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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>(+/-)-&#945;-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>CuSO4 &#215; 5H2O</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&#181;m</td>
			<td>SARSTEDT</td>
			<td>83.3942.101</td>
			<td>for Basal Medium</td>
		</tr>
		<tr>
			<td>Forceps&#160;</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&#160;</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>MnCl2 x 4H2O</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>NaSeO3 x 5H2O</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&#160;</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&#160;</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&#181;m</td>
			<td>MILLEX GP</td>
			<td>SLGP033RS</td>
			<td>for supplements</td>
		</tr>
		<tr>
			<td>T3&#160;</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/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/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/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/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/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

Research

JoVE Journal

Neuroscience

8.4K Views.  University of Tübingen. 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.

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...