Name: Organotypic Culture of Adult Rabbit Retina (Video) | JoVE Journal
Uploaded: 2007-04-29T00:00:00.000+00:00
Duration: 10 min 12 s
Description: Learn organotypic culture of adult rabbit retina for neuroscience research using particle-mediated gene transfer. This method enables GFP visualization of retinal ganglion cell morphology and subcellular dynamics.

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5 minutes

Making gene gun bullets (GOLD)
<ol>
<li>Make 100X PVP (0.5mg/ml) in 100% EtOH.</li>
<li>Weigh out 30mg of BioRad 1.6 Micron microcarrier gold and place it into a 1.5 ml eppendorf tube. </li>
<li>Calculate the amount of plasmid needed to give a concentration of 0.5-3 &micro;g plasmid /mg gold, and place the plasmid into the tube with the gold microcarriers. Combine several plasmids if needed.</li>
<li>Bring volume of plasmid to 100 &micro;l with distilled water.</li>
<li>Add 100 &micro;l of 0.05M spermidine into the tube. Vortex tube 30 seconds.</li>
<li>Vortex 1 minute at full speed. Sonicate for 3-5 minutes..</li>
<li>Dry an appropriate length of Tefzel tubing in a Biorad PDS-1000/He apparatus for 15 minutes.</li>
<li>After sonication, vortex for 1-2 minutes at full speed. Reduce speed slowly to allow the tube to be opened while vortexing.</li>
<li>Add 100 &micro;l of 1 M CaCl2 slowly to the tube drop wise while vortexing.</li>
<li>Leave tube at room temperature for 10 minutes.</li>
<li>Make 5 Micron filter. <ol>
<li>Take two 5ml syringes without needles.</li>
<li>Remove the plunger, and detach the black caps. Taking small scissors, cut off a part of the black cap to give a ring. Repeat on the other plunger to net two rings.</li>
<li>Sandwich a 5 Micron (Small Parts, Inc. nylon filter, &frac12; inch diameter) filter between the two rings, and push the sandwich into the tip of syringe. </li>
<li>Dangle syringe over a 50ml conical flask.</li>
</ol></li>
<li>After 10 minutes, vortex the tube for 30 seconds.</li>
<li>Centrifuge the tube for 30 seconds and save the pellet.</li>
<li>Add 1ml of 100% EtOH to pellet. Use pipette tip to break up the pellet, and vortex 30 seconds. Spin 30 seconds and remove EtOH. Repeat 2 more times.</li>
<li>Add 1ml of 100% EtOH to the pellet (break up with tip). Place this into the 5 Micron filter and let it flow through by gravity into the 50ml conical tube. Use more EtOH as needed to wash out tube or help with flow through.</li>
<li>Spin the 50ml tube at 2000rpm in centrifuge for 5-10 minutes. Remove supernatant, save pellet.</li>
<li>Depending on the desired density of microcarriers needed, add 800&micro;l-1.5ml 100% EtOH and corresponding 8 &micro;l-15 &micro;l PVP solution.</li>
<li>Swirl and immediately go to the Biorad PDS-1000/He apparatus.</li>
<li>Biorad PDS-1000/He apparatus <ol>
<li>Disconnect the tube connecting the N2 gas tank to the drying tube</li>
<li>Take out drying Tefzel tubing from machine</li>
<li>Insert one end into the 50ml conical tube, and attach a syringe to the other end of the tubing. Suck brown slurry into the middle of the tube.</li>
<li>Insert tubing back into machine.</li>
<li>Wait (without rotation) for 4 minutes</li>
<li>After 4 minutes, suck off EtOH slowly and carefully, making sure not to disturb microcarriers.</li>
<li>Rotate the tubing for 1 minute.</li>
<li>After 1 minute, connect the tube connecting the N2 gas tank to the drying tube and let tubing dry for 15 minutes.</li>
</ol></li>
<li>After 15 minutes, cut the tubing to bullet size to be used immediately or stored at 4&deg;C in an exsiccator. Bullets can be stored up to 3 months.</li>
</ol>
Preparation of gene-gun
<ol>
<li>Place bullets into a cartridge. Each piece of retina can be shot with 1-3 bullets.</li>
<li>Insert the cartridge into the gene-gun and attach the gun to a helium source. Set the pressure to 110 Psi.</li>
</ol>
Preparation of media
<ol>
<li>Dissection medium 
<table style="width: 229px; height: 157px;" border="0">
<tbody>
<tr>
<td>&nbsp;1 bottle Ames solution (Sigma, 8.8g) </td>
</tr>
<tr>
<td>&nbsp;1.9 gm sodium bicarbonate</td>
</tr>
<tr>
<td>&nbsp;10 ml 1% pen/strep/L-Glutamine (1:100) (Gibco/Invitrogen)</td>
</tr>
<tr>
<td>&nbsp;990 ml distilled water to 1L</td>
</tr>
<tr>
<td>-------------------------------</td>
</tr>
<tr>
<td>&nbsp;1 Liter</td>
</tr>
</tbody>
</table>
 </li>
<li>Filter the dissection medium using a 0.22 &micro;m filter. </li>
<li>Incubation medium (500ml) 
<table style="width: 283px; height: 157px;" border="0">
<tbody>
<tr>
<td>485 ml of filtered dissection media</td>
</tr>
<tr>
<td>5 ml of 1% N2 supplement (Gibco/Invitrogen)</td>
</tr>
<tr>
<td>10ml 1% horse serum (Sigma)</td>
</tr>
<tr>
<td>500 &micro;l of phenol red</td>
</tr>
<tr>
<td>----------------------------------------------------</td>
</tr>
<tr>
<td>&nbsp;0.5 L</td>
</tr>
</tbody>
</table>
 </li>
<li>Filter the incubation medium using a 0.22 &micro;m filter. </li>
<li>Bubble O2 into the dissection medium for 15 minutes before beginning the retina harvesting protocol. </li>
</ol>
Preparation of incubation chambers for retina
<ol>
<li>Under a hood, fill several 60 mm deep dishes (incubation chambers, Nunc) with 25 ml of incubation medium. The number of dishes depends on the number of retinas needed.</li>
<li>Place a filter stand into each deep dish. Only the very tips of the stand should not be submerged.</li>
<li>Place all deep dishes into the incubator until the retinas are ready.</li>
<li>Fill two 100 mm petri dishes and bring to the dissection bench.</li>
</ol>
Preparation of rabbit retina
<ol>
<li>Sacrifice a rabbit according to established protocols. The following does not have to be performed under a hood.</li>
<li>With angled scissors, insert it behind the eye into the eye socket of the rabbit, cutting the membranes and optical nerve that are attached to the eye.</li>
<li>Gently lift out the eye and wash it with oxygenated dissection media.</li>
<li>Place the eye (cornea facing up) into the well of an eye slicer and close the lid.</li>
<li>Place the blade horizontally right under the lid. In one smooth motion, draw the blade across the eye to cut off the cornea, leaving an eye cup.</li>
<li>Using forceps, remove the retina from the eye slicer well and place it onto a filter paper.</li>
<li>Remove the vitreous and lens by clamping an area near the severed optic nerve and pulling backwards on a filter paper. Repeat several times until almost all the vitreous is removed and the eye cup looks deflated.</li>
<li>Place eye cup into a petri dish with dissection media to wash.</li>
<li>Cut the eye cup into the desired number of pieces (5 pieces maximum).</li>
<li>Take one piece of eye cup and place it under the microscope.</li>
<li>Clamp the sclera and choroid lightly together on the edge of retina using forceps. With the other hand, use a fine brush to brush right between the choroid and retina.</li>
<li>Using short, gentle motions, tease the retina off the choroid until it becomes detached.</li>
<li>Cut 1 inch off a sterile transfer pipette. Use this to suck up the retina and place into a petri dish of incubation medium to wash.</li>
<li>Using another sterile transfer pipette with 1 inch cut off, carefully transfer the retina onto a 0.4 &micro;m Millicell filter (Millipore, Cat No: PICMORG50).</li>
<li>Use a brush to orient the retina with the ganglion cell side facing upwards.</li>
<li>Flatten out the retina by brushing lightly on the edges of the retina. Minimize touching the ganglion cell layer with the brush as much as possible.</li>
<li>Remove excess medium on filter with a transfer pipette.</li>
<li>Using forceps, transfer the filter onto a modified suctioner. Draw on the suctioner 2-3 times to remove all the incubation medium from the filter.</li>
</ol>
Gene gunning and incubation of retina
<ol>
<li>Under a hood, place each of the retina-containing-filters onto the filter stands in the 60 mm deep dishes (incubation chambers).</li>
<li>Ensure that the medium is in contact with the underside of the filter and that the medium does not spill over the sides of the filter onto the retina. The filter should act as an interface between the retina and medium.</li>
<li>Shoot each piece of retina with two bullets. Place gene gun directly above tissue, about 3-4 cm away.</li>
<li>Place all incubation chambers onto a shaker in the 35 - 37&deg;C incubator.</li>
<li>Replace with incubation medium every day. The retina can be incubated for a maximum of 6 days.</li>
</ol>

