g., pigs) and may be resolved by increasing the proteinase K concentration.
Conducting organotypic retinal explant cultures is a complex procedure that requires adequate training and experience. Lack of training can lead to variability in the quality of the retinal explants. For these reasons, it is important to monitor and verify viability and reproducibility, characterizing, for instance, the rate of cell death with the TUNEL assay. The use of an antibiotic-free medium makes the retinal explants vulnerable to contamination by bacteria and fungi. To minimize this risk, we recommend that particular care is taken to work under truly aseptic conditions. Another limitation of in vitro&#160;retinal culturing are differences in physiochemical environment when compared to the in vivo retina (e.g., choroidal and retinal blood supply, oxygen and glucose levels, intraocular pressure, composition of the vitreous). A continuous perfusion system, perhaps embedded into a dedicated bioreactor32&#160;could make this model closer to the in vivo condition. Furthermore, the axotomy of the optic nerve during retinal dissection will lead to ganglion cell death, that can induce stress responses8. Therefore, it is recommendable that the explant be left to adapt to culturing conditions for at least 2 days in vitro before it is subjected to a specific manipulation or treatment.
The described method is usually performed on immature retinal tissues, which may survive well for 4 weeks in vitro7,33. However, the procedure is tailorable to a variety of applications, including culturing of adult retina. Although different published approaches describe the isolation of the adult retina without its RPE34,35, the incubation with papain solution for up to 1 h at 37 &#176;C before dissection allows the RPE to stay attached to the retina even when derived from an adult mouse36.
The serum-free medium and the chemically defined in vitro environment provide for an entirely defined and reproducible manipulation of the experimental conditions. Therefore, organotypic retinal explant cultures are valuable tools in the field of ophthalmology and neuroscience, and have been used for studying retinal diseases37, retina development38,39, retinal stem cell therapy40, genetic modifications41, and pharmacological screening. As a specific example of drug testing, here we used retinal explant cultures to test a cGMP analogue (CN003), known to reduce photoreceptor cell death in animal models for inherited retinal disease23&#160;(Figure 3B). Another possible application of the technique is described in&#160;Figure 3C, which illustrates how the precise control of the tissue environment can be exploited to emulate diabetic conditions24. Because of the preservation of tissue architecture over the entire culturing period, organotypic retinal explant cultures are also suitable for electrophysiological studies. Neuronal functionality on retinal explants have been investigated using patch-clamp recording42&#160;and multi-electrode-array (MEA) recording33,43. The latter allows recording of electrical activity of neuronal populations at the same time and has been exploited to characterize photoreceptor and ganglion cell functionality in culture conditions. In a broader perspective, the organotypic explant culture systems can also be applied in pre-clinical research, where explant cultures were used to test the therapeutic efficacy of hypothermia44.
The organotypic explant culturing technique is relatively simple to perform and, when compared to corresponding in vivo experiments, is less expensive and time-consuming, and avoids the ethical concerns related to live animal studies. The precise control over experimental conditions and the preservation of RPE and tissue complexity make the method a valuable tool to improve our knowledge on retinal physiology and pathophysiology and enable numerous experimental applications.

In ophthalmic research, a variety of models are available to study the retina, including primary retinal cell cultures, retina-derived cell lines, retinal organoids, and in vivo animal models1,2,3,4,5. However, each of these models suffers from drawbacks. For instance, cells grow in isolation while the retina is a complex network with a multitude of cell-to-cell interactions. Thus, the behavior of isolated cell cultures is likely to be artificial compared to that observed in a whole tissue. This problem can in part be remedied using in vitro differentiated retinal organoids, which can be used to study development and basic biology6. Yet, as of today, retinal organoid generation still is time-consuming, labor-intensive, and suffers from reproducibility issues, requiring substantial further development work before organoids can be used for translational retinal research. Finally, studies on live animals, while arguably the model that comes closest to the requirements of ophthalmic research, are associated with strong ethical concerns. A good compromise between the efficiency of cell culture systems and the real-life situation of in vivo animal models are organotypic retinal explant cultures. Such cultures also reduce animal suffering since no in vivo interventions are performed.
Several methods have been described for culturing retinal explants from different species5,7,8. Our protocol describes a technique for the isolation of the mouse neuroretina together with its retinal pigment epithelium (RPE). This technique will also be suitable for rat retinal cultures9. The culture of neuroretina&#160;together with its RPE is of major importance for success. The RPE performs essential functions for the retina: transport of nutrients, ions, water, absorption of light and protection against photooxidation, re-isomerization of all-trans-retinal into 11-cis-retinal, which is crucial for the visual cycle, phagocytosis of shed photoreceptor membranes, and secretion of essential factors for the structural integrity of the retina10.&#160;Maintaining the RPE allows a successful development of photoreceptor outer and inner segments, keeping the retina viable for a longer time11. The procedure described below preserves the histotypic and physiological characteristics of the retina for at least two weeks12. Moreover, culturing the organotypic retinal explants in serum-free, antibiotic-free medium avoids the presence of unknown substances and enables a straightforward interpretation of the results12.
Organotypic retinal explant cultures have been essential for improving our knowledge on retinal development and degeneration7,13,14. We show here that they are also a useful tool for pharmacological screening and that they can be employed to model a variety of retinal diseases, including diabetic retinopathy.

