In this video, we describe a protocol for Organotypic culture of full thickness adult cy and retina. This cartoon shows the overall procedure. First, remove the anterior segment of the adult pig eye.
Use a six millimeter tree, find blade to make a full thickness explan from the posterior pole. Gently attach a piece of sterile paper to the ganglion cell side of the retina. Carefully peel the retina from the retinal pigment Epithelium.
Place the filter paper retina complex photoreceptor side up onto a cell culture dish insert, which is resting on a custom made stand inside a cultured dish. Well, the stand improves the oxygen and nutrient supply for the retina by allowing more space for the medium below the specimen To make a stand, a five millimeter pipette tip was cut off near the base to produce a six millimeter long hollow cylinder, and then pieces were removed from the side of the cylinder to create the three legs. The insert is then placed inside the stand to create a stable complex.
The culture insert and base complex is assembled, placed inside a culture dish and filled with medium to cover the filter of the cell culture.Insert. Sterile culture filters are prepared by Autoclaving Watman filter paper. Porcine eyes are obtained from a local slaughterhouse within three hours of animal sacrifice.
These are the tools required for the dissection. A dissecting microscope is used to better visualize the retina. The extraneous tissue is trimmed off the eyeball.
The eye is dipped in Betadine and washed off with sterile irrigating solution. BSSA two by two gauze is moistened. The optic optic nerve is carefully cut off.
A four to five millimeter incision is made with a blade. Curved scissors are used to make a circumferential cut near the junction between the ciliary body and the neural retina. The anterior segment along with the vitreous are removed leaving the neural retina in the posterior pole.
A six millimeter tree fine is used to make full thickness explan through the posterior pole. A sterile filter paper is carefully applied onto the ganglion cell side of the retina. Once the filter paper is fully moistened, the retina separated from the underlying RP layer by peeling off the filter paper, The filter paper retina complexes, and placed just underneath the level of the culture medium in the prepared culture dish insert.
Here is the culture setup. Here are the culture conditions. After four days of culture, the porcine retinal explan remains flat on the filter paper.
Here's a four day old retinal explan without filter paper po hand retinal was fixed and sectioned either by a cryostat or a viome. After fixation, the retina separated from the filter paper using a spatula, excess fluid is removed for frozen sections. Make square block frame using dental wax.
Place it around the six millimeter retinal explan and submerge everything in tissue tech OCT compound. After the sample block is completely frozen, it is placed on a cryostat sample platform. Vertically my own the sample platform to make sure that the block is vertical to the blade.
Remove the dental wax and start to do the sectioning at the desired thickness place the sections on gelatin treated slides to cut the retina with the vibrating microtome. The explant is embedded in 4%agar. The 4%agar is melted in a boiling water bath and poured into a small plastic container.
When cooled down to about 50 degrees Celsius, the retina sample is then dipped into the melted agar using a spatula. The agar block is solidified at four degrees Celsius and cut into a small cube. With the retina in the middle, the block is glued to the stage of a vibram.
Using crazy glue, the retina is mounted such that it is a 90 degree angle to the Vibram blade. The 50 micron thick sections were transferred underwater onto a glass slide using a soft brush immunohistochemistry. For synaptic vesicle protein two glial fibrillary acidic protein is carried out by using any standard protocol.
The sections were also stained with propidium iodide. For nuclear visualization, the immuno labeled sections are viewed with a confocal microscope. Here are the immuno labeled crass dead sections.
At times zero, at four days, at seven days, the structure of the retina section still remains intact. Cultured pig retina has the potential for many different types of studies. There are many similarities between pig and human retina.
Therefore, when high quality human retina becomes readily available, presumably similar techniques can be applied to studying its morphology and functionality. For more details, please refer to the manuscript.