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<li>Stacy, R. C., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+relationship+between+cholinergic+amacrine+cell+processes+and+ganglion+cell+dendrites+of+the+mouse+retina%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Developmental relationship between cholinergic amacrine cell processes and ganglion cell dendrites of the mouse retina.</a> J Comp Neurol. 456, 154-166 (2003).</li>
<li>Strettoi, E., Pignatelli, V., Rossi, C., Porciatti, V., Falsini, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remodeling+of+second-order+neurons+in+the+retina+of+rd%2Frd+mutant+mice%5BTitle%5D)+AND+%22Vision+Res%22%5BJournal%5D)">Remodeling of second-order neurons in the retina of rd/rd mutant mice.</a> Vision Res. 43, 867-877 (2003).</li>
<li>Pignatelli, V., Strettoi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bipolar+cells+of+the+mouse+retina%3A+a+gene+gun%2C+morphological+study%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Bipolar cells of the mouse retina: a gene gun, morphological study.</a> J Comp Neurol. 476, 254-266 (2004).</li>
<li>Connaughton, V. P., Graham, D., Nelson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+morphological+classification+of+horizontal%2C+bipolar%2C+and+amacrine+cells+within+the+zebrafish+retina%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina.</a> J Comp Neurol. 477, 371-385 (2004).</li>
<li>Edqvist, P. H., Hallbook, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newborn+horizontal+cells+migrate+bi-directionally+across+the+neuroepithelium+during+retinal+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Newborn horizontal cells migrate bi-directionally across the neuroepithelium during retinal development.</a> Development. 131, 1343-1351 (2004).</li>
<li>Jakobs, T. C., Libby, R. T., Ben, Y., John, S. W., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+ganglion+cell+degeneration+is+topological+but+not+cell+type+specific+in+DBA%2F2J+mice%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice.</a> J Cell Biol. 171, 313-325 (2005).</li>
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</ol>