<table><tbody><tr><td>Biotin</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>(+/-)-&amp;alpha;-LipoicAcid (=Thiotic acid)</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>CDP-Choline-Na</td><td>Sigma</td><td>30290</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt; &amp;times; 5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>C8027</td><td></td></tr><tr><td>DL-Tocopherol</td><td>Sigma</td><td>T1539</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E0135</td><td></td></tr><tr><td>FCS</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Filtropur BT100, 1L, 0.2&amp;micro;m</td><td>SARSTEDT</td><td>83.3942.101</td><td>for Basal Medium</td></tr><tr><td>Forceps&amp;nbsp;</td><td>F.S.T</td><td>15003-08</td><td></td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G8540</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma</td><td>I6634</td><td></td></tr><tr><td>L-CysteineHCl</td><td>Sigma</td><td>C7477</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2 &lt;/sub&gt;x 4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M5005</td><td></td></tr><tr><td>Na-pyruvate</td><td>Sigma</td><td>P3662</td><td></td></tr><tr><td>NaSeO&lt;sub&gt;3 &lt;/sub&gt;x 5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Ophthalmic microscope scaping spoon</td><td>F.S.T.</td><td>10360-13</td><td></td></tr><tr><td>Progesteron</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>MP Biomedicals</td><td>21935025</td><td>44 mAnson U/mg</td></tr><tr><td>R16</td><td>Gibco</td><td>07491252A</td><td></td></tr><tr><td>Retinol</td><td>Sigma</td><td>R7632</td><td></td></tr><tr><td>Retinyl acetate&amp;nbsp;</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Scissors</td><td>F.S.T</td><td>15004-08</td><td></td></tr><tr><td>Sterile filter 0.22&amp;micro;m</td><td>MILLEX GP</td><td>SLGP033RS</td><td>for supplements</td></tr><tr><td>T3&amp;nbsp;</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>Tocopherylacetate</td><td>Sigma</td><td>T1157</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T1283</td><td></td></tr><tr><td>Transwell permeable supports</td><td>Corning</td><td>3412</td><td></td></tr><tr><td>Vitamin B1</td><td>Sigma</td><td>T1270</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma</td><td>V6629</td><td></td></tr><tr><td>Vitamin C</td><td>Sigma</td><td>A4034</td><td></td></tr></tbody></table>

long-term, serum-free cultivation of organotypic mouse retina explants with intact retinal pigment epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

