Abstract
Purpose :
The porcine retina has a similar structure and characteristics compared to the human, and thus, it constitutes a suitable model for experimentation and testing of treatments for retinal diseases. Prolonged survival and excitability of the retina by culturing ex vivo allow more extensive analysis of treatment effects.
Methods :
Porcine retinas were isolated from pig eyes and cultured in either B-27-supplemented Neurobasal A medium, Dulbecco’s Modified Eagle’s Medium (DMEM) or a serum-free DMEM and F12 50:50 mixture for nine days. On day eight retinas were fixated and stained with Draq5 and RbPMS-antibody to assess structural integrity. Viability was determined on day nine by a non-toxic resazurin-based assay, by measuring absorbance at 570 nm. To examine excitability of the tissue, a microelectrode array (MEA) was used to detect extracellular compound action potentials by stimulating by-phasically with 1 msec pulses of increasing voltages (0.5V, 0.8V, 1V and 1.5V) at 10 Hz through a carbon electrode chip. To determine whether recorded spikes were biological in origin, tetrodotoxin (TTX), a selective voltage-gated sodium channel blocker, was applied to the tissue.
Results :
The porcine retinas maintained a reduction capacity for nine days in culture, while a distinct layering of photoreceptors and retinal ganglion cells was still observable after immunostaining. Media composition had no clear effect on viability or excitability of the retinas. At 1.5V stimulation the MEA measured clear spiking from the nine-day-old tissue albeit at a greatly reduced spiking rate and amplitude (200 μV) compared with freshly dissected tissue.
Conclusions :
Isolated retinas cultured for nine days remain metabolically active and can fire action potentials upon electrical stimulation, thus still exhibiting important in vivo characteristics. Future optimizations will likely further increase survivability of the retinas. Hence, genetical modifications influencing the excitability of the retina could be tested in this model, for example optogenetics.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.