1. What can I do with this method?
<ul>
<li>See cell morphology clearly.</li>
<li>Label cells to record from them.</li>
<li>Overexpress proteins, like PSD95, but also other proteins that
modulate the cell's response, e.g. ion channels, receptors.</li>
<li>Express inhibitory RNAs.</li>
</ul>
2. How long can I keep adult retina?
<ul>
<li>4 days is no problem for adult rabbit. After six days the cells
look "sick", i.e. the axons retract, you see swelling of dendrites
and the recor...

organotypic culture of adult rabbit retina

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Hi, my name is, And today I'll show you the incubation methods of a adult rabbit retina and the subsequent gene.Great. Washing several signs. I think it also dries out the retina.Yeah, that's right. That's right, that's right.Yeah. So you mean all absolve water from the cell so that dries out.Yeah, I think, yeah. So tissue, that's fine. Do you Oh, it's okay.It's okay. So now gang sell side up very front. This, let's first introduce ourselves.Yeah, I'm Tiana. I'm Amari. You've already met.Yeah.So what can I do with this method? Well, you can culture adult webinars. And since you can also gene gun it and express for instance GFP, it's very useful to see the morphology of sickle cells very clearly.So you can do anatomy on these cells or also you can label cells with DS red or with GFP to find them for later recording, amp recording for instance. It is also possible to express, for instance, synaptic marker proteins like PSD 95 GFP, and also other proteins which you might be interested in studying in the retina, like ion channels. It could also be used to express inhibitory RNAs.Yeah.So what are the limitations of this na, Well, I think probably the most important is the time. You cannot keep the retina for longer than six days. I would say we don't trust the retina after six days anymore.This is because of, this is mized spread Probably, yeah. And now if you just want to express GFP, which comes on after 24 or 48 hours, that's no problem at all. But in some cases, for instance, if you wanna express inhibitory RNAs, now these effects may take four days or even more to develop.And you have to keep these timelines in frame. In, in, in. You have to, to, to make sure that you will see your effect in a day in, in a time which is shorter than six days because you can't reliably keep it longer.So this may or may not be possible with your, with the experiment you are, you are planning to do. But this certainly is the biggest limitation. It is medium term, let's say.So some, some RN ai work within four days, but the others work more.Right. Maybe it's takes five or six days or more. In that case, it's not suitable for this kind of a wrong may.Yeah, may not be possible. I should say perhaps that during the time we use for incubation, that is up to six days. We do not see any signs of re reorganization of the retina.Really the retinas do look normal morphologically.Okay. So six days morphology is good, but some visual carry, it looks unreliable.Physiologically.Yes. And also you start to see swelling of den, something like that.So no, what I meant with the retina looks normal is the layer structure of the retina is still intact. And in principle, some of the cells are still life responsive. It might be that the photoreceptors die or degenerate after six days too.So that might explain why we can't reliably record life responses anymore. But by and large, mostly the retina looks normal.Okay. Any other points Or, okay.Okay.

organotypic-culture-of-adult-rabbit-retina

Research

JoVE Journal

Biology

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.

In vivo-like Organotypic Murine Retinal Wholemount Culture

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.

Organotypic Culture of Full-thickness Adult Porcine Retina

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 µm) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

A Step by Step Protocol for Subretinal Surgery in Rabbits

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

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

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

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

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.

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

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 epithelium-choroid (RPE-choroid) and sclera.

Characterization of a Novel Human Organotypic Retinal Culture Technique

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

Obtaining High-Quality Cryosections of Whole Rabbit Eye

Learn organotypic culture of adult rabbit retina for neuroscience research using particle-mediated gene transfer. This method enables GFP visualization of retinal ganglion cell morphology and subcellular dynamics.

Organotypic Culture of Adult Rabbit Retina

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