After following the protocol, dissected and cultured retinal explants preserve their normal tissue architecture, with distinct layers, from the RPE to the ganglion cell layer (GCL), as shown in&#160;Figure 2. Outer nuclear layer (ONL) and inner nuclear layer (INL) size remained mostly stable for 2-3 weeks, with a slowly progressing cell loss and gradual thinning of these layers becoming more and more apparent if the culturing period is prolonged to 4 weeks and beyond. In the GCL, in contrast, because of the axotomy of the optic nerve, a marked thinning is usually observed within the first 4 days of culturing. Afterwards, the remaining cell population in the GCL (mostly displaced amacrine cells) will continue to be viable for another 3-4-weeks17,18,19.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61868/61868fig02.jpg" /> 
Figure 2: Cell types found in retinal explant culture.&#160;Retinal explant culture at P11 derived from&#160;rd1&#160;mutant (A) and WT animals (B) showing nuclear staining with DAPI (left, blue), rod photoreceptors (center, red) and M&#252;ller cells (right, green). Nuclear staining highlights all the major cellular layers of the retina such as, retinal pigment epithelium (RPE), outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Specific cell types in the nuclear layers, such as rods and M&#252;ller cells, are immunolabeled with alpha arrestin26&#160;and glutamine synthetase27&#160;antibodies, respectively. (C)&#160;Shows full length section of a whole&#160;rd1&#160;mouse retina, with DAPI staining highlighting the consistency, integration, and development of the retina. These retinas were cultured for 6 days. Procedure description: Retina and RPE derived from&#160;rd1&#160;or WT animals were isolated at P5 and cultured as described in the protocol, until P11 with a medium change every 48 h. Cultures were fixed with 4% PFA at P11 and cryosectioned.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig02large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure</a>
Serum-free medium and the sustained in vitro environment allow to have full control over the experimental conditions. Here, we provide two examples for specific applications of this protocol. 
The first example illustrates the possibility to use retinal explants for drug testing or screening purposes. Organotypic retinal explant cultures were prepared from wild-type (WT) and&#160;rd1&#160;mouse models. The latter is a well characterized model for retinal degeneration15. In the&#160;rd1&#160;mouse retina, ONL degeneration is triggered by abnormally high levels of cGMP in rod photoreceptors6,20. Excessive cGMP causes increased activity of cyclic nucleotide gated ion channels (CNGCs) and cGMP-dependent protein kinase (PKG), leading to cell death21. The treatment of&#160;rd1&#160;mouse retinas with a structural analogue to cGMP (cyclic nucleotide #3; CN003), which targets both PKG and CNGC, was tested. After explantation at P5, the treatment paradigm described in Figure 3A was followed. Explant cultures were fixed with 4% PFA at P11 and prepared for cryosectioning (Figure 3A). To assess cell death of histological sections from treated, non-treated (NT), and WT specimens, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay22&#160;was performed. The analysis of TUNEL labeled cells showed a high percentage of dying cells in the ONL of the&#160;rd1&#160;untreated specimens, while CN003 protected&#160;rd1&#160;mouse photoreceptors when applied at a concentration of 50 &#181;M23&#160;(Figure 3B).
A frequent complication of diabetes is diabetic retinopathy, a blinding disease which is difficult to faithfully reproduce in animal models5. The second example highlights the use of organotypic retinal explant cultures to characterize retinal cell viability under conditions emulating type-2 diabetes mellitus (T2DM)24. Here, we used 20 mM of the glycolysis inhibitor 2-deoxy-glucose (2-DG)25&#160;and administered it to the culture medium for 24 h from P10 to P11. We show that subjecting WT retinal explants to such in vitro simulated diabetic conditions leads to extensive neuronal cell death of the retina (Figure 3C). This paradigm in turn may then be used, for instance, to study degenerative mechanisms or to test retinoprotective treatments in a diabetes context.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61868/61868fig03.jpg" /> 
Figure 3: Two examples for applications of organotypic retinal explant cultures.&#160;(A) Procedure description: Retina and RPE derived from&#160;rd1&#160;or WT animals were isolated at post-natal day (P) 5 and cultured as described above, with a medium change every 48 h. At P7 and P9, the spent medium was discarded and fresh CM containing active compound at a concentration of 50 &#181;M was added to the plate. Cultures were fixed with 4% PFA at P11 and cryosectioned. (B) Compound testing on&#160;rd1&#160;retina. Sections were obtained from WT, treated (003), and non-treated (NT) organotypic retinal explant cultures. TUNEL assay (red) was used as a marker for cell death. Nuclear staining with DAPI (blue). The quantification shown in bar graph illustrates the percentages of dying cells in WT,&#160;rd1&#160;NT, and&#160;rd1&#160;003 condition. Treatment with compound 003 significantly reduced&#160;rd1&#160;photoreceptor cell death. (C) Simulation of type-2 diabetes mellitus (T2DM) on WT retina using 2-deoxy-glucose (2-DG) treatment. TUNEL assay was performed on NT and T2DM specimens. The quantification indicates a highly significant increase in cell death.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig03large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure</a>
Table S1.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table S1 - Basal Medium.doc" target="_blank">Please click here to download the table</a>
Table S2.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table S2 - Supplements.doc" target="_blank">Please click here to download the table</a>
Table S3.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table S3 - Complete Medium.doc" target="_blank">Please click here to download the table</a>

Watch this Scientific Journal Video about Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium at JoVE.com

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

long-term-serum-free-cultivation-organotypic-mouse-retina-explants

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K Views.  University of Tübingen. Het protocol beschrijft organotypische explants van het neuro-netvliesvan muizen , gekweekt samen met het retinale pigmentepitheel (RPE), in R16 gedefinieerd medium, vrij van serum en antibiotica. Deze methode is relatief eenvoudig uit te voeren, goedkoper en tijdrovend in vergelijking met in vivo experimenten en kan worden aangepast aan tal van experimentele toepassingen.

Video: Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Developmental Biology

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Biology

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Medicine

Organotypic Retinal Explant Cultures from Macaque Monkey

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Retinal explants obtained from wild-type macaques were cultured in vitro. Retinal degeneration and the cGMP-PKG signaling pathway was induced using the PDE6 inhibitor zaprinast. cGMP accumulation in the explants at different zaprinast concentrations was verified using immunofluorescence.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

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