Medicine
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Characterization of a Novel Human Organotypic Retinal Culture Technique
Chapters
Summary June 9th, 2021
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.
Transcript
This technique preserves all retinal layers and cell types in situ, making it more clinically relevant compared to animal and in vitro models. The main advantage of this protocol is that it minimizes retinal integrity disruption during tissue handling, which is essential when comparing healthy and diseased retina. Retinal diseases can be mimicked by culturing the tissue under specific conditions.
This allows for testing of new drug therapies. The dissection and sample collection procedures are complex. Therefore, visual demonstration is critical to help the readers understand the written protocol and conduct the procedure successfully.
Demonstrating the procedure will be Charisse Kuo, a second year PhD student from my laboratory. Prepare the culture medium containing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 and a one times antibiotics and antimycotics mixture. First, place 500 microliters of medium in a 24-well plate and equilibrate it in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.
Having the media ready prior to explant extraction avoids dislodging of the retina upon subsequent addition. For extracting retinal explants, begin by placing the eyecup on a Petri dish with the iris and lens facing upwards and the ONH contacting the Petri dish. Hold the eyecup steady at the limbus using forceps and detach the iris and lens by making small cuts circumferentially along the outer edge of the limbus.
Remove the iris and lens carefully, making sure to avoid disturbing the retina. Identify the ONH using a bright white light source and incise at the four quadrants towards the ONH rotating the Petri dish for easier handling. Spread and flatten the eyecup carefully.
Apply the forceps to the sclera instead of the retina to avoid disrupting the retinal integrity. Remove the plate containing the prepared media from the incubator. Place a surgical trephine on the retina in a region without retinal folds and press hard to penetrate the sclera, which should generate a cracking sound as you break through the Petri dish.
Rotate the trephine to ensure the sclera has been penetrated fully such that the retinal explant is now separated from the rest of the sample. Apply the forceps at the sclera and transfer the sandwich retinal explant to the culture medium. Culture the collected sandwich retinal explants at 37 degrees Celsius for up to 72 hours in a humidified 5%carbon dioxide incubator.
Hematoxylin and eosin staining showed that the sandwich retinal explants preserved integrity and a distinct lamellae structure from the GCL to the ONL with compact nuclei in the inner and outer nuclear layers. Disrupted retinal integrity was found at the edges of the same sample where the retina had detached from the underlying RPE choroid and sclera showing reduced retinal thickness and loss of nuclei in the INL and ONL. No TUNEL-positive cell nuclei, which marks cellular apoptosis, were found in retinal explants cultured in basal conditions, demonstrating sustained cellular vitality even after 72 hours.
GFAP labeling was restricted to the GCL and the IPL without glial fibrosis, a sign of pathology which would be identified as extended expression of GFAP into the ONL. Vimentin was highly expressed from the ganglion cell layer to the outer nuclear layer, showing preserved Mueller cell integrity as seen in normal retinal tissues. Luminex magnetic assay showed that the IL-18, VEGF, IL-6, and IL-8 levels increased significantly above baseline in the sandwich retinal explant cultures in high glucose and pro-inflammatory cytokines after 24 hours.
After 72 hours, IL-18 and VEGF levels remained significantly increased, but no significant difference was found in IL-6 and IL-8 levels. When performing this procedure, leave sufficient residual vitreous, which will weigh the retina down so that it does not detach from the RP choroid and float in the culture media. The cultured retina can be further characterized using immunohistochemistry and histology, while the inflammatory cytokines released into the cultured medium can be measured using a Luminex magnetic assay.
This model is more clinically translatable compared to animal and in vitro models, which renders more suitable for testing the efficacy of novel therapeutic drugs for retinal diseases.